Current Status on Biochemistry and Molecular Biology of Microbial Degradation of Nicotine

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 125385 15 pageshttpdxdoiorg1011552013125385

Review ArticleCurrent Status on Biochemistry and Molecular Biology ofMicrobial Degradation of Nicotine

Raman Gurusamy and Sakthivel Natarajan

Department of Biotechnology School of Life Sciences Pondicherry University Puducherry 605014 India

Correspondence should be addressed to Sakthivel Natarajan puns2005gmailcom

Received 18 August 2013 Accepted 14 October 2013

Academic Editors N Ercal and T Niu

Copyright copy 2013 R Gurusamy and S Natarajan This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Bioremediation is one of the most promising methods to clean up polluted environments using highly efficient potent microbesMicrobes with specific enzymes and biochemical pathways are capable of degrading the tobacco alkaloids including highly toxicheterocyclic compound nicotine After the metabolic conversion these nicotinophilic microbes use nicotine as the sole carbonnitrogen and energy source for their growth Various nicotine degradation pathways such as demethylation pathway in fungipyridine pathway in Gram-positive bacteria pyrrolidine pathway and variant of pyridine and pyrrolidine pathways in Gram-negative bacteria have been reported In this review we discussed the nicotine-degrading pathways of microbes and their enzymesand biotechnological applications of nicotine intermediate metabolites

1 Introduction

Tobacco (Nicotiana Solanaceae family) is mainly cultivatedin Brazil China Cuba India and USA An annual produc-tion of 67million tonnes of tobacco has been reported Chinais the largest tobacco producer (396) followed by India(83) Brazil (70) and the USA (46) [1] India is thethird largest tobacco consumer (275 million) in the worldfollowed by China and USA [2] It is anticipated that thetobacco industry would produce 300274 tonnes of nicotinewastes every year [3] Nicotine usually accounts for morethan 90 of the whole plant alkaloid fraction in commercialtobacco Nicotiana tabacum [4] The entire or part of thetobacco leaf was used as raw material for tobacco productssuch as cigarette cigars chewing tobacco and snuff

In the year 2000 it was estimated that 49 million deathsoccur due to smoking [5] By the year 2020 it is also expectedto exceed 9 million deaths annually [5] Due to the increasedusage of tobacco products the industry generated solidand liquid tobacco wastes containing high concentrationsof nicotine [6] The tobacco industries produce tobaccowaste with an average content of nicotine of 18 g per kg

of dry weight [7] The Environmental Protection Agency(EPA) has classified these nonrecyclable powdery tobaccowastes as Toxic Release Inventory (TRI) chemicals [8] Whenthe concentration of nicotine content exceeds more than005 (ww) it is designated as ldquotoxic and hazardousrdquo bythe European Union Regulations (EUR) [7] These tobaccowastes are dumped on the groundwithout proper storage andprocessing [7 8] Nicotine dissolves easily in water leadingto the contamination of the ground water [7 8] Hence thenicotine-contaminated water disturbs the ecological balanceof soil [7 8] Therefore it is important to remove nicotinefrom tobacco polluted soil and water [7 8]

2 Nicotine

Nicotine 3-(1-methyl-2-pyrrolidinyl) pyridine is a hetero-cyclic compound with a pyridine and a pyrrolidine ringmoiety Nicotine is a pale yellow to dark brown liquid withslight fishy odorwhenwarm It is water-solubleThe chemicalformula of nicotine is C

10H14N2 Its molecular weight is

162234 with melting point ndash79∘C and boiling point 247∘C

2 The Scientific World Journal

Nicotine is present up to 2 to 8of the drymass of the tobaccoleaves [9]

21 Effect of Nicotine on Human Beings Nicotine is a haz-ardous compound that causes tobacco related lung cancerand peripheral arterial disease [10] Although more than4000 substances are present in the tobacco cigarette smokenicotine is the major substance [11 12] Nicotine has ablood half-life period of approximately 2 h and causes severevascular diseases [11 12] Nicotine can cause cancer genemutation and malformation [13] An array of toxic nicotineintermediate metabolites such as N1015840-nitrosonornicotine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone cotinine andN-nitrosamine causes tobacco lung cancer [14 15] Neuro-toxin developmental effects of nicotine can naturally affect avariety of cellular processes such as generation of oxidativeradicals apoptosis and hyperplasia of cell enhancing geneexpression to secrete hormones and regulation of enzymaticactivity [13 16 17] The lungs rapidly absorb 90 of thenicotine that is present in the cigarette smoke inhaled byhuman beings [18] Nicotine in the human body can easilypass the biological membranes and blood-brain barrier andincreases heart rate mean arterial blood pressure andmimicsthe venous endothelial dysfunction [15 19 20]

Nicotine is an additive substance that can lead to nicotinedependence and addictive behavior in human populations[43] Nicotine dependence is complex multidimensionaltrait that involves psychological physiological behavioraland social factors [44] Nicotine the primary psychoactiveingredient of tobacco contributes to physical dependenceby acting on nicotinic acetylcholine receptors in the centralnervous system and leads to the release of neurotransmitters(eg dopamine and serotonin) which produce reinforcingeffects by activating the mesocorticolimbic dopamine system[45]

3 Microbial Degradation of Nicotine

Physical and chemical methods are available to degrade nico-tine in the tobaccoThesemethods are often time-consumingexpensive and involve solvent extraction procedures [46]Bioremediation is one of the promising methods to clean uppolluted environments using microbes [47ndash49] Briski et al[50] reported that aerobic composting is an effective methodto reduce 80 of nicotine and 50 of the volume andmass oftobacco solid wastes in 16 dMeher et al [51] used a techniquecalled biomethanation that removed 60 of nicotine 756of chemical oxygen demand and 80 of biological oxygendemand from the tobacco wastes Biological method employsa variety of nicotine-degrading bacteria and fungi [41 46 51]These ecofriendly biological methods are extensively usedin wastewater treatment due to its high efficiency and lowcost Microbes that degrade nicotine are reported to adapt topolluted environment easily [52 53]Native strains of bacteriaand fungi that live in the tobacco environment have the abilityto degrade nicotine [54] These microbes utilize nicotine assole carbon nitrogen and energy source for their growth

[6] Batham [55] reported that the nitrate content of the soilincreased due to the microbial degradation of nicotine

31 Optimal Conditions of Nicotine-Degrading BacteriaNicotine degradation experiments are carried out with dif-ferent culture media such as distilled water minimal saltmedium inorganic salt medium and basal salt medium(BSM)The culture media influence the nicotine degradationefficiency Wang et al [28] reported that 3 gL nicotine wasfully degraded in 5 h when the degradation experiment wascarried out with 005M sodium phosphate buffer (pH 70)However it tookmore than 8 h to complete degradationwhencarried out with distilled water The optimal culture condi-tions and nicotine degradation efficiency of nicotinophilicbacteria vary from each other Most of the bacteria growat 30∘C pH range of 64 to 75 and degrade maximumconcentration of nicotine up to 6 gL [2 5] Huang et al[56] reported that the rate of nicotine degradation efficiencywas high at 37∘C when compared to 30∘C The optimalconditions and nicotine degradation efficiency of variousnicotine-degrading bacteria are presented in Table 1

32 Effect of Trace Elements Carbon and Nitrogen Sources onNicotine Degradation Trace elements and other carbon andnitrogen sources play a significant role in the biodegradationof nicotine [26] The presence of (NH

4)2SO4in the nicotine

containing medium could decrease the nicotine degradationefficiency [26] The nicotine degradation rate was increasedin the presence of yeast extract glucose and Tween 80in tobacco waste extract (TWE) containing media [57]However the nicotine degradation rate was dependent onthe concentration of yeast extract and Tween 80 in the TWEmedium [27] In contrast the medium containing glucoseand (NH

4)2SO4reduced the efficiency of nicotine degrada-

tion [32] ZnSO4sdot7H2O and NiCl

2sdot6H2O had no influence on

nicotine degradation whereas Na2MoO4and CuCl

2sdot4H2O

inhibited the rate of nicotine degradation [39] Glucose is animportant carbon source that promotes bacterial cell growthand improves the rate of nicotine degradation [24 34 39]Theconcentration of glucose above 10 gL inhibited the nicotinedegradation by Pseudomonas sp ZUTSKD [34] Ramanet al [2] reported that 1 gL dextrose increased nicotinedegradation rate In contrast glucose is not influenced in thenicotine degradation rate when the experiment was carriedout in solid-state fermentation process [39 56] Jiang et al[36] reported that the nicotine degradation rate was slowin the presence of glucose Conversely P stutzeri ZCJ couldnot utilize carbon sources such as sucrose and maltose andnitrogen source namely NaNO

2 for their growth which in

turn inhibits nicotine degradation [39]

33 Effect of Nicotine-Degrading Bacteria on Tobacco Leavesand Tobacco Wastes Nicotine-degrading bacteria played animportant role in the improvement of quality of tobaccoleaves during aging (fermentation) process These bacteriaaccumulatedmore in younger tobacco leaves when comparedto aged leaves [56] The desirable flavor taste and smokingproperties of tobacco remained unaltered when treated with

The Scientific World Journal 3

Table1Th

eoptim

alcond

ition

snicotin

edegradatio

neffi

ciencyand

degradationpathwayso

fnicotine-degradingbacteria

Microorganism

Medium

Optim

alcond

ition

s(pHandTemperature)

Nicotined

egradatio

neffi

ciency

(gL)

Pathway

Reference

Fung

iPellicularia

filam

entosa

JTS-208

Synthetic

ND

004

20d

Dem

ethylatio

n[21]

Cunn

ingham

ellaechinu

lata

IFO-444

4Synthetic

ND

05413

dDem

ethylatio

n[21]

Aspergillus

oryzae

112822

Synthetic

6528∘C

21240h

Avaria

ntof

pyrid

inea

ndpyrrolidine

[22]

Gram-positive

bacteria

Arthrobacternicotin

ophagum

Nsp

Synthetic

68to

7020to

25∘C

16256

hPy

ridine

[23]

Arthrobacter

spH

F-2

Synthetic

7030∘C

07043h

ND

[24]

Gram-negativeb

acteria

Pseudomonas

sp41

Synthetic

6430∘C

13024

hPy

rrolidine

[25]

Pseudomonas

spH

F-1

Synthetic

65to

7530∘C

13025

hPy

rrolidine

[26]

Ochrobactr

uminterm

edium

DN2

Synthetic

30∘C

05036h

ND

[27]

Pputid

aS16

Synthetic

7030∘C

30010

hPy

rrolidine

[28]

Pseudomonas

spN

ic22

Synthetic

6530

to34∘C

28952h

ND

[29]

Pputid

aJ5

Synthetic

30∘C

39748h

ND

[30]

RhodococcusspY2

2Synthetic

7028∘C

10052

hND

[31]

Agrobacte

rium

tumefa

ciens

S33

Synthetic

7030∘C

50018

hAvaria

ntof

pyrid

inea

ndpyrrolidine

[32]

Gram-negativeb

acteria

Ensifer

spN

7Synthetic

7028∘C

33548h

ND

[33]

Pseudomonas

spZ

UTS

KDSynthetic

7530∘C

15512h

ND

[34]

Pseudomonas

spH

ZN6

Synthetic

7030∘C

05012

hPy

rrolidine

[35]

ShinellaspH

ZN1

Synthetic

7030∘C

0509h

ND

[36]

AcinetobacterspTW

Synthetic

7030∘C

10012h

ND

[37]

Sphingom

onas

spT

YSynthetic

7030∘C

10018h

ND

[37]

Pseudoxanthomonas

sp5ndash52

Synthetic

7028∘C

10028

hND

[38]

Pstu

tzeri

Synthetic

7537∘C

25036h

ND

[39]

Sinorhizobium

sp5ndash28

Synthetic

7028∘C

15028

hND

[38]

Ochrobactrum

sp4ndash4

0Synthetic

7028∘C

05028h

ND

[38]

Pplecoglossicid

aTN

D35

Synthetic

7030∘C

3012h

Avaria

ntof

pyrrolidine

[2]

NDnot

detected


Table 2 The nicotine degradation efficiency of different nicotine-degrading bacteria on tobacco leaves and tobacco wastes

Bacterium Source Duration(timetemperature)

Nicotine(mgg)

Nicotine degradation() Reference

Micrococcus nicotianae Tobacco leaves NM NM 083 [40]Debaryomyces nicotianae Tobacco leaves NM NM 045 [40]Cellulomonas sp Tobacco leaves NM NM 1500 [41]A oxidans 120572-2 Tobacco waste 212 h 700 NM [7]A oxidans pAO1 Tobacco waste 125 h 3400 NM [7]P putida Tobacco waste 40 h 3300 NM [7]P putida J5 Tobacco leaves 7 d 28 1172 [30]Pseudomonas sp Nic 22 Tobacco leaves 30∘C NM 3310 [29]Ensifer sp N7 Tobacco leaves NM 41 1600 [42]Arthrobacter sp Tobacco leaves NM NM 2800 to 3900 [33]Pseudoxanthomonas sp 5ndash52 Tobacco leaves NM NM 4720 [38]Sinorhizobium sp 5ndash28 Tobacco leaves NM NM 7250 [38]Ochrobactrum sp 4ndash40 Tobacco leaves NM NM 5150 [38]P stutzeri ZCJ Tobacco leaves 7 d37∘C NM 3224 [39]P plecoglossicida Tobacco leaves 10 h 13 8800 [2]P plecoglossicida Tobacco wastes 10 h 5 9610 [2]NM not mentioned

nicotinophilicPseudomonas [6 29] Li et al [42] reported thatArthrobacter sp successfully reduced 28 to 39 of nicotineduring flue curing of tobacco leaves and enhanced thequality of tobacco leaf The nicotine degradation efficiency ofdifferent nicotine-degrading bacteria on tobacco leaves andtobacco wastes is listed in Table 2

The two major bacterial species that degrade nico-tine are Pseudomonas and Arthrobacter [6] Furthermorerecently a new nicotine-degrading strain Arthrobacter spstrain M2012083 was sequenced by Illumina High-Seq 2000which represented the first sequenced nicotine-degradingArthrobacter strain [58] Tang et al [59] recently sequenceda novel bacterial strain Pseudomonas geniculata N1 whichrepresents the first sequence of the Pseudomonas geniculategroup

34 Pigment Production during Nicotine BiodegradationDuring nicotine degradation different colours were pro-duced The Gram-positive bacteria A nicotinovorans uti-lized nicotine and changed to yellow pigments and furtherturned carmine during nicotine degradation [60ndash62] Incontrast Hylin [23] reported that A nicotinovorans didnot produce any pigments during nicotine degradationArthrobacter sp produced blue violet colour during nicotinedegradation due to the transformation of nicotine into236-trihydroxypyridine The colour change was due to thepresence of oxygen and the absence of metabolic catalyzingenzymes [63 64] The hydroxylated pyridine ring dimerizedand formed a blue pigment during nicotine degradation byArthrobacter sp [62] Interestingly during nicotine degrada-tion the Gram-negative bacteria such as Pseudomonas spShinella sp HZN1 and Acinetobacter sp ND12 changed thecolour of the nicotine medium to green and oxidized to

blue gray and finally to brown [2 26 31 32 36 42 57]The formation of green colour was mainly dependent uponthe concentration of nicotine [2 32 36] P geniculata N1produced golden yellow pigment on the plate during nicotinedegradation [59] However no pigment was observed duringnicotine degradation by P putida and Ensifer sp N7 [30 33]

35 Microbial Degradation Procedure Standard biodegrada-tion methods were employed to treat tobacco wastes [30]Initially the tobacco wastes are sterilized under UV lightThe resting cells of biodegrading strain are inoculated intotobaccowastesThen the tobaccowastes are baked at 60∘C for6 days crushed to a powder and passed through a sieve withmuslin clothThe tobacco powder is extracted with methanoland the concentration of nicotine is determined as describedin [30]

4 Biochemical Pathways That MediateNicotine Biodegradation

Biodegradation of nicotine by A nicotinovorans and Aoxidans has been reported [65 66] These Gram-positivebacteria followed pyridine pathway The nicotine interme-diate metabolites of pyridine pathway were identified andcharacterized [67] The microbial degradation of nicotinediffers among the different strains of species [54] Previousresearch suggests that the encoding genes that mediatenicotine degradation are not only located on the bacterialchromosomes but also present in the plasmids [65 6668] Plasmid-borne genes (160Kb) in A nicotinovorans areresponsible for nicotine degradation [65 66] Similarlynicotine-degrading genes are located on the outside of thechromosomes of P convexa [68] The bacteria such as


Arthrobacter sp (Gram-positive) followed pyridine pathwaywhich attacked the pyridine ring of the nicotine duringdegradation The Gram-negative bacteria Pseudomonas spinitially attacked pyrrolidine ring and followed pyrrolidinepathway Fungi employed the demethylation pathway thatdemethylates methyl group in the pyrrolidine ring of thenicotine Surprisingly Agrobacterium sp followed a newvariant pathway of pyridine and pyrrolidine for nicotinedegradation Several nicotine-degrading genes have beenreported [6 45 69]

41 Pyridine Pathways of Nicotine Biodegradation Pyridinepathways of nicotine degradation by A nicotinovoransNocardioides sp JS614 are presented in Figure 1 A nicoti-novorans harbors a 160Kb plasmid which is responsiblefor nicotine degradation [65 66] These bacteria initiallyattacked 6th position of carbon in the pyridine ring ofnicotine by hydroxylation to introduce hydroxyl groupand formed 6-hydroxynicotine (6-HN) This hydroxyla-tion step was catalyzed out by nicotine dehydrogenase(NDH) It is a heterotrimeric molybdenum enzyme callednicotine accepter oxidoreductase [70ndash73] This NDH is aheterotrimeric enzyme of xanthine dehydrogenase familycontaining dinucleotide form of molybdopterin cofactor aflavin adenine dinucleotide (FAD) and two iron-sulphurclusters [74] This 6-HN was oxidized to form an opticallyinactive 6-hydroxy-N-methlymyosmine (6-HMM) by oxi-dation of pyrrolidine ring at the 2nd position of carbonInterestingly this step was catalyzed by two enzymes namely6-hydroxy-L-nicotine oxidase (6-HLNO) and 6-hydroxy-D-nicotine oxidase (6HDNO) The dimeric form of 6-HLNOand monomeric form of 6HDNO contained FAD noncova-lently bound to 47 kDa and covalently to 49 kDa polypeptiderespectively [75] The third step was followed by hydrationof 6-HMM that spontaneously leads to the opening of thepyrrolidine ring and tautamerization of ketone moiety toform 6-hydroxypseudooxynicotine (6-HPON) [76] The 2ndposition of the carbon of pyridine ring of this metabolite wasfurther hydroxylated by ketone oxidase or ketone dehydroge-nase enzyme similar to nicotine dehydrogenase to form 26-dihydroxypseudooxynicotine (26-DHPON) [77]The subse-quent cleavage of side chain of 26-DHPON to form 120574-N-methylaminobutyrate (MGABA) and 26-dihydroxypyridine(26-DHP) by the action of 2-6-dihyroxypseudooxynicotinehydrolase (26-DHPONH) [61 78ndash80] 26-DHP was furtherhydroxylated to form 236-trihydroxypyridine (23-6-THP)by the addition of hydroxyl group to the 3rd positionof carbon in the pyridine ring This step was catalyzedby an enzyme 26-dihydroxypyridine-3-hydroxylase (26-DHPH) in a strictly nicotinamide adenine dinucleotidehydrogen (NADH)-dependentmanner Dimeric flavoproteinof this enzyme is tightly bound noncovalently to a FADsubunit and inhibited by 23-dihydroxypyridine and 26-dimethoxypyridine Each subunit consists of 397 amino acidsandmass of 434 kDawith addition of one FADmolecule [81]In the presence of oxygen spontaneous oxidation of 236-THP and dimerization of this hydroxylated pyridine ringmoiety formed nicotine blue colour [63] However Sachelaru

et al [82] reported that mobA gene that encodes MobAprotein with molybdenum cofactor cytidylate transferasewas responsible for formation of nicotine blue colour in Anicotinovorans

The degradation of MGABA was regulated by clus-ter of genes encoded by plasmid pAO1 This clusterof genes contained purU-mabO-folD operon that tran-scribed only in the presence of nicotine and was regu-lated by transcriptional activator pmfR [83] MGABA wasfurther degraded into two pathways The first pathwaystarted with an enzyme 120574-N-methylaminobutyrate oxidase(MABO) which catalyzedMGABA to form 120574-aminobutyrate(GABA) and methylenetetrahydrofolate The methylenegroup of methylenetetrahydrofolate was further oxidizedby two enzymes namely methylene-tetrahydrofolate dehy-drogenasecyclohydrolase (FolD) and formyltetrahydrofo-late deformylase (PurU) to formaldehyde [77] These twoenzymes are nicotinamide adenine dinucleotide hydrogenphosphate (NADPH) dependent and formaldehyde maybe assimilated in the Embden-Meyerhof pathway [84 85]Another metabolite GABA metabolized into an ammoniaand succinic semialdehyde (Ssa) by monoamine oxidase(MAO) is an amine oxidase (AO) enzyme family Ssa isfurther oxidized to succinic acid by an enzyme called nicoti-namide adenine dinucleotide phosphate (NADP+) depen-dent succinic semialdehyde dehydrogenase (SsaDH) [86 87]This succinic acid entered into citric acid cycle and thiscatabolic pathway of 120574-aminobutyrate was normally foundin bacteria [88ndash90] A newly discovered second degradationpathway ofMGABA is deaminated into Ssa andmethylamineby amine oxidase (AO) with reduction of FAD to FADH

2

It produced succinic acid which entered citric acid cycle[83 91]

42 Pyrrolidine Pathways of Nicotine BiodegradationThe extrachromosomal genes of Pseudomonas sp thatdegrades organic pollutants such as octane camphortoluene methyl benzoate salicylate naphthalene andxylene are responsible for nicotine degradation [68] Apurple crystalline substance N-methylmyosmine wasisolated from nicotine containing medium during thedegradation of nicotine by A oxydans [92 93] Thetransformation of intermediate metabolites of nicotinesuch as 3-nicotinoylpropionic acid pseudooxynicotine andN-methylmyosmine produced by Pseudomonas sp has beenreported [25 94ndash96] Thacker et al [68] reported that Pconvexa Pc1 degraded nicotine to 25-dihydroxypyridine (25-DHP) via pseudooxynicotine 3-succinoyl pyridine (SP)and 6-hydroxy-3-succinoyl pyridine (HSP) by nicotine-degrading enzyme 25-dihydroxypyridine oxygenase Pplecoglossicida TND35 degraded nicotine to 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) via N-methylmyosmine[2]

The predominant nicotine-degrading Gram-negativePseudomonas sp followed pyrrolidine pathway [97]However the mechanism of this pathway has been studiedpoorly [98] Pseudomonas sp followed mainly four differentpathways of pyrrolidine (Figure 2) Pyrrolidine pathway


N

N

NN

N

NHO

N

O

NNH

O

HO

O

O

O

N

N

N

NDH

KOKDH

N

NOO

O

MABO

H H

O

Nicotine

(Ssa)

2H

6-HLNO6-HDLO

26-DHPONH

2H26-DHPH

+

Formaldehyde

FolDPurU

+

MAO

Methylamine

AO

SsaDH

Spontaneous

Spontaneous

Ammonia

H2O

H2O

HO

HO

HO

HO

HO

HO

HOHO

HOOHOH

6-Hydroxy-nicotine (6-HN)

O2

O2

6-Hydroxy-N-methylmyosmine (6-HMM)

6-Hydroxypseudooxynicotine (6-HPON)

OH

OH

OH

HO

OH

HO

OH

OH

26-Dihydroxypseudooxynicotine (26-DHPON)

26-Dihydroxypyridine (26-DHP)120574-Methylaminobutyrate (MGABA)

236-Trihyroxypyridine (236-THP)

Nicotine blue

NH2

120574-Aminobutyrate (GABA) Methylene tetrahydrofolateCH2TH4

H2NCH3

Citric acid cycle

NH3

Succinic semialdehyde

Embden-Meyerhof pathway

Succinic acid

O2

NH

2HH2O

H2O

CH3

CH3

CH3

Figure 1 The pyridine pathway of nicotine degradation by A nicotinovorans and Nocardioides sp NDH nicotine dehydrogenase 6-HLNO6-hydroxy-L-nicotine oxidase 6HDNO 6-hydroxy-D-nicotine oxidase KO ketone oxidase KDH ketone dehydrogenase 26-DHPONH2-6-dihyroxypseudooxynicotine hydrolase 26-DHPH 26-dihydroxypyridine-3-hydroxylase MABO 120574-N-methylaminobutyrate oxidaseMAO monoamine oxidase AO amine oxidase FolD methylenetetrahydrofolate dehydrogenasecyclohydrolase PurU formyltetrahydrofo-late deformylase SsaDH succinic semialdehyde dehydrogenase


NN

HO

O

O

O

NicA

NN

N

O

O

N

N

NHO

N

OHO

O

PNAO

OO

OO O

OOH

N

HO

O

SAPD

NN

NN

NNH

NNH

NN OH

NN O

Nicotine

HspA

Methylamine

Pseudooxynicotine

Spontaneous

SsaDH

3-(34-Dihydro-2H-pyrrol-5-yl) pyridine

Nicotyrine

Nornicotine

Myosmine

1-Methyl-5-(pyridin-3-yl) pyrrolidin-2-ol

Cotinine

1

2

3

4

5

HspB

CH3

CH3

HO

HO

HO

HOHO

OH

OH

OH

OH

OH

N-Methylmyosmine

3-Succinoylsemialdehyde pyridine (SAP)

H2NCH3

3-Succinoyl pyridine (SP)

6-Hydroxy-3-succinoyl pyridine (HSP)

25-Dihydroxypyridine (25-DHP) Succinic semialdehyde (Ssa)

H2N

Maleic acid Pyruvic acid

Citric acid cycle

Succinic acidMaleamic acid

CH3

H2O

Figure 2 Various pyrrolidine pathways of nicotine degradation followed by 1 Pseudomonas sp HZN6 and P putida S16 2 Pseudomonas spHF-1 3 Pseudomonas sp HF-1 and Pseudomonas sp Nic22 4 Pseudomonas sp HF-1 Pseudomonas sp Nic22 and Pseudomonas sp CS3 5Pseudomonas sp CS3 NicA nicotine oxidoreductase PNAO pseudooxynicotine amine oxidase SAPD 3-succinoylsemialdehyde pyridinedehydrogenase HspA and HspB 6-hydroxy-3-succinoyl pyridine hydroxylase


of nicotine degradation of P putida S16 has been reported[28] (Figure 2) The intermediate metabolites and itsnicotine-degrading genes of pyrrolidine pathways ofother Pseudomonas sp have not been fully elucidated orthoroughly characterized Ruan et al [26] reported thatthe Pseudomonas sp HF-1 followed three other incompletepyrrolidine pathways such as nicotine to cotinine nicotyrineand nornicotine (Figure 2) However Pseudomonas sp Nic22degraded nicotine via cotinine and nornicotine pathways ofpyrrolidine [29]

The pyrrolidine pathway of P putida S16 initially attackedthe pyrrolidine ring of nicotine to give N-methylmyosmineby the formation of a double bond NicA enzyme wasinvolved in the first step of dehydrogenation and it wasbelieved that nicA gene encodes nicotine oxidoreductase thatplays an important role in the initial steps of pyrrolidinepathway and involved in the degradation of nicotine toSP [99] The reversible second step was carried out in thepresence of water and a double bond of N-methylmyosminewas spontaneously hydrolyzed to form pseudooxynicotinea direct precursor of a potent tobacco-specific lung car-cinogen [99ndash101] This carcinogenic intermediate metabo-lite pseudooxynicotine was further dehydrogenated to givemethylamine and SP by NicA enzyme Tang et al [99]hypothesized that two unstable compounds were producedduring the conversion of pseudooxynicotine to SP The 6thposition of carbon of SP was further hydrolyzed to yieldHSP by an unknown enzyme The hsp gene encodes anenzyme 6-hydroxy-3-succinoyl pyridine hydroxylase (HSPhydroxylase) The hsp gene attacked on the 3rd positionof HSP to form 25-DHP and succinic semialdehyde (Ssa)[28 97 102]The 25-DHPwas further degraded to maleamicacid which deaminates by hydrolysis and produces maleicacid [103] The cleavage of 5th and 6th position of carbonof maleic acid gave pyruvic acid which entered into citricacid cycle [102] On the other side Ssa easily convertedinto succinic acid by succinic semialdehyde dehydrogenase(SsaDH) enzyme that is widely present in Pseudomonas sp[104ndash106]

Interestingly the two genes hspA and hspB that encodeHSP hydroxylase are involved in the conversion of HSP to25-DHP [97 99 107] The hydroxylase enzyme is involvedin oxidation reactions in which one of the two atoms ofmolecular oxygen is integrated into the substrate and anotheris used to oxidize NADH or NADPH [108ndash110] In P putidaS16 4879 bp nic gene cluster encoded three open readingframes (ORF) namely ORF1 (1853 bp) ORF2 (936 bp) andORF3 (582 bp) The ORF1 encoded nicA gene and ORF2encoded hspA gene whereas the function of remaining ORF3is unknown [97 99 107] Nevertheless the newly identifiedhspB gene was located on 30Kb of DNA away from thenic gene cluster [107] The molecular mass of HspA has38 kDa and NADH dependent while HspB has 40 kDa adimer and a prosthetic group FAD dependent The deletionof hspB gene in the mutant strain could not degrade HSPsuggesting that hspB gene plays an important role in theconversion of HSP to 25-DHP [107] The molecular massof flavin adenine mononucleotide (FMN) dependent enzymeNicA is approximately 65 kDa that degraded nicotine into

N-methylmyosmine pseudooxynicotine and SP in P putidaS16 These three nicotine intermediate metabolites were con-firmed by electrospray ionization quadrupole time of flightmass spectrometry (ESI-Q-TOF-MS) analysis [99] All threegenes have been cloned and overexpressed in Escherichiacoli [97 99 107] However the Pseudomonas sp Nic22bacteria followed other pyrrolidine pathways that producedmyosmine 231015840-dipyridyl and cotinine during biodegrada-tion of nicotine [29] (Figure 2) Shinella sp HZN1 producedthree nicotine intermediate metabolites during biodegrada-tion which were characterized and identified as cotininemyosmine and nicotyrine using gas chromatography-massspectrometry (GC-MS) analysis [36] Four major nicotineintermediate metabolites pseudooxynicotine SP an unstablecompound 3-succinoylsemialdehyde pyridine (SAP) andHSP and three nicotine-degrading genes sir A2 pao andsap were identified in Pseudomonas sp HZN6 [35 98] SirA2 protein was encoded by Sir A2 and a sulfurtransferasehomologue gene is responsible for the degradation of SP [98]An unstable nicotine intermediate metabolite SAP producedduring the conversion of pseudooxynicotine by pseudooxyn-icotine amine oxidase (PNAO) was encoded by pna geneThis enzyme oxidatively deaminates pseudooxynicotine bynoncovalently bound FAD O

2 and H

2O and forms SAP

methylamine and H2O2 Another gene sap encodes NADP+

dependent enzyme 3-succinoylsemialdehyde pyridine dehy-drogenase (SAPD) dehydrogenates SAP to SP [35] Anothernicotine-degrading bacterium Pseudomonas CS3 producedthree new nicotine intermediate metabolites [111] (Figure 2)In the initial step demethylation of nicotine forms a metabo-lite 3-(34-dihydro-2H-pyrrol-5-yl) pyridine Concurrentlyfurther degradation was initiated by hydroxylation of 2ndposition of pyrrolidine ring of nicotine to form 1-methyl-5-(3-pyridyl) pyrroline-2-ol which is further transformed tocotinine

Wang et al [112] identified nicotine-degrading genehsp in the plasmid pMF1 (21 Kb) of Pseudomonas spHF-1 and reported that nicotine degradation is regulatedby plasmid not chromosomal DNA Ketopantoate hydrox-ymethyltransferase encoded by panB gene of P putida J5 isinvolved in nicotine catabolism Pyruvic acid the end productof pyrrolidine pathway is a precursor of ketoisovaleratewhich undergoes catabolism using the enzyme ketopantoatehydroxymethyltransferase leading to synthesis of vitaminpantothenate [113]

Raman et al [2] recently reported that P plecoglossicidaTND35 followed a variant of pyrrolidine pathway whichis different from pathways of other bacteria and fungi(Figure 3) Strain TND35 oxidized pyrrolidine ring moietyof nicotine to form N-methylmyosmine This intermediatewas further hydroxylated at 2nd position of pyrrolidine ringto form a new cotinine analogue metabolite 23-dihydro-1-methyl-5-(pyridin-3-yl)-1H-pyrrol-2-ol (IM2) In additionthis metabolite was demethylated and hydroxyl group of 23-dihydro-1-methyl-5-(pyridin-3-yl)-1H-pyrrol-2-ol was fur-ther oxidized to formanother new cotinine analoguemetabo-lite 5-(pyridin-3-yl)-1H-pyrrol-2(3H)-one (IM3) Concur-rently 23-dihydro-1-methyl-5-(pyridin-3-yl)-1H-pyrrol-2-olwas further oxidized and releasedmethylamineThis reaction


N

N

N

N

N

N

N

NH

O

N

O

N

NN

Nicotine

23-Dihydro-1-methyl-5-

pyridine

N-Methylmyosmine

4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)

2(3H)-one5-(Pyrin-3-yl)-1H-pyrrol-

(pyridin-3-yl)-1H-pyrrol-2-ol

OH

OH

35-Bis(1-methylpyrrolidineminus2-yl)

Figure 3 A variant pyrrolidine pathway of nicotine biodegradationby P plecoglossicida TND35

further leads to opening of the pyrrolidine ring to forman end product 4-hydroxy-1-(3-pyridyl)-1-butanone (IM4)Interestingly new metabolite 35-bis (1-methylpyrrolidin-2-yl) (IM5) was observed during nicotine degradation Thismay be due to the cleavage of bond between pyridine andpyrrolidine ring of the nicotine This N-methyl pyrrolidinering attacked the pyridine moiety of nicotine and formed thismetabolite

43 Variant Pathway of Pyridine and Pyrrolidine for NicotineBiodegradation ThebacteriumA tumefaciens S33 followed avariant of pyridine and pyrrolidine pathway (Figure 4) Thisbacterium partially shared both the pathways and producedits intermediate metabolites [114] Similarly this bacteriumalso produced bright green colour initially and oxidized tobrown colour at concentration above 3 gL nicotine Theproduction of colour depends upon the concentration ofnicotine oxygen content and pH of the medium [114] Path-way of pyridine intermediate metabolites 6-HN 6-HMMand 6-HPON and pyrrolidine pathway metabolites HSPand 25-DHP were identified by ultraviolet-visible (UV-Vis)spectroscopy thin layer chromatography (TLC) high perfor-mance liquid chromatography (HPLC) gas chromatography-high resolution-mass spectrometry (GC-HR-MS) and ESI-Q-TOF-MS analyses during nicotine degradation of A

O

O

N

O

O

N

N

N

N

N

N

O

N

N

NDH

HSP hydroxylase

Citric acid cycle

Succinic acid

Nicotine

2H

6-HLNO

Spontaneous

Methylamine+

Unknown steps

H2O

H2O

HO

HO

HO

HO

HO

OHHO OH

OH


O2


NH


6-Hydroxyminus3-succinoyl pyridine (HSP)

H2NCH3

25-Dihydroxypyridine (25-DHP)

CH3

Figure 4 A variant of pyridine and pyrrolidine pathway of nicotinedegradation by A tumefaciens S33 NDH nicotine dehydroge-nase 6-HLNO 6-hydroxy-L-nicotine oxidase HSP hydroxylase 6-hydroxy-3-succinoyl pyridine hydroxylase

tumefaciens S33 The catabolic nicotine-degrading enzymesNDH 6-HLNO and HSP hydroxylase were also observedin this bacterium The initial step of pyridine pathwayfollowed hydroxylation of nicotine to 6-HN by NDH Thismetabolite was further oxidized and transformed into 6-HMM by 6-HLNO The occurrence of spontaneous hydrol-ysis of 6-HMM into 6-HPON makes entry into pyrrolidinepathway The metabolite 6-HPON is dehydrogenated andspontaneous hydrolysis leads to the production of HSPand removal of methylamine However similar mechanism


was also observed during catabolic degradation of chlo-rine and aromatic amine 2-phenethylamine [115 116] TheHSP was further catabolized into 25-DHP In contrast N-methylmyosmine pseudooxynicotine and SPwere not foundduring this degradation [114]

44 Demethylation Pathway for Nicotine BiodegradationFungi such as M gypseum P filamentosa JTS-208 andC echinulata have been reported for nicotine degrada-tion These fungi are involved in the initial step demethy-lation of nicotine that leads to the formation of nor-nicotine [21 117] (Figure 5) Recently Meng et al [22]reported nicotine intermediate metabolites namely nor-nicotine N-methylnicotinamide 23-DHP 2-hydroxy-N-methylnicotinamide acetic acid carbomic acid and suc-cinic acid on the basis of TLC GC-MS nuclear magneticresonance (NMR) and Fourier transform infrared (FT-IR)analyses and proposed a hypothetical demethylation pathwayof nicotine degradation by A oryzae 112822 (Figure 5) Theprimary step of this hypothetical pathway is the eliminationof the removal of methyl group in the pyrrolidine ring ofnicotine to form nornicotine which is further converted intomyosmine by the formation of double bond in the pyrroli-dine ring (Figure 5) The subsequent cleavage of pyrrolidinering resulted in the formation of unknown intermediatemetaboliteThe hydrolytic attack on the postulated unknownintermediate metabolite resulted in N-methylnicotinamideand acetic acidTheN-methylnicotinamidewas hydroxylatedto form 2-hydroxy-N-methylnicotinamide which was catab-olized to a new nicotine intermediate metabolite 23-DHPwith the formation of aminomethyl The aminomethyl wasfurther transformed into carbomic acid The opening of ringin 23-DHP leads to the formation of succinic acid whichenters into citric acid cycle [22] Nevertheless no catabolicenzymes were identified during the degradation of nicotineby fungi

5 Biotechnological Applications of NicotineIntermediate Metabolites

Biotransformation or biocatalysis involves the use of micro-organisms to catalyze the conversion of one metabolite intoanotherThesemetabolites were catalyzed bywholemicrobialcells cellular extracts or enzymes [118] Biotransformationis a promising tool used in the synthesis of bulk chemicalsfor synthesis of pharmaceutical food and agrochemicalingredients in the industry [119] Nicotine is used as a startingmaterial for the biocatalytic production of functionalizedpyridines from renewable sources [120] The easiest andfriendliest ways of biotransformation approach were usedto transfer toxic nicotine into valuable compounds such asHSP and DHP [110 119 121 122] Biotransformation inter-mediates of nicotine are widely used in anticancer therapiesantimalarial and analgesics drug development and treatmentof Parkinsonrsquos disease hypertension and disorders of centralnervous system [123] Nicotine intermediate metabolites areprecursors in the synthesis of drug such as analogues ofepibatidine an extremely effective analgesic molecule that is

N

N

N

NH

N

O

N

O

OH

N

OHO

O

N

N

Nicotine

Nornicotine

Succinic acid

Carbamic acid

Acetic acid

Myosmine

1

2

Aminomethyl

HO

OH

OH

NH

NH

N-Methylnicotinamide

CH3COOH

2-Hydroxy-N-methylnicotinamide


CH3-NH-COOH

NH2-COOH

CH3

CH3

Figure 5 Demethylation pathway of nicotine degradation by fungi1 A oryzae 11282 2 M gypseum P filamentosa JTS-208 and Cechinulata

used in pharmaceutical industry [121] Hydroxylated pyridineintermediates are used as precursors for the synthesis of drugsand instecticides via chemical methods [114]The biologicallyactive metabolites 25- or 35-disubstituted pyridines arecatabolized from 6HLN and HSP which is used for thesynthesis of insecticide imidacloprid SIB-1508Y is an anti-Parkinsonrsquos agent [119 121 122] The important nicotineintermediate metabolite 25-DHP can be used as the initialmaterial for the chemical synthesis of universal precursoraminolevulinic acid This precursor is used to synthesize


plant growth regulators herbicides and drugs used in cancertherapies and to synthesize porphyrins such as heme andchlorophyll [110] The biotransformation nicotine intermedi-ate metabolite HPB has been widely used as a biomarker fortobacco related lung cancer studies [2]

6 Conclusions and Future Perspectives

Environmental pollution is one of the major problems inthe world Tobacco industries produced enormous amountof nicotine The nonreadily degradable nicotine causes envi-ronmental problems and human healthwhen directly enteredinto soil Major microbes degrade the toxic compoundnicotine In this paper we have discussed all the metabolicpathways and the genes involved in nicotine degradationThese microbes produce various intermediate metaboliccompounds of pharmaceutical importance during nicotinedegradation Bioremediation is one of the promising toolsused to convert the toxic compounds into valuable com-pounds These nicotine-degrading microbes can be usedfor bioremediation of nicotine-polluted environments Largescale production of these intermediatemetabolites of nicotinecould be of great use in pharmaceutical industries

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by UGC-Major Project and UGC-SAP Programme coordinated by Professor N Sakthivel andUGC-Rajiv Gandhi National Fellowship awarded to Dr GRaman

References

[1] ERC Statistics International PLC World Cigarettes 2005 ERCStatistics International PLC Newmarket UK 2005

[2] G Raman KMohan VManohar andN Sakthivel ldquoBiodegra-dation of nicotine by a novel nicotine-degrading bacteriumPseudomonas plecoglossicida TND35 and its new biotransfor-mation intermediatesrdquo Biodegradation 2013

[3] J A Dani D Ji and F Zhou ldquoSynaptic plasticity and nicotineaddictionrdquo Neuron vol 31 no 3 pp 349ndash352 2001

[4] L Bush W P Hempfling and H Burton ldquoBiosynthesis ofnicotine and related compoundsrdquo in Analytical Determinationof Nicotine and Related Compounds and Their Metabolites J WGorrod and P Jacob III Eds pp 13ndash44 Elsevier AmsterdamThe Netherlans 1999

[5] F A Sloan and H Gelband Cancer Control Opportunities inLow- and Middle-Income Countries Institute of Medicine (US) Committee on Cancer Control in Low- and Middle-IncomeCountries National Academies Press Washington DC USA

[6] H Li X Li Y Duan K Zhang and J Yang ldquoBiotransformationof nicotine by microorganism the case of Pseudomonas spprdquoApplied Microbiology and Biotechnology vol 86 no 1 pp 11ndash172010

[7] M Civilini C Domenis N Sebastianutto and M de BertoldildquoNicotine decontamination of tobacco agro-industrial wasteand its degradation by micro-organismsrdquo Waste Managementand Research vol 15 no 4 pp 349ndash358 1997

[8] T E Novotny and F Zhao ldquoConsumption and productionwaste another externality of tobacco userdquo Tobacco Control vol8 no 1 pp 75ndash80 1999

[9] D W Armstrong X Wang and N Ercal ldquoEnantiomeric com-position of nicotine in smokeless tobacco medicinal productsand commercial reagentsrdquo Chirality vol 10 pp 587ndash591 1998

[10] T EThorgeirsson F Geller P Sulem et al ldquoA variant associatedwith nicotine dependence lung cancer and peripheral arterialdiseaserdquo Nature vol 452 no 7187 pp 638ndash642 2008

[11] B T Hawkins T J Abbruscato R D Egleton et al ldquoNicotineincreases in vivo blood-brain barrier permeability and alterscerebral microvascular tight junction protein distributionrdquoBrain Research vol 1027 no 1-2 pp 48ndash58 2004

[12] K Cogo M Franz Montan C D C Bergamaschi E DAndrade P L Rosalen and F C Groppo ldquoIn vitro evaluationof the effect of nicotine cotinine and caffeine on oral microor-ganismsrdquo Canadian Journal of Microbiology vol 54 no 6 pp501ndash508 2008

[13] D Yildiz ldquoNicotine its metabolism and an overview of itsbiological effectsrdquo Toxicon vol 43 no 6 pp 619ndash632 2004

[14] K D Brunnemann B Prokopczyk M V Djordjevic andD Hoffmann ldquoFormation and analysis of tobacco-specific N-nitrosaminesrdquo Critical Reviews in Toxicology vol 26 no 2 pp121ndash137 1996

[15] J A Campain ldquoNicotine potentially a multifunctional carcino-genrdquo Toxicological Sciences vol 79 no 1 pp 1ndash3 2004

[16] D Yildiz N Ercal and D W Armstrong ldquoNicotine enan-tiomers and oxidative stressrdquo Toxicology vol 130 no 2-3 pp155ndash165 1998

[17] D Qiao F J Seidler and T A Slotkin ldquoOxidative mechanismscontributing to the developmental neurotoxicity of nicotine andchlorpyrifosrdquo Toxicology and Applied Pharmacology vol 206no 1 pp 17ndash26 2005

[18] Y D Zhang C R Luo H L Wang and G F Lu ldquoAdvances inmicrobial degradation of nicotine and its applicationrdquo Tobaccoand Science Technology vol 12 pp 3ndash7 2003

[19] H Schievelbein ldquoNicotine resorption and faterdquo PharmacologyandTherapeutics vol 18 no 2 pp 233ndash248 1982

[20] M Sabha J E Tanus-Santos J C Y Toledo M CittadinoJ C Rocha and H Moreno Jr ldquoPharmacodynamics anddrug action transdermal nicotinemimics the smoking-inducedendothelial dysfunctionrdquo Clinical Pharmacology andTherapeu-tics vol 68 no 2 pp 167ndash174 2000

[21] S Uchida S Maeda and T Kisaki ldquoConversion of nicotine intonornicotine andN-methylmyosmine by fungirdquoAgricultural andBiological Chemistry vol 47 no 9 pp 1949ndash1953 1983

[22] X J Meng L L Lu G F Gu and M Xiao ldquoA novel pathwayfor nicotine degradation by Aspergillus oryzae 112822 isolatedfrom tobacco leavesrdquo Research in Microbiology vol 161 no 7pp 626ndash633 2010

[23] J W Hylin ldquoMicrobial degradation of nicotine I Morphologyand physiology of Achromobacter nicotinophagum n sprdquo Jour-nal of Bacteriology vol 76 no 1 pp 36ndash40 1958

[24] A Ruan H Min and W Zhu ldquoStudies on biodegradation ofnicotine by Arthrobacter sp strain HF-2rdquo Journal of Environ-mental Science and Health B vol 41 no 7 pp 1159ndash1170 2006


[25] E Wada and K Yamasaki ldquoMechanism of microbial degrada-tion of nicotinerdquo Science vol 117 no 3033 pp 152ndash153 1953

[26] A Ruan H Min X Peng and Z Huang ldquoIsolation andcharacterization of Pseudomonas sp strain HF-1 capable ofdegrading nicotinerdquo Research in Microbiology vol 156 no 5-6pp 700ndash706 2005

[27] Y J Yuan Z X Lu L J Huang X M Bie F X Lu andY Li ldquoOptimization of a medium for enhancing nicotinebiodegradation byOchrobactrum intermediumDN2rdquo Journal ofApplied Microbiology vol 101 no 3 pp 691ndash697 2006

[28] S N Wang Z Liu H Z Tang J Meng and P Xu ldquoCharac-terization of environmentally friendly nicotine degradation byPseudomonas putida biotype A strain S16rdquo Microbiology vol153 no 5 pp 1556ndash1565 2007

[29] C Chen X Li J Yang X Gong B Li and K Zhang ldquoIsolationof nicotine-degrading bacterium Pseudomonas sp Nic22 andits potential application in tobacco processingrdquo InternationalBiodeterioration and Biodegradation vol 62 no 3 pp 226ndash2312008

[30] HWei L Lei Z Xia S Liu P Liu andX Liu ldquoCharacterisationof a novel aerobic nicotine-biodegrading strain of Pseudomonasputidardquo Annals of Microbiology vol 58 no 1 pp 41ndash45 2008

[31] X Gong J Yang Y Duan et al ldquoIsolation and characterizationof Rhodococcus sp Y22 and its potential application to tobaccoprocessingrdquo Research in Microbiology vol 160 no 3 pp 200ndash204 2009

[32] S N Wang Z Liu and P Xu ldquoBiodegradation of nicotine by anewly isolated Agrobacterium sp strain S33rdquo Journal of AppliedMicrobiology vol 107 no 3 pp 838ndash847 2009

[33] L Lei W Zhang H Wei Z Xia and X Liu ldquoCharacterizationof a novel nicotine-degrading Ensifer sp strainN7 isolated fromtobacco rhizosphererdquo Annals of Microbiology vol 59 no 2 pp247ndash252 2009

[34] W Zhong C Zhu M Shu et al ldquoDegradation of nicotinein tobacco waste extract by newly isolated Pseudomonas spZUTSKDrdquo Bioresource Technology vol 101 no 18 pp 6935ndash6941 2010

[35] J Qiu Y Ma Y Wen L Chen L Wu and W Liu ldquoFunctionalidentification of two novel genes from Pseudomonas sp strainHZN6 involved in the catabolism of nicotinerdquoApplied Environ-mantal Microbiology vol 78 pp 2154ndash2160 2012

[36] H J Jiang Y Ma G J Qiu F L Wu and S L ChenldquoBiodegradation of nicotine by a novel Strain Shinella sp HZN1isolated fromactivated sludgerdquo Journal of Environmental Scienceand Health B vol 46 no 8 pp 703ndash708 2011

[37] M Wang G Yang X Wang Y Yao H Min and Z LuldquoNicotine degradation by two novel bacterial isolates of Acine-tobacter sp TW and Sphingomonas sp TY and their responsesin the presence of neonicotinoid insecticidesrdquoWorld Journal ofMicrobiology and Biotechnology vol 27 no 7 pp 1633ndash16402011

[38] G Ma L Lei Z Xia X Gong W Zhou and J YangldquoDiversity and phylogenetic analyses of nicotine-degradingbacteria isolated from tobacco plantation soilsrdquo African Journalof Microbiological Research vol 6 pp 6392ndash6398 2012

[39] L Zhao C Zhu Y Gao et al ldquoNicotine degradation enhance-ment by Pseudomonas stutzeri ZCJ during aging process oftobacco leavesrdquo World Journal of Microbiology and Biotechnol-ogy vol 28 no 5 pp 2077ndash2086 2012

[40] G Giovannozzi-Sermanni ldquoIndustrial experiments of fermen-tation with addition of microbic culturesrdquo Tobacco vol 51 pp6ndash15 1947

[41] L E Gravely E Lawrence V L Geiss L Vernon and F CharlesldquoProcess for reduction of nitrate and nicotine content of tobaccoby microbial treatmentrdquo US Patent No 4557280 1978

[42] H Li Y Duan G Ma L Lei K Zhang and J Yang ldquoIsolationand characterization of Acinetobacter sp ND12 capable ofdegrading nicotinerdquo African Journal of Microbiology Researchvol 5 no 11 pp 1335ndash1341 2011

[43] T Niu C Chen N Jiatong et al ldquoNicotine dependence andits familial aggregation in Chineserdquo International Journal ofEpidemiology vol 29 no 2 pp 248ndash252 2000

[44] S M Zbikowski G E Swan and J B McClure ldquoCigarettesmoking and nicotine dependencerdquo Medical Clinics of NorthAmerica vol 88 no 6 pp 1453ndash1465 2004

[45] P Dome J Lazary M P Kalapos and Z Rihmer ldquoSmokingnicotine and neuropsychiatric disordersrdquo Neuroscience andBiobehavioral Reviews vol 34 no 3 pp 295ndash342 2010

[46] A A Lenkey ldquoNicotine removal process and product producedthereby mixing with alkaline agent in aerobic environmentrdquoUnited States Patent No 4848373 1989

[47] V P Beskoski G Gojgicc-Cvijovic J Milic et al ldquoEx situbioremediation of a soil contaminated bymazut (heavy residualfuel oil) a field experimentrdquo Chemosphere vol 83 pp 34ndash402011

[48] U Langer L Bohme and F Bohme ldquoClassification of soilmicroorganisms based on growth properties a critical view ofsome commonly used termsrdquo Journal of Plant Nutrition and SoilScience vol 167 no 3 pp 267ndash269 2004

[49] K Brenner L You and F H Arnold ldquoEngineering microbialconsortia a new frontier in synthetic biologyrdquo Trends inBiotechnology vol 26 no 9 pp 483ndash489 2008

[50] F Briski N Horgas M Vukovic and Z Gomzi ldquoAerobiccomposting of tobacco industry solid waste-simulation of theprocessrdquo Clean Technologies and Environmental Policy vol 5pp 295ndash301 2003

[51] K K Meher A M Panchwagh S Rangrass and K GGollakota ldquoBiomethanation of tobacco wasterdquo EnvironmentalPollution vol 90 no 2 pp 199ndash202 1995

[52] C G Schmit K Jahan K H Schmit E Debik and VMahendraker ldquoActivated sludge and other aerobic suspendedculture processesrdquo Water Environment Research vol 81 no 10pp 1127ndash1193 2009

[53] S Mace and J Mata-Alvarez ldquoUtilization of SBR technology forwastewater treatment an overviewrdquo Industrial and EngineeringChemistry Research vol 41 no 23 pp 5539ndash5553 2002

[54] H Eberhardt ldquoThe biological degradation of nicotine bynicotinophilic microorganismsrdquo Beitrage zur TabakforschungInternational vol 16 pp 119ndash129 1995

[55] H N Batham ldquoNitrification in soil IIrdquo Soil Science vol 24 pp187ndash203 1927

[56] J Huang J Yang Y Duan et al ldquoBacterial diversities on unagedand aging flue-cured tobacco leaves estimated by 16S rRNAsequence analysisrdquoAppliedMicrobiology and Biotechnology vol88 no 2 pp 553ndash562 2010

[57] Y J Yuan Z X Lu N Wu L J Huang F X Lu and X M BieldquoIsolation and preliminary characterization of a novel nicotine-degrading bacterium Ochrobactrum intermedium DN2rdquo Inter-national Biodeterioration and Biodegradation vol 56 no 1 pp45ndash50 2005

[58] Y Yao H Tang H Ren H Yu L Wang and P Xu ldquoGenomesequence of a nicotine-degrading strain of Arthrobacterrdquo Jour-nal of Bacteriology vol 194 pp 5714ndash5715 2012


[59] H Tang H Yu C Tai et al ldquoGenome sequence of a novelnicotine-degrading strain Pseudomonas geniculataN1rdquo Journalof Bacteriology vol 194 p 3553 2012

[60] G Giovannozzi-Sermanni ldquoArthrobacter nicotianae a newtype ofArthrobacter causing nicotine degradationrdquoCoersta vol3 p 2595 1959

[61] R L Gherna S H Richardson and S C Rittenberg ldquoThebacterial oxidation of nicotine VI The metabolism of 26-dihydroxypseudooxynicotinerdquo The Journal of Biological Chem-istry vol 240 no 9 pp 3669ndash3674 1965

[62] C Cobzaru and R Brandsch ldquoSteps in uncovering the keyenzyme in the degradation of the pyridine ring in theArthrobac-ter nicotinovoransrdquo Genetics and Molecular Biology vol 4 pp1ndash9 2008

[63] P E Holmes S C Rittenberg and H J KnackmussldquoThe bacterial oxidation of nicotine 8 Synthesis of 236-trihydroxypyridine and accumulation and partial characteriza-tion of the product of 26-dihydroxypyridine oxidationrdquo TheJournal of Biological Chemistry vol 247 no 23 pp 7628ndash76331972

[64] G L Igloi and R Brandsch ldquoSequence of the 165-kilobasecatabolic plasmid pAO1 from Arthrobacter nicotinovorans andidentification of a pAO1-dependent nicotine uptake systemrdquoJournal of Bacteriology vol 185 no 6 pp 1976ndash1989 2003

[65] R Brandsch and K Decker ldquoIsolation and partial characteri-zation of plasmid DNA from Arthrobacter oxidansrdquo Archives ofMicrobiology vol 138 no 1 pp 15ndash17 1984

[66] K Decker and R Brandsch ldquoFlavoproteins with a covalenthistidyl(N3)-8120572-riboflavin linkagerdquo BioFactors vol 3 no 2 pp69ndash81 1991

[67] S Schenk A Hoelz B Krauszlig and K Decker ldquoGene structuresand properties of enzymes of the plasmid-encoded nicotinecatabolism ofArthrobacter nicotinovoransrdquo Journal ofMolecularBiology vol 284 no 5 pp 1323ndash1339 1998

[68] R Thacker O Rorvig P Kahlon and I C Gunsalus ldquoNICa conjugative nicotine-nicotinate degradative plasmid in Pseu-domonas convexardquo Journal of Bacteriology vol 135 no 1 pp289ndash290 1978

[69] R Brandsch ldquoMicrobiology and biochemistry of nicotinedegradationrdquo Applied Microbiology and Biotechnology vol 69no 5 pp 493ndash498 2006

[70] L I Hochestein and S C Rittenberg ldquoThe bacterial oxidationof nicotine I Nicotine oxidation by cell-free preparationsrdquoTheJournal of Biological Chemistry vol 234 no 1 pp 151ndash155 1959

[71] L I Hochestein and S C Rittenberg ldquoThe bacterial oxidationof nicotine II The isolation of the first oxidative productand its identification as (1)-6-hydroxynicotinerdquo The Journal ofBiological Chemistry vol 234 no 1 pp 156ndash160 1959

[72] S Grether-Beck G L Igloi S Pust E Schilz K Decker andR Brandsch ldquoStructural analysis and molybdenum-dependentexpression of the pAO1-encoded nicotine dehydrogenase genesof Arthrobacter nicotinovoransrdquoMolecular Microbiology vol 13no 5 pp 929ndash936 1994

[73] J R Andreesen and S Fetzner ldquoThe molybdenum-containinghydroxylases of nicotinate isonicotinate and nicotinerdquo MetalIons in Biological Systems vol 39 pp 405ndash430 2002

[74] P Hanzelmann and O Meyer ldquoEffect of molybdate andtungstate on the biosynthesis of CO dehydrogenase andthe molybdopterin cytosine-dinucleotide-type of molybdenumcofactor in Hydrogenophaga pseudoflavardquo European Journal ofBiochemistry vol 255 no 3 pp 755ndash765 1998

[75] S Schenk and K Decker ldquoHorizontal gene transfer involvedin the convergent evolution of the plasmid-encoded enan-tioselective 6-hydroxynicotine oxidasesrdquo Journal of MolecularEvolution vol 48 no 2 pp 178ndash186 1999

[76] J W A Koetter and G E Schulz ldquoCrystal structure of 6-hydroxy-D-nicotine oxidase fromArthrobacter nicotinovoransrdquoJournal of Molecular Biology vol 352 no 2 pp 418ndash428 2005

[77] C B Chiribau C Sandu M Fraaije E Schiltz and RBrandsch ldquoA novel 120574-N-methylaminobutyrate demethylatingoxidase involved in catabolism of the tobacco alkaloid nicotineby Arthrobacter nicotinovorans pAO1rdquo European Journal ofBiochemistry vol 271 no 23-24 pp 4677ndash4684 2004

[78] F A Gries K Decker and M Bruhmuller ldquoUber denAbbau des Nicotins durch Bakterienenzyme V Der Abbau desL-6-Hydroxynicotins zu [120574-Methylamino-propyl]-[6-hydroxy-pyridyl-(3)]-ketonsrdquo Hoppe-Seylerrsquos Zeitschrift PhysiologisheChemie vol 325 pp 229ndash241 1961

[79] F A Gries K Decker H Eberwein and M BruhmullerldquoUber den Abbau des Nicotins durch Bakteienenzyme VI Dieenzymatische Umwandlung des (120574-Methylamino-propyl)-[6-hydroxy- pyridyl-(3)]-ketonsrdquoBiochemische Zeitschrift vol 335pp 285ndash302 1961

[80] P Sachelaru E Schiltz G L Igloi and R Brandsch ldquoAn120572120573-fold C-C bond hydrolase is involved in a central step ofnicotine catabolism by Arthrobacter nicotinovoransrdquo Journal ofBacteriology vol 187 no 24 pp 8516ndash8519 2005

[81] N Treiber and G E Schulz ldquoStructure of 26-dihydroxypyri-dine 3-hydroxylase from a nicotine-degrading pathwayrdquo Jour-nal of Molecular Biology vol 379 no 1 pp 94ndash104 2008

[82] P Sachelaru E Schiltz and R Brandsch ldquoA functional mobAgene for molybdopterin cytosine dinucleotide cofactor biosyn-thesis is required for activity and holoenzyme assembly ofthe heterotrimeric nicotine dehydrogenases of Arthrobacternicotinovoransrdquo Applied and Environmental Microbiology vol72 no 7 pp 5126ndash5131 2006

[83] C B Chiribau C Sandu G L Igloi and R BrandschldquoCharacterization of PmfR the transcriptional activator of thepAO1-borne purU-mabO-folD operon of Arthrobacter nicoti-novoransrdquo Journal of Bacteriology vol 187 no 9 pp 3062ndash30702005

[84] P R Levering D J Binnema J P van Dijken and W HarderldquoEnzymatic evidence for a simultaneous operation of two one-carbon assimilation pathways during growth ofArthrobacter P1on cholinerdquo FEMSMicrobiology Letters vol 12 no 1 pp 19ndash251981

[85] X Zhang J H Fuller and W S McIntire ldquoCloning sequenc-ing expression and regulation of the structural gene for thecoppertopa quinone-containing methylamine oxidase fromArthrobacter strain P1 a gram-positive facultative methy-lotrophrdquo Journal of Bacteriology vol 175 no 17 pp 5617ndash56271993

[86] K Bartsch A von Johnn-Marteville and A Schulz ldquoMolecularanalysis of two genes of the Escherichia coli gab clusternucleotide sequence of the glutamatesuccinic semialdehydetransaminase gene (gabT) and characterization of the succinicsemialdehyde dehydrogenase gene (gabD)rdquo Journal of Bacteri-ology vol 172 no 12 pp 7035ndash7042 1990

[87] K L Chambliss D L Caudle D D Hinson et al ldquoMolecularcloning of the mature NAD+-dependent succinic semialde-hyde dehydrogenase from rat and human cDNA Isolationevolutionary homology and tissue expressionrdquo The Journal ofBiological Chemistry vol 270 no 1 pp 461ndash467 1995


[88] S Dover and Y S Halpern ldquoUtilization of c-aminobutyric acidas the sole carbon and nitrogen source by Escherichia coli K-12mutantsrdquo Journal of Bacteriology vol 109 no 2 pp 835ndash8431972

[89] E Metzer and Y S Halpern ldquoIn vivo cloning and character-ization of the gabCTDP gene cluster of Escherichia coli K-12rdquoJournal of Bacteriology vol 172 no 6 pp 3250ndash3256 1990

[90] E Niegemann A Schulz and K Bartsch ldquoMolecular organi-zation of the Escherichia coli gab cluster nucleotide sequenceof the structural genes gabD and gabP and expression of theGABA permease generdquo Archives of Microbiology vol 160 no 6pp 454ndash460 1993

[91] P Ganas P Sachelaru M Mihasan G L Igloi and R Brand-sch ldquoTwo closely related pathways of nicotine catabolism inArthrobacter nicotinovorans and Nocardioides sp strain JS614rdquoArchives of Microbiology vol 189 no 5 pp 511ndash517 2008

[92] A Wenusch ldquoFurther study of a biological decompositionof nicotinerdquo Zeitschrift fur Lebensmittel-Untersuchung und -Forschung vol 84 pp 498ndash501 1942

[93] H Bucherer ldquoUber den mikrobiellen Abbau von GiftstoffenI Mitteilung Uber den mikrobiellen Abbau von NikotinrdquoZentralblatt fur Bakteriology vol 105 pp 166ndash173 1942

[94] T Tabuchi ldquoMicrobial degradation of nicotine and nicotinicacid I Isolation of nicotine-decomposing bacteria and theirmorphological and physiological propertiesrdquo Journal of Agricul-tural Chemical Society of Japan vol 28 pp 806ndash810 1955

[95] T Tabuchi ldquoMicrobial degradation of nicotine and nicotinicacid II Isolation of nicotine-decomposing bacteria and theirmorphological and physiological propertiesrdquo Journal of Agricul-tural Chemical Society of Japan vol 29 pp 219ndash221 1955

[96] T Tabuchi ldquoMicrobial degradation of nicotine and nicotinicacid III Isolation of nicotine-decomposing bacteria and theirmorphological and physiological propertiesrdquo Journal of Agricul-tural Chemical Society of Japan vol 29 pp 222ndash225 1955

[97] H Tang S Wang L Ma et al ldquoA novel gene encoding 6-hydroxy-3-succinoylpyridine hydroxylase involved in nicotinedegradation by Pseudomonas putida strain S16rdquo Applied andEnvironmental Microbiology vol 74 no 5 pp 1567ndash1574 2008

[98] J Qiu Y Ma L Chen L Wu Y Wen and W Liu ldquoA sirA-likegene sirA2 is essential for 3-succinoyl-pyridine metabolism inthe newly isolated nicotine-degrading Pseudomonas sp HZN6strainrdquo Applied Microbiology and Biotechnology vol 92 no 5pp 1023ndash1032 2011

[99] H Tang L Wang X Meng et al ldquoNovel nicotine oxidor-eductase- encoding gene involved in nicotine degradation byPseudomonas putida strain S16rdquo Applied and EnvironmentalMicrobiology vol 75 no 3 pp 772ndash778 2009

[100] S S Hecht B J Hochalter P W Villalta and S E Murphy ldquo21015840-hydroxylation of nicotine by cytochrome P450 2A6 and humanliver microsomes formation of a lung carcinogen precursorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 97 no 23 pp 12493ndash12497 2000

[101] J Hukkanen P Jacob and N L Benowitz ldquoMetabolism anddisposition kinetics of nicotinerdquo Pharmacological Reviews vol57 no 1 pp 79ndash115 2005

[102] J Kaiser Y Feng and J Bollag ldquoMicrobial metabolism of pyri-dine quinoline acridine and their derivatives under aerobicand anaerobic conditionsrdquoMicrobiological Reviews vol 60 no3 pp 483ndash498 1996

[103] E J Behrman and R Y Stanier ldquoThe bacterial oxidation ofnicotinic acidrdquoThe Journal of Biological Chemistry vol 228 no2 pp 923ndash945 1957

[104] D M Callewaert M S Rosemblatt K Suzuki and T T TchenldquoSuccinic semialdehyde dehydrogenase from a Pseudomonasspecies I Purification and chemical propertiesrdquoThe Journal ofBiological Chemistry vol 248 no 17 pp 6009ndash6013 1973

[105] M S Rosemblatt D M Callewaert and T T Tchen ldquoSuccinicsemialdehyde dehydrogenase from a Pseudomonas species IIPhysical and immunochemical properties of the enzymerdquo TheJournal of Biological Chemistry vol 248 no 17 pp 6014ndash60181973

[106] M SanchezM A Alvarez R Balana andA Garrido-PertierraldquoProperties and functions of two succinic-semialdehyde dehy-drogenases fromPseudomonas putidardquoBiochimica et BiophysicaActa vol 953 pp 249ndash257 1988

[107] H Tang Y Yao D Zhang et al ldquoA novel NADH-dependent andFAD-containing hydroxylase is crucial for nicotine degradationby Pseudomonas putidardquo The Journal of Biological Chemistryvol 286 no 45 pp 39179ndash39187 2011

[108] D Baitsch C Sandu R Brandsch and G L Igloi ldquoGenecluster on pAO1 of Arthrobacter nicotinovorans involved indegradation of the plant alkaloid nicotine cloning purificationand characterization of 26-dihydroxypyridine 3-hydroxylaserdquoJournal of Bacteriology vol 183 no 18 pp 5262ndash5267 2001

[109] R Hirschberg and J C Ensign ldquoOxidation of nicotinic acid bya Bacillus species source of oxygen atoms for the hydroxylationof nicotinic acid and 6-hydroxynicotinic acidrdquo Journal ofBacteriology vol 108 no 2 pp 757ndash759 1971

[110] H Nakano M Wieser B Hurh et al ldquoPurification char-acterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescensTN5rdquo EuropeanJournal of Biochemistry vol 260 no 1 pp 120ndash126 1999

[111] H H Wang B Yin X X Peng et al ldquoBiodegradation of nico-tine by newly isolated Pseudomonas sp CS3 and itsmetabolitesrdquoJournal of Applied Microbiology vol 112 no 2 pp 258ndash2682012

[112] MWangG YangHMin andZ Lv ldquoAnovel nicotine catabolicplasmid pMH1 in Pseudomonas sp strain HF-1rdquo CanadianJournal of Microbiology vol 55 no 3 pp 228ndash233 2009

[113] HWei L Lei S Liu Z Xia X Liu and P Liu ldquoPanB is involvedin nicotine metabolism in Pseudomonas putidardquo InternationalBiodeterioration and Biodegradation vol 63 no 8 pp 988ndash9922009

[114] SWang H Huang K Xie and P Xu ldquoIdentification of nicotinebiotransformation intermediates byAgrobacterium tumefaciensstrain S33 suggests a novel nicotine degradation pathwayrdquoApplied Microbiology and Biotechnology vol 95 pp 1567ndash15782012

[115] E Diaz A Ferrandez M A Prieto and J L Garcıa ldquoBiodegra-dation of aromatic compounds by Escherichia colirdquo Microbiol-ogy and Molecular Biology Review vol 65 pp 523ndash569 2001

[116] L Xun andCMWebster ldquoAmonooxygenase catalyzes sequen-tial dechlorinations of 2 4 6-trichlorophenol by oxidative andhydrolytic reactionsrdquo The Journal of Biological Chemistry vol279 no 8 pp 6696ndash6700 2004

[117] R D Sindelar J P Rosazza and C F Barfknecht ldquoN-demethylation of nicotine and reduction of nicotine-1rsquo-N-oxide by Microsporum gypseumrdquo Applied and EnvironmentalMicrobiology vol 38 no 5 pp 836ndash839 1979

[118] O Ghisalba H P Meyar and R Wohlgemuth ldquoIndustrialbiotransformationrdquo in Encyclopedia of Industrial BiotechnologyBioprocess Bioseparation and Cell Technology M C FlickingerEd pp 1ndash18 Wiley 2010


[119] S N Wang P Xu H Z Tang J Meng X L Liu and C QMa ldquolsquoGreenrsquo route to 6-hydroxy-3-succinoyl-pyridine from (S)-nicotine of tobacco waste by whole cells of a Pseudomonas sprdquoEnvironmental Science and Technology vol 39 no 17 pp 6877ndash6880 2005

[120] A Schmid J S Dordick B Hauer A Kiener M Wubboltsand B Witholt ldquoIndustrial biocatalysis today and tomorrowrdquoNature vol 409 no 6817 pp 258ndash268 2001

[121] T F Spande H M Garraffo M W Edwards H J C YehL Pannell and J W Daly ldquoEpibatidine a novel (chloropy-ridyl)azabicycloheptane with potent analgesic activity from anecuadoran poison frogrdquo Journal of the American ChemicalSociety vol 114 no 9 pp 3475ndash3478 1992

[122] J Roduit A Wellig and A Kiener ldquoRenewable functionalizedpyridines derived from microbial metabolites of the alkaloid(5)-nicotinerdquo Heterocycles vol 45 no 9 pp 1687ndash1702 1997

[123] D A Rathbone and N C Bruce ldquoMicrobial transformation ofalkaloidsrdquo Current Opinion in Microbiology vol 5 no 3 pp274ndash281 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Nicotine is present up to 2 to 8of the drymass of the tobaccoleaves [9]

21 Effect of Nicotine on Human Beings Nicotine is a haz-ardous compound that causes tobacco related lung cancerand peripheral arterial disease [10] Although more than4000 substances are present in the tobacco cigarette smokenicotine is the major substance [11 12] Nicotine has ablood half-life period of approximately 2 h and causes severevascular diseases [11 12] Nicotine can cause cancer genemutation and malformation [13] An array of toxic nicotineintermediate metabolites such as N1015840-nitrosonornicotine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone cotinine andN-nitrosamine causes tobacco lung cancer [14 15] Neuro-toxin developmental effects of nicotine can naturally affect avariety of cellular processes such as generation of oxidativeradicals apoptosis and hyperplasia of cell enhancing geneexpression to secrete hormones and regulation of enzymaticactivity [13 16 17] The lungs rapidly absorb 90 of thenicotine that is present in the cigarette smoke inhaled byhuman beings [18] Nicotine in the human body can easilypass the biological membranes and blood-brain barrier andincreases heart rate mean arterial blood pressure andmimicsthe venous endothelial dysfunction [15 19 20]

Nicotine is an additive substance that can lead to nicotinedependence and addictive behavior in human populations[43] Nicotine dependence is complex multidimensionaltrait that involves psychological physiological behavioraland social factors [44] Nicotine the primary psychoactiveingredient of tobacco contributes to physical dependenceby acting on nicotinic acetylcholine receptors in the centralnervous system and leads to the release of neurotransmitters(eg dopamine and serotonin) which produce reinforcingeffects by activating the mesocorticolimbic dopamine system[45]

3 Microbial Degradation of Nicotine

Physical and chemical methods are available to degrade nico-tine in the tobaccoThesemethods are often time-consumingexpensive and involve solvent extraction procedures [46]Bioremediation is one of the promising methods to clean uppolluted environments using microbes [47ndash49] Briski et al[50] reported that aerobic composting is an effective methodto reduce 80 of nicotine and 50 of the volume andmass oftobacco solid wastes in 16 dMeher et al [51] used a techniquecalled biomethanation that removed 60 of nicotine 756of chemical oxygen demand and 80 of biological oxygendemand from the tobacco wastes Biological method employsa variety of nicotine-degrading bacteria and fungi [41 46 51]These ecofriendly biological methods are extensively usedin wastewater treatment due to its high efficiency and lowcost Microbes that degrade nicotine are reported to adapt topolluted environment easily [52 53]Native strains of bacteriaand fungi that live in the tobacco environment have the abilityto degrade nicotine [54] These microbes utilize nicotine assole carbon nitrogen and energy source for their growth

[6] Batham [55] reported that the nitrate content of the soilincreased due to the microbial degradation of nicotine

31 Optimal Conditions of Nicotine-Degrading BacteriaNicotine degradation experiments are carried out with dif-ferent culture media such as distilled water minimal saltmedium inorganic salt medium and basal salt medium(BSM)The culture media influence the nicotine degradationefficiency Wang et al [28] reported that 3 gL nicotine wasfully degraded in 5 h when the degradation experiment wascarried out with 005M sodium phosphate buffer (pH 70)However it tookmore than 8 h to complete degradationwhencarried out with distilled water The optimal culture condi-tions and nicotine degradation efficiency of nicotinophilicbacteria vary from each other Most of the bacteria growat 30∘C pH range of 64 to 75 and degrade maximumconcentration of nicotine up to 6 gL [2 5] Huang et al[56] reported that the rate of nicotine degradation efficiencywas high at 37∘C when compared to 30∘C The optimalconditions and nicotine degradation efficiency of variousnicotine-degrading bacteria are presented in Table 1

32 Effect of Trace Elements Carbon and Nitrogen Sources onNicotine Degradation Trace elements and other carbon andnitrogen sources play a significant role in the biodegradationof nicotine [26] The presence of (NH

4)2SO4in the nicotine

containing medium could decrease the nicotine degradationefficiency [26] The nicotine degradation rate was increasedin the presence of yeast extract glucose and Tween 80in tobacco waste extract (TWE) containing media [57]However the nicotine degradation rate was dependent onthe concentration of yeast extract and Tween 80 in the TWEmedium [27] In contrast the medium containing glucoseand (NH

4)2SO4reduced the efficiency of nicotine degrada-

tion [32] ZnSO4sdot7H2O and NiCl

2sdot6H2O had no influence on

nicotine degradation whereas Na2MoO4and CuCl

2sdot4H2O

inhibited the rate of nicotine degradation [39] Glucose is animportant carbon source that promotes bacterial cell growthand improves the rate of nicotine degradation [24 34 39]Theconcentration of glucose above 10 gL inhibited the nicotinedegradation by Pseudomonas sp ZUTSKD [34] Ramanet al [2] reported that 1 gL dextrose increased nicotinedegradation rate In contrast glucose is not influenced in thenicotine degradation rate when the experiment was carriedout in solid-state fermentation process [39 56] Jiang et al[36] reported that the nicotine degradation rate was slowin the presence of glucose Conversely P stutzeri ZCJ couldnot utilize carbon sources such as sucrose and maltose andnitrogen source namely NaNO

2 for their growth which in

turn inhibits nicotine degradation [39]

33 Effect of Nicotine-Degrading Bacteria on Tobacco Leavesand Tobacco Wastes Nicotine-degrading bacteria played animportant role in the improvement of quality of tobaccoleaves during aging (fermentation) process These bacteriaaccumulatedmore in younger tobacco leaves when comparedto aged leaves [56] The desirable flavor taste and smokingproperties of tobacco remained unaltered when treated with


Table1Th

eoptim

alcond

ition

snicotin

edegradatio

neffi

ciencyand



Microorganism

Medium

Optim

alcond

ition

s(pHandTemperature)

Nicotined

egradatio

neffi

ciency

(gL)

Pathway

Reference

Fung

iPellicularia

filam

entosa

JTS-208

Synthetic

ND

004

20d

Dem

ethylatio

n[21]

Cunn

ingham

ellaechinu

lata

IFO-444

4Synthetic

ND

05413

dDem

ethylatio

n[21]

Aspergillus

oryzae

112822

Synthetic

6528∘C

21240h

Avaria

ntof

pyrid

inea

ndpyrrolidine

[22]

Gram-positive

bacteria

Arthrobacternicotin

ophagum

Nsp

Synthetic

68to

7020to

25∘C

16256

hPy

ridine

[23]

Arthrobacter

spH

F-2

Synthetic

7030∘C

07043h

ND

[24]

Gram-negativeb

acteria

Pseudomonas

sp41

Synthetic

6430∘C

13024

hPy

rrolidine

[25]

Pseudomonas

spH

F-1

Synthetic

65to

7530∘C

13025

hPy

rrolidine

[26]

Ochrobactr

uminterm

edium

DN2

Synthetic

30∘C

05036h

ND

[27]

Pputid

aS16

Synthetic

7030∘C

30010

hPy

rrolidine

[28]

Pseudomonas

spN

ic22

Synthetic

6530

to34∘C

28952h

ND

[29]

Pputid

aJ5

Synthetic

30∘C

39748h

ND

[30]

RhodococcusspY2

2Synthetic

7028∘C

10052

hND

[31]

Agrobacte

rium

tumefa

ciens

S33

Synthetic

7030∘C

50018

hAvaria

ntof

pyrid

inea

ndpyrrolidine

[32]

Gram-negativeb

acteria

Ensifer

spN

7Synthetic

7028∘C

33548h

ND

[33]

Pseudomonas

spZ

UTS

KDSynthetic

7530∘C

15512h

ND

[34]

Pseudomonas

spH

ZN6

Synthetic

7030∘C

05012

hPy

rrolidine

[35]

ShinellaspH

ZN1

Synthetic

7030∘C

0509h

ND

[36]

AcinetobacterspTW

Synthetic

7030∘C

10012h

ND

[37]

Sphingom

onas

spT

YSynthetic

7030∘C

10018h

ND

[37]

Pseudoxanthomonas

sp5ndash52

Synthetic

7028∘C

10028

hND

[38]

Pstu

tzeri

Synthetic

7537∘C

25036h

ND

[39]

Sinorhizobium

sp5ndash28

Synthetic

7028∘C

15028

hND

[38]

Ochrobactrum

sp4ndash4

0Synthetic

7028∘C

05028h

ND

[38]

Pplecoglossicid

aTN

D35

Synthetic

7030∘C

3012h

Avaria

ntof

pyrrolidine

[2]

NDnot

detected




Nicotine(mgg)















2





N

N

NN

N

NHO

N

O

NNH

O

HO

O

O

O

N

N

N

NDH

KOKDH

N

NOO

O

MABO

H H

O

Nicotine

(Ssa)

2H

6-HLNO6-HDLO

26-DHPONH

2H26-DHPH

+

Formaldehyde

FolDPurU

+

MAO

Methylamine

AO

SsaDH

Spontaneous

Spontaneous

Ammonia

H2O

H2O

HO

HO

HO

HO

HO

HO

HOHO

HOOHOH


O2

O2



OH

OH

OH

HO

OH

HO

OH

OH




Nicotine blue

NH2


H2NCH3

Citric acid cycle

NH3



Succinic acid

O2

NH

2HH2O

H2O

CH3

CH3

CH3



NN

HO

O

O

O

NicA

NN

N

O

O

N

N

NHO

N

OHO

O

PNAO

OO

OO O

OOH

N

HO

O

SAPD

NN

NN

NNH

NNH

NN OH

NN O

Nicotine

HspA

Methylamine

Pseudooxynicotine

Spontaneous

SsaDH


Nicotyrine

Nornicotine

Myosmine


Cotinine

1

2

3

4

5

HspB

CH3

CH3

HO

HO

HO

HOHO

OH

OH

OH

OH

OH

N-Methylmyosmine


H2NCH3




H2N


Citric acid cycle


CH3

H2O







2 and H

2O and forms SAP






N

N

N

N

N

N

N

NH

O

N

O

N

NN

Nicotine


pyridine

N-Methylmyosmine




OH

OH





O

O

N

O

O

N

N

N

N

N

N

O

N

N

NDH

HSP hydroxylase

Citric acid cycle

Succinic acid

Nicotine

2H

6-HLNO

Spontaneous

Methylamine+

Unknown steps

H2O

H2O

HO

HO

HO

HO

HO

OHHO OH

OH


O2


NH



H2NCH3


CH3








N

N

N

NH

N

O

N

O

OH

N

OHO

O

N

N

Nicotine

Nornicotine

Succinic acid

Carbamic acid

Acetic acid

Myosmine

1

2

Aminomethyl

HO

OH

OH

NH

NH


CH3COOH



CH3-NH-COOH

NH2-COOH

CH3

CH3









Acknowledgments


References







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Th

eoptim

alcond

ition

snicotin

edegradatio

neffi

ciencyand



Microorganism

Medium

Optim

alcond

ition

s(pHandTemperature)

Nicotined

egradatio

neffi

ciency

(gL)

Pathway

Reference

Fung

iPellicularia

filam

entosa

JTS-208

Synthetic

ND

004

20d

Dem

ethylatio

n[21]

Cunn

ingham

ellaechinu

lata

IFO-444

4Synthetic

ND

05413

dDem

ethylatio

n[21]

Aspergillus

oryzae

112822

Synthetic

6528∘C

21240h

Avaria

ntof

pyrid

inea

ndpyrrolidine

[22]

Gram-positive

bacteria

Arthrobacternicotin

ophagum

Nsp

Synthetic

68to

7020to

25∘C

16256

hPy

ridine

[23]

Arthrobacter

spH

F-2

Synthetic

7030∘C

07043h

ND

[24]

Gram-negativeb

acteria

Pseudomonas

sp41

Synthetic

6430∘C

13024

hPy

rrolidine

[25]

Pseudomonas

spH

F-1

Synthetic

65to

7530∘C

13025

hPy

rrolidine

[26]

Ochrobactr

uminterm

edium

DN2

Synthetic

30∘C

05036h

ND

[27]

Pputid

aS16

Synthetic

7030∘C

30010

hPy

rrolidine

[28]

Pseudomonas

spN

ic22

Synthetic

6530

to34∘C

28952h

ND

[29]

Pputid

aJ5

Synthetic

30∘C

39748h

ND

[30]

RhodococcusspY2

2Synthetic

7028∘C

10052

hND

[31]

Agrobacte

rium

tumefa

ciens

S33

Synthetic

7030∘C

50018

hAvaria

ntof

pyrid

inea

ndpyrrolidine

[32]

Gram-negativeb

acteria

Ensifer

spN

7Synthetic

7028∘C

33548h

ND

[33]

Pseudomonas

spZ

UTS

KDSynthetic

7530∘C

15512h

ND

[34]

Pseudomonas

spH

ZN6

Synthetic

7030∘C

05012

hPy

rrolidine

[35]

ShinellaspH

ZN1

Synthetic

7030∘C

0509h

ND

[36]

AcinetobacterspTW

Synthetic

7030∘C

10012h

ND

[37]

Sphingom

onas

spT

YSynthetic

7030∘C

10018h

ND

[37]

Pseudoxanthomonas

sp5ndash52

Synthetic

7028∘C

10028

hND

[38]

Pstu

tzeri

Synthetic

7537∘C

25036h

ND

[39]

Sinorhizobium

sp5ndash28

Synthetic

7028∘C

15028

hND

[38]

Ochrobactrum

sp4ndash4

0Synthetic

7028∘C

05028h

ND

[38]

Pplecoglossicid

aTN

D35

Synthetic

7030∘C

3012h

Avaria

ntof

pyrrolidine

[2]

NDnot

detected




Nicotine(mgg)















2





N

N

NN

N

NHO

N

O

NNH

O

HO

O

O

O

N

N

N

NDH

KOKDH

N

NOO

O

MABO

H H

O

Nicotine

(Ssa)

2H

6-HLNO6-HDLO

26-DHPONH

2H26-DHPH

+

Formaldehyde

FolDPurU

+

MAO

Methylamine

AO

SsaDH

Spontaneous

Spontaneous

Ammonia

H2O

H2O

HO

HO

HO

HO

HO

HO

HOHO

HOOHOH


O2

O2



OH

OH

OH

HO

OH

HO

OH

OH




Nicotine blue

NH2


H2NCH3

Citric acid cycle

NH3



Succinic acid

O2

NH

2HH2O

H2O

CH3

CH3

CH3



NN

HO

O

O

O

NicA

NN

N

O

O

N

N

NHO

N

OHO

O

PNAO

OO

OO O

OOH

N

HO

O

SAPD

NN

NN

NNH

NNH

NN OH

NN O

Nicotine

HspA

Methylamine

Pseudooxynicotine

Spontaneous

SsaDH


Nicotyrine

Nornicotine

Myosmine


Cotinine

1

2

3

4

5

HspB

CH3

CH3

HO

HO

HO

HOHO

OH

OH

OH

OH

OH

N-Methylmyosmine


H2NCH3




H2N


Citric acid cycle


CH3

H2O







2 and H

2O and forms SAP






N

N

N

N

N

N

N

NH

O

N

O

N

NN

Nicotine


pyridine

N-Methylmyosmine




OH

OH





O

O

N

O

O

N

N

N

N

N

N

O

N

N

NDH

HSP hydroxylase

Citric acid cycle

Succinic acid

Nicotine

2H

6-HLNO

Spontaneous

Methylamine+

Unknown steps

H2O

H2O

HO

HO

HO

HO

HO

OHHO OH

OH


O2


NH



H2NCH3


CH3








N

N

N

NH

N

O

N

O

OH

N

OHO

O

N

N

Nicotine

Nornicotine

Succinic acid

Carbamic acid

Acetic acid

Myosmine

1

2

Aminomethyl

HO

OH

OH

NH

NH


CH3COOH



CH3-NH-COOH

NH2-COOH

CH3

CH3









Acknowledgments


References







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Nicotine(mgg)















2





N

N

NN

N

NHO

N

O

NNH

O

HO

O

O

O

N

N

N

NDH

KOKDH

N

NOO

O

MABO

H H

O

Nicotine

(Ssa)

2H

6-HLNO6-HDLO

26-DHPONH

2H26-DHPH

+

Formaldehyde

FolDPurU

+

MAO

Methylamine

AO

SsaDH

Spontaneous

Spontaneous

Ammonia

H2O

H2O

HO

HO

HO

HO

HO

HO

HOHO

HOOHOH


O2

O2



OH

OH

OH

HO

OH

HO

OH

OH




Nicotine blue

NH2


H2NCH3

Citric acid cycle

NH3



Succinic acid

O2

NH

2HH2O

H2O

CH3

CH3

CH3



NN

HO

O

O

O

NicA

NN

N

O

O

N

N

NHO

N

OHO

O

PNAO

OO

OO O

OOH

N

HO

O

SAPD

NN

NN

NNH

NNH

NN OH

NN O

Nicotine

HspA

Methylamine

Pseudooxynicotine

Spontaneous

SsaDH


Nicotyrine

Nornicotine

Myosmine


Cotinine

1

2

3

4

5

HspB

CH3

CH3

HO

HO

HO

HOHO

OH

OH

OH

OH

OH

N-Methylmyosmine


H2NCH3




H2N


Citric acid cycle


CH3

H2O







2 and H

2O and forms SAP






N

N

N

N

N

N

N

NH

O

N

O

N

NN

Nicotine


pyridine

N-Methylmyosmine




OH

OH





O

O

N

O

O

N

N

N

N

N

N

O

N

N

NDH

HSP hydroxylase

Citric acid cycle

Succinic acid

Nicotine

2H

6-HLNO

Spontaneous

Methylamine+

Unknown steps

H2O

H2O

HO

HO

HO

HO

HO

OHHO OH

OH


O2


NH



H2NCH3


CH3








N

N

N

NH

N

O

N

O

OH

N

OHO

O

N

N

Nicotine

Nornicotine

Succinic acid

Carbamic acid

Acetic acid

Myosmine

1

2

Aminomethyl

HO

OH

OH

NH

NH


CH3COOH



CH3-NH-COOH

NH2-COOH

CH3

CH3









Acknowledgments


References







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






2





N

N

NN

N

NHO

N

O

NNH

O

HO

O

O

O

N

N

N

NDH

KOKDH

N

NOO

O

MABO

H H

O

Nicotine

(Ssa)

2H

6-HLNO6-HDLO

26-DHPONH

2H26-DHPH

+

Formaldehyde

FolDPurU

+

MAO

Methylamine

AO

SsaDH

Spontaneous

Spontaneous

Ammonia

H2O

H2O

HO

HO

HO

HO

HO

HO

HOHO

HOOHOH


O2

O2



OH

OH

OH

HO

OH

HO

OH

OH




Nicotine blue

NH2


H2NCH3

Citric acid cycle

NH3



Succinic acid

O2

NH

2HH2O

H2O

CH3

CH3

CH3



NN

HO

O

O

O

NicA

NN

N

O

O

N

N

NHO

N

OHO

O

PNAO

OO

OO O

OOH

N

HO

O

SAPD

NN

NN

NNH

NNH

NN OH

NN O

Nicotine

HspA

Methylamine

Pseudooxynicotine

Spontaneous

SsaDH


Nicotyrine

Nornicotine

Myosmine


Cotinine

1

2

3

4

5

HspB

CH3

CH3

HO

HO

HO

HOHO

OH

OH

OH

OH

OH

N-Methylmyosmine


H2NCH3




H2N


Citric acid cycle


CH3

H2O







2 and H

2O and forms SAP






N

N

N

N

N

N

N

NH

O

N

O

N

NN

Nicotine


pyridine

N-Methylmyosmine




OH

OH





O

O

N

O

O

N

N

N

N

N

N

O

N

N

NDH

HSP hydroxylase

Citric acid cycle

Succinic acid

Nicotine

2H

6-HLNO

Spontaneous

Methylamine+

Unknown steps

H2O

H2O

HO

HO

HO

HO

HO

OHHO OH

OH


O2


NH



H2NCH3


CH3








N

N

N

NH

N

O

N

O

OH

N

OHO

O

N

N

Nicotine

Nornicotine

Succinic acid

Carbamic acid

Acetic acid

Myosmine

1

2

Aminomethyl

HO

OH

OH

NH

NH


CH3COOH



CH3-NH-COOH

NH2-COOH

CH3

CH3









Acknowledgments


References







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


N

N

NN

N

NHO

N

O

NNH

O

HO

O

O

O

N

N

N

NDH

KOKDH

N

NOO

O

MABO

H H

O

Nicotine

(Ssa)

2H

6-HLNO6-HDLO

26-DHPONH

2H26-DHPH

+

Formaldehyde

FolDPurU

+

MAO

Methylamine

AO

SsaDH

Spontaneous

Spontaneous

Ammonia

H2O

H2O

HO

HO

HO

HO

HO

HO

HOHO

HOOHOH


O2

O2



OH

OH

OH

HO

OH

HO

OH

OH




Nicotine blue

NH2


H2NCH3

Citric acid cycle

NH3



Succinic acid

O2

NH

2HH2O

H2O

CH3

CH3

CH3



NN

HO

O

O

O

NicA

NN

N

O

O

N

N

NHO

N

OHO

O

PNAO

OO

OO O

OOH

N

HO

O

SAPD

NN

NN

NNH

NNH

NN OH

NN O

Nicotine

HspA

Methylamine

Pseudooxynicotine

Spontaneous

SsaDH


Nicotyrine

Nornicotine

Myosmine


Cotinine

1

2

3

4

5

HspB

CH3

CH3

HO

HO

HO

HOHO

OH

OH

OH

OH

OH

N-Methylmyosmine


H2NCH3




H2N


Citric acid cycle


CH3

H2O







2 and H

2O and forms SAP






N

N

N

N

N

N

N

NH

O

N

O

N

NN

Nicotine


pyridine

N-Methylmyosmine




OH

OH





O

O

N

O

O

N

N

N

N

N

N

O

N

N

NDH

HSP hydroxylase

Citric acid cycle

Succinic acid

Nicotine

2H

6-HLNO

Spontaneous

Methylamine+

Unknown steps

H2O

H2O

HO

HO

HO

HO

HO

OHHO OH

OH


O2


NH



H2NCH3


CH3








N

N

N

NH

N

O

N

O

OH

N

OHO

O

N

N

Nicotine

Nornicotine

Succinic acid

Carbamic acid

Acetic acid

Myosmine

1

2

Aminomethyl

HO

OH

OH

NH

NH


CH3COOH



CH3-NH-COOH

NH2-COOH

CH3

CH3









Acknowledgments


References







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


NN

HO

O

O

O

NicA

NN

N

O

O

N

N

NHO

N

OHO

O

PNAO

OO

OO O

OOH

N

HO

O

SAPD

NN

NN

NNH

NNH

NN OH

NN O

Nicotine

HspA

Methylamine

Pseudooxynicotine

Spontaneous

SsaDH


Nicotyrine

Nornicotine

Myosmine


Cotinine

1

2

3

4

5

HspB

CH3

CH3

HO

HO

HO

HOHO

OH

OH

OH

OH

OH

N-Methylmyosmine


H2NCH3




H2N


Citric acid cycle


CH3

H2O







2 and H

2O and forms SAP






N

N

N

N

N

N

N

NH

O

N

O

N

NN

Nicotine


pyridine

N-Methylmyosmine




OH

OH





O

O

N

O

O

N

N

N

N

N

N

O

N

N

NDH

HSP hydroxylase

Citric acid cycle

Succinic acid

Nicotine

2H

6-HLNO

Spontaneous

Methylamine+

Unknown steps

H2O

H2O

HO

HO

HO

HO

HO

OHHO OH

OH


O2


NH



H2NCH3


CH3








N

N

N

NH

N

O

N

O

OH

N

OHO

O

N

N

Nicotine

Nornicotine

Succinic acid

Carbamic acid

Acetic acid

Myosmine

1

2

Aminomethyl

HO

OH

OH

NH

NH


CH3COOH



CH3-NH-COOH

NH2-COOH

CH3

CH3









Acknowledgments


References







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






2 and H

2O and forms SAP






N

N

N

N

N

N

N

NH

O

N

O

N

NN

Nicotine


pyridine

N-Methylmyosmine




OH

OH





O

O

N

O

O

N

N

N

N

N

N

O

N

N

NDH

HSP hydroxylase

Citric acid cycle

Succinic acid

Nicotine

2H

6-HLNO

Spontaneous

Methylamine+

Unknown steps

H2O

H2O

HO

HO

HO

HO

HO

OHHO OH

OH


O2


NH



H2NCH3


CH3








N

N

N

NH

N

O

N

O

OH

N

OHO

O

N

N

Nicotine

Nornicotine

Succinic acid

Carbamic acid

Acetic acid

Myosmine

1

2

Aminomethyl

HO

OH

OH

NH

NH


CH3COOH



CH3-NH-COOH

NH2-COOH

CH3

CH3









Acknowledgments


References







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


N

N

N

N

N

N

N

NH

O

N

O

N

NN

Nicotine


pyridine

N-Methylmyosmine




OH

OH





O

O

N

O

O

N

N

N

N

N

N

O

N

N

NDH

HSP hydroxylase

Citric acid cycle

Succinic acid

Nicotine

2H

6-HLNO

Spontaneous

Methylamine+

Unknown steps

H2O

H2O

HO

HO

HO

HO

HO

OHHO OH

OH


O2


NH



H2NCH3


CH3








N

N

N

NH

N

O

N

O

OH

N

OHO

O

N

N

Nicotine

Nornicotine

Succinic acid

Carbamic acid

Acetic acid

Myosmine

1

2

Aminomethyl

HO

OH

OH

NH

NH


CH3COOH



CH3-NH-COOH

NH2-COOH

CH3

CH3









Acknowledgments


References







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






N

N

N

NH

N

O

N

O

OH

N

OHO

O

N

N

Nicotine

Nornicotine

Succinic acid

Carbamic acid

Acetic acid

Myosmine

1

2

Aminomethyl

HO

OH

OH

NH

NH


CH3COOH



CH3-NH-COOH

NH2-COOH

CH3

CH3









Acknowledgments


References







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Acknowledgments


References







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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Current Status on Biochemistry and Molecular Biology of Microbial Degradation of Nicotine

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