Recent advances in production and biotechnological applications of thermostable and alkaline...

Mini-reviewReceived: 7 May 2013 Revised: 1 July 2013 Accepted article published: 13 July 2013 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.4170

Recent advances in production andbiotechnological applications of thermostableand alkaline bacterial lipasesLimpon Bora,∗ Dibakar Gohain and Reshmi Das

Abstract

Microbial lipases occupy a vital position with respect to their industrial applications and they are studied extensively.Thermophilic microorganisms are potential sources of thermostable alkaline lipases, which have been isolated from variousnatural origins. Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common.Thermostable alkaline lipases have commercial value and find applications in various industrial and biotechnological sectorssuch as additives in detergents, additives in food industries, environmental bioremediations and in molecular biology. Thelatest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directedevolution and exploring natural communities by the metagenomic approach. Therefore, thermostable alkaline lipases are theenzymes of choice for many biotechnologists, microbiologists, biochemists, environmentalists and biochemical engineers. Inthe present review, we discuss some novel sources along with recent advances in fermentation conditions, substrate conditionsand biotechnological applications of thermostable alkaline bacterial lipases.c© 2013 Society of Chemical Industry

Keywords: thermostable alkaline lipase; thermophiles; statistical approach; biotechnological applications

INTRODUCTIONLipases are emerging as the leading biocatalysts with provenindustrial potential for contributing to the multi-billion dollarunder-exploited lipid technology-based bio-industry and havebeen used in in situ lipid metabolism and ex situ multi-facetedindustrial applications.1 Lipases are triaclyglycerol acylhydrolases(EC 3.1.1.3) that catalyse the hydrolysis of triaclyglycerol to glyceroland fatty acids. Besides hydrolysis, lipase can catalyse a wide rangeof reactions including inter-esterification, alcoholysis, acidolysis,esterification and aminolysis (Fig. 1).2 They also express otheractivities, such as phospholipase, isophospholipase, cholesterolesterase, cutinase, amidase and other esterase-type activities.3

Lipases contain a common folding pattern, like other hydrolases,which is known as the α/β hydrolase fold.4 The canonical α/βhydrolase fold consists of a central, mostly parallel β sheet ofeight strands with the second strand anti-parallel (Fig. 2). Theparallel strands β3–β8 are connected by α helices, which pack oneither side of the central β sheet. The β sheet has a left-handedsuper-helical twist so that the surface of the sheet covers abouthalf a cylinder and the first and last strands cross each other atan angle of 90◦.5 The X-ray structure of Pseudomonas aeruginosalipase reveals the conserved α/β hydrolase fold of lipase (Fig. 3).The active site of the α/β hydrolase fold enzymes consists ofthree catalytic residues: a nucleophilic residue (serine, cysteine oraspartate), a catalytic acid residue (aspartate or glutamate), and ahistidine residue, always in this order in the amino acid sequence.4

Microbial lipases have versatile industrial potential and are gainingattention due to their stability in extreme temperature, pH andorganic solvents. Lipases are naturally produced by several plants,animals and microorganisms. Some important lipase-producing

bacterial genera are Bacillus, Pseudomonas, Burkholderia, etc.6 andfungal genera include Aspergillus, Penicillium, Rhizopus, Candida,etc.7 Different species of yeasts belonging to seven differentgenera Zygosaccharomyces, Pichia, Lachancea, Kluyveromyces,Saccharomyces, Candida and Torulaspora are also reported toproduce lipase.8

Lipases are industrially important biocatalysts because they canact on a variety of substrates promoting a broad range of bio-catalytic reactions.9 Lipases from different sources show differentsubstrate specificities.10 Lipase activity can be divided into typo-selectivity (ability to hydrolyse a particular type of fatty acid ester),regioselectivity (ability to hydrolyse carboxylic ester groups atsn-1 and sn-3 positions as compared to sn-2 position) and stereo-selectivity (ability to differentiate between two enantiomers ina racemic substrate).11 Stereo-isomers are molecules having thesame molecular structure with different spatial arrangementsof the atoms whereas enantiomers are isomers which arenon-superimposable mirror images of each other.12 The stereo-specificity of a lipase is determined by the structure of thesubstrate, interaction at the active site and also by the reactionconditions.13 Lipases are capable of recognising enantiomers aswell as enantiotropic groups of prochiral molecules.14 The lipaseexists in two conformations (open and closed) in solution.15 The

∗ Correspondence to: Limpon Bora, Department of Molecular Biology andBiotechnology, Tezpur University, Tezpur 784028, Assam, India, E-mail:[email protected] or [email protected]

Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur,784028, Assam, India

J Chem Technol Biotechnol (2013) www.soci.org c© 2013 Society of Chemical Industry

www.soci.org L. Bora, D. Gohain and R. Das

Figure 1. Reactions catalysed by lipase.

two states are in free equilibrium favouring the closed form insolution. When an interface is introduced, the adsorption of thelipase will change the overall equilibrium in favour of the openconformation.16 The enantio-selectivity largely depends upon thechemical properties of the substrate, the origin of the lipase and theexperimental conditions used in the reaction.17 A lipase moleculecan differentiate between different stereo-isomeric groups presentin a prochiral molecule, e.g. porcine pancreatic lipase, rabbitgastric lipase, and human gastric lipase exhibit similar stereo-specificity, but they differ with sterically non-equivalent estergroups containing one single Triacylglyceride (TAG) molecule18,19

(Fig. 4).Thermostable alkaline lipases isolated and reported were of

diverse form based on their specificity and applicability20 andthis makes them ideal tools in industrial and chemical processeswhere relatively high reaction temperatures and organic solventsare used. The importance of thermostable alkaline lipases couldbe observed by the great number of published articles recently.In fact, over the last few years, there has been a progressiveincrease in the number of publications with the development ofnewer techniques and advances in molecular biology related toindustrial applications of lipase-catalysed reactions, performedin common organic solvents, ionic liquids, or even in non-conventional solvents. The reviews in the recent years focussedon microbial lipases, their production, biochemical properties and

different aspects but specifically never focussed on thermostablealkaline bacterial lipases and their different perspectives. In theseendeavours there is a great need to compile detailed informationon bacterial thermostable alkaline lipases related to potentialindustrial applications, which will in turn provide a direction ofresearch into thermostable alkaline lipases.

Hence, in this review, we have made an effort to organisethe information and work in recent times on thermostablealkaline bacterial lipases. Besides discussing the reason for thethermostability and alkaline character of the lipases, possibleimprovements and a number of expected developments arealso suggested in the article. The present review is focussedon lipase production, discussing the microorganisms, substratesand process operations used in this specific field in recent times.

SOURCES OF BACTERIAL THERMOSTABLEAND ALKALINE LIPASESLipases are ubiquitous in nature and are produced by severalplants, animals and microorganisms. Lipases of microbialorigin represent the most widely used class of enzymes inbiotechnological applications and organic chemistry. Most lipasesthat are derived from mesophilic microorganisms can act in awide range of pH values but are mostly unstable at temperaturesabove 70 ◦C. Bacterial lipases generally have temperature optima

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Figure 2. Canonical fold of α/β hydrolases. α helices are indicated by cylinders, and β strands are indicated by shaded arrows. Position of the active-siteresidues is shown by a solid circle; the nucleophile is the residue after β strand 5, the Asp/Glu residue is after β strand 7, and the histidine residue is in theloop between β8 and αF.4

Figure 3. The X-ray structure of Pseudomonas aeruginosa lipase.5

in the range of 30–65◦C21 and pH optima in the range of4.0–11.0.6 Microorganisms living at temperatures above 70 ◦C(extreme thermophiles), however, are an interesting source ofthermostable lipases. They are, in general, superior to the normallipases, because they produce proteins with unique propertiesand show reasonable activity even at high temperature and inthe presence of organic solvents and detergents.22 Though very

little information is available about the lipolytic enzyme systemsof extreme thermophiles and akaliphiles, especially from strictlyanaerobic bacteria, their enzymes are expected to be a potent

and powerful tool in industrial biotransformation processes.23–25

In recent literature bacterial species such as Bacillus pumilus,26

Burkholderia multivorans,27,28 Geobacillus sp.29 etc. are reported toproduce thermostable alkaline lipases. A review of most recent

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Figure 4. Enantiomer differentiation in lipase and other related reactions.16

potential bacterial species producing thermostable alkaline lipaseis reported in Table 1.

THERMOPHILIC BACTERIA AND THEIRHABITATThermophiles are a group of heat-loving microbes which thrive athigh temperature, usually greater than 45 ◦C. They are inhabitantsof various ecological niches such as deep sea hydrothermalvents, terrestrial hot springs and other extreme geographicalsites, including volcanic sites, tectonically active faults as well asdecaying matter such as the compost and deep organic landfills.46

In the literature, it is reported that thermophilic lipase-producingbacterial strains are isolated from hot springs,34,43 volcanic sites47

marine sediments,38 wastes48 etc. In hot springs, the temperatureis above 45 ◦C, and it is kept constant by continual volcanicactivity. Besides temperature, other environmental parameterssuch as pH, available energy sources, ionic strength and nutrientsinfluence the diversity of thermophilic microbial populations.The best known and well-studied geothermal areas are in NorthAmerica (Yellowstone National Park), Iceland, New Zealand,Japan, Italy, and countries of the former Soviet Union. Hot springsare situated throughout the length and breadth of India, suchas Manikaran in Himachal Pradesh, Soldhar and Ringigad inUttranchal, Bukreshwar in West Bengal, India,49 and Dirang andTawang in Arunachal Pradesh.50 A review of the habitat of the

Table 1. Bacterial strains producing thermostable and alkaline lipase

Microorganism

Optimum

temperature

(◦C)

Optimum

pH Reference

Acinetobacter sp. EH28 50 10.0 30

Aneurinibacillusthermoaerophilus HZ

65 7.0 31

Bacillus cereus C7 60 8.0 32

Burkholderia cepaciaATCC 25416

60 11 33

Bacillus sp. 50 10.0 34

Bacillus sp. LBN-4 65 8.0 35

Bacillus sp. 55 7.0 36

Bacillus subtilis EH 37 60 8.0 37

Bacillus licheniformes 45 9.0 38

Bacillus alcalophilusB-M20

60 10.6 39

Bacillus smithi BTMS11 50 8.0 40

Bacillus coagulans BTS-3 55 8.5 41

Bacillusstearothermophilus

70 6.0 42

Bacillus thermoleovoransCCR11

60 9.0 and 10.0 43

Burkholderia multivoransPSU-AH130

55 8.0 28

Caldanaerobactersubterraneus DSM15242

75 7.0 21

Staphylococcus aureus 55 8.0 44

StreptomycesthermocarboxydusME168

50 8.5 45

ThermoanaerobacterthermohydrosulfuricusDSM 7021

75 8.0 21

most recent potential thermophilic bacterial species producinglipase are mentioned in Table 2.

ALKALIPHILIC BACTERIA AND THEIR HABITATAlkaliphiles are the microorganisms which are tolerant to highalkaline conditions and can grow at the pH range of 9.0–10.0.51

Alkaliphiles can be isolated from normal environments such as gar-den soil, household wastes etc., but the population of alkaliphiles ishigher in alkaline environments. Soda lakes are the natural alkalinehabitats of alkaliphilic bacteria. The water of soda lakes remainssaturated with sodium salts, especially chloride, carbonate andbicarbonate, with the pH generally around 10 due to high levels ofsodium carbonate.52 Such lakes are distributed all over the worldin countries such as the USA, South Africa, Kenya, China, India,Egypt, Mongolia etc. Some of the most recent lipase producingalkaliphilic bacteria and their habitat are mentioned in Table 3.

MOLECULAR ADAPTATION TO HIGHTEMPERATURE AND ALKALINE CONDITIONSThermophiles have evolved various strategies to survive athigh temperatures and developed adaptive mechanisms toperform their metabolic functions by incorporating uniquefeatures in their proteins and membranes. From an evolutionary



Table 2. Some thermophilic lipase-producing bacteria and theirsources

Bacterial strain Source of isolation Reference

Bacillus licheniformes Marine sediments ofparangipettai coast(latitude 11◦ 46′ Nlongitude 79◦ 46′ E),Tamilnadu

38

Bacillus sp. Hot spring of Dirangdistrict of ArunachalPradesh, India

34

Bacillus sp. LBN-4 Hot springs of Kitpi inTawang district ofArunachal Pradesh,India

35


Petrol from an oil refineryin Al-Zarqa city, Jordan

42


‘El Carrizal’ hot springs inVeracruz, Mexico

43


Chinese hot spring inTengcong

21

Geobacillus sp. TW1 Hot spring in Xiamen ofChina

29


Solar Lake 21

Table 3. Some alkaliphilic lipase producing bacteria and theirsources

Bacterial strain Source of isolation Reference

Bacillus sp. KBDL4 Bogoria soda lake in Kenya 53

Halomonas desiderata Bogoria soda lake in Kenya 53

Fodinicurvata sediminis Salt mine in Yunan (China) 54

Fodinicurvata fenggangensis Salt mine in Yunan (China) 54

perspective, current views suggest that the last universalcommon ancestor was mesophilic or moderately thermophilicand that extant extremophiles have subsequently colonised harshenvironments.55 The possibility of survival and occurrence ofthermophilic bacterial strains in high temperature habitats such ashot springs, is the presence of more saturated and straight chainfatty acids in the membrane lipids of these thermophiles whichallows them to grow at temperature by providing the right degreeof fluidity needed for membrane function. Such lipids are rich instable ether bonds making them thermo-tolerant or heat resistant.This implies that thermophilic organisms are able to grow at hightemperature due to the chemical stability of their membranelipids.56 Also the fatty acids provide a hydrophobic environmentfor the cells and keep the cells rigid enough to live at elevatedtemperatures. However, the exact mechanism of the survival andoccurrence of these thermophilic bacterial strains is known to bethe presence of type I DNA topoisomerase, which causes positivesuper-coiling and may stabilise the DNA. The histone-like proteinspresent inside the cell binds DNA, and protects the DNA fromdenaturation. Heat shock proteins, chaperons, are also likely toplay a role in stabilising and refolding proteins as they begin todenature. Thermophiles also tolerate high temperature by usingincreased interaction such as electrostatic, disulfide bridge andhydrophobic interactions.56 The presence of chaperones which

refold denatured proteins increases the stability of thermophilicproteins in thermophiles.57 Also thermophilic proteins appear tobe smaller and in some cases more basic, which may also result inincreased stability.58

The molecular electric charges and consequently molecularinteractions and functions are directly related to media pH, thusany changes in medium pH affect many biological functions suchas enzymatic processes, signalling pathways and transportationsof various components across the cytoplasmic membrane andcell wall.59 Alkaliphiles require sodium ions for normal growthand metabolism. Alkaliphiles adapt themselves with some uniqueadaptation mechanisms so that they can thrive in alkaline habitatssuch as soda lakes. Aerobic alkaliphiles generally maintain theircytoplasmic pH about 2 units lower than ambient pH, anaerobesaround 1 pH unit.60,61 Sodium ions are necessary to keep theintracellular H+ level high50,62 with release of sodium ions coupledwith the import of protons against both the gradients by Na+/H+

antiporters.63 Many membranes in alkaliphiles are reported tocontain sodium-coupled ATPases in place of proton-coupledATPases.64,65 It could be stated that the pH of the culture mediumplays critical role for the optimal physiological performance of thebacterial cells and the transport of various nutrient componentsacross the cell membrane thus maximising the alkaline level ofmedia for optimum production which was envisaged from theresults obtained.

FERMENTATION CONDITIONS OFTHERMOSTABLE AND ALKALINE BACTERIALLIPASES AND STRATEGIES FORIMPROVEMENTHighly thermostable alkaline lipases are mostly produced bythermophilic bacteria. Some mesophilic bacteria are also reportedto produce moderately thermostable alkaline lipases. To meetthe growing demand of industries, the development of low-cost medium and growth conditions are required for theproduction of lipases. Bacterial growth and enzyme production aremainly influenced by fermentation conditions, media compositionand physicochemical parameters such as incubation period,temperature, pH, agitation, dissolved oxygen, nitrogen source,carbon source, inducers, metal ions etc. Both solid statefermentation and submerged fermentation can be used forproduction of thermostable alkaline lipases, but submergedfermentation is the most preferred one because of greatercontrol on growth conditions. A list of some thermostable alkalinelipase producing bacteria and their production parameters aregiven in Table 4. Almost all the thermostable alkaline lipasepurification processes pare based on multi-step processes. Theprocesses used for protein purification, such as filtration, saltingout, dialysis and various chromatographic techniques, are used incombinations for enzyme purification. Some techniques employedin the purification of thermostable alkaline lipases are givenin Table 5. The significance of purified thermostable alkalinelipases has been increasing every day as their use in a number ofapplications increases, such as in the pharmaceutical and cosmeticindustries, food industries, detergent industries etc. The propertiesof thermostable alkaline lipases can be studied in terms of optimumpH, optimum temperature, effect of metal ions and chelatingagents, nature and concentration of substrate, organic solventsand stabilising agents, inhibitors etc. The bacterial thermostablealkaline lipases have an optimum activity at specific temperature



Table 4. Thermostable alkaline lipase-producing bacteria and their production parameters

Microbe

Incubation

period (h)

Growth

temperature

(◦C)

Growth

pH Nitrogen source Carbon source Inducer Reference

Acinetobacter sp.EH28

240 37 NM Tryptone and yeast extract Tributyrin NM 30

AneurinibacillusthermoaerophilusHZ

48 60 7.5 Peptone Olive oil NM 31

Bacillus cereus C7 24 30 8.0 Peptone Starch NM 32

Burkholderia cepaciaATCC 25416

48 30 7.0 Yeast extract Maltose Olive oil 33

Bacillus sp. 48 50 NM Inorganic Olive oil NM 34

Bacillus sp. LBN-4 30 50 NM Peptone Starch Olive oil 35

Bacillus sp. 72 55 7.0 NM Olive oil NM 36

Bacillus subtilis EH 37 240 37 NM Tryptone, yeast extract Tributyrin NM 37

Bacillus licheniformes 72 55 9.0 Soyabean extract Starch Peanut oil cake 38

Bacillus alcalophilusB-M20

72 60 10.6 SSY medium SSY medium NM 39

Bacillus smithi BTMS11

18 28 ± 2 NM Zo Bells marine broth Zo Bells marine broth NM 40

Bacillus coagulansBTS-3

48 55 8.5 Peptone, yeast extract Olive oil NM 41


72 45 NM Yeast extract Olive oil NM 42

BacillusthermoleovoransCCR11

18 55 6.5 Nutrient broth Nutrient broth Olive oil 43

BurkholderiamultivoransPSU-AH130

72 45 7.5 Tryptone, yeast extract Palm oil NM 28


32 65 7.2 Peptone, yeast extract NM NM 21

Staphylococcusaureus

24 37 7.2 YTTS medium YTTS medium NM 44

StreptomycesthermocarboxydusME168

72 45 7.2 Malt extract, Yeast extract Glucose Olive oil 45

Thermoanaerobacterthermohydrosulfu-ricus DSM7021

32 65 7.2 Peptone, yeast extract NM NM 21

NM, not mentioned; SSY medium, soybean starch yeast extract medium; YTTS medium, yeast extract, tryptone, Tween 80 medium.

and pH, but they are stable at a wide range of temperatures,pH and have broad substrate specificity. Some thermostablealkaline lipases along with their optimum pH and temperatureare mentioned in Table 6. Due to the stability at broad ranges oftemperature and pH the thermostable alkaline lipases have greatindustrial potential.

Olive mill waste (OMW) is an important industrial effluent whichcontains simple and complex sugars that might be a basis forfermentation processes.66,67 In addition, OMW generally containsvariable quantities of residual oil. For these reasons, OMW couldbe a potential candidate as a suitable liquid growth medium formicroorganisms producing lipolytic enzymes.68 Lipolytic mouldsare among the primary colonisers of OMW during its storage.69

The design and optimisation of fermentation conditions is ofimportance in biological processes in order to increase the yieldand productivity and improve a system without increasing thecost.70 This classical method is time consuming, and may also

lead to misinterpretation of results. To overcome these problems,the emphasis has shifted towards medium optimisation usingresponse surface methodology. The factorial design of a limitedset of variables is advantageous in relation to the conventionalmethod of manipulation of a single parameter per trial, it helpsin building models and in evaluating the effective factors tostudy interaction, and select optimum conditions of variables for adesired response.71 Recently, a number of statistical experimentaldesigns with response surface methodology have been employed

for optimising conditions which the most widely used.70–72

RECENT ADVANCES IN THE FIELD OFTHERMOSTABLE AND ALKALINE BACTERIALLIPASEThe demand of thermostable and alkaline bacterial lipase isincreasing day by day. To meet the increasing demand, both



Table 5. Purification of thermostable and alkaline lipases

Microorganism Chromatography technique

Purification

(fold)

Yield

(%) Reference

Acinetobacter sp. EH28 Hydrophobic interaction (phenyl Sepharose) 24.2 47 30

Aneurinibacillus thermoaerophilusHZ

Gel filtration (Sephadex-G75) 9.3 48.7 31

Burkholderia cepacia ATCC 25416 Ion exhange(DEAE- SepharoseFF column) 4.9 4.8 33

Bacillus sp. Gel filtration (Sephadex G-100 column) NM NM 34

Bacillus sp. Hydrophobic interaction (phenyl Sepharose CL-4Bcolumn)

8.6 13.0 36

Bacillus subtilis EH 37 Hydrophobic interaction (phenyl Sepharose) 17.8 16.0 37

Bacillus licheniformes Ion exchange (DEAE cellulose) 3.06 25.0 38

Gel filtration (Sephadex G-50) 3.6 19.0

Bacillus alcalophilus B-M20 Gel filtration (Sephadex G-100) 111.0 5.0 39

Bacillus smithi BTMS 11 Ion exchange (DEAE cellulose) 4.33 0.1 40

Bacillus coagulans BTS-3 Ion exchange (DEAE Sepharose) 40.0 2.5 41

Bacillus stearothermophilus NM NM NM 42

Bacillus thermoleovorans CCR11 NM NM NM 43


Ion exchange (DEAE-Toyopearl) 9.4 28.8 28

Gel filtration (Sephadex G-150) 21.6 12.1

Caldanaerobacter subterraneus DSM15242

Hydrophobic interaction (phenyl Sepharose) 36.3 30.0 21

Gel filtration (Superdex 200) 93.6 8.1

Staphylococcus aureus Hydrophobic interaction (phenyl Sepharose CL-4Bcolumn)

3.62 74.91 44

Gel filtration (Superose-12) 6.76 20

Streptomyces thermocarboxydusME168

Gel filtration (Superdex 200) 9.6 20.3 45

Thermoanaerobacterthermohydrosulfuricus DSM 7021

Hydrophobic interaction (phenyl Sepharose) 49.0 48.3 21

Gel filtration (Superdex 200) 108.7 13.0

NM, not mentioned.

quality and quantity of lipase need to be increased. Recentadvances in recombinant DNA technologies, high-throughputtechnologies, genomics and proteomics have fuelled thedevelopment of new catalysts and biocatalytic processes. So, itbecame necessary to use modern biotechnological approachesfor improving both quality and quantity of lipase. TheLipase Engineering Database (<http://www.led.uni-stuttgart.de/)provides structure–sequence–function relationships of lipaseinformation and the α/β fold hydrolase-related protein.73

However, only 28 three-dimensional structures of microbiallipases have been solved and the crystal structures of somelipases have not been discovered.74 So, together with traditionalmicrobiological screening methods, various molecular biologicaltools such as protein engineering, recombinant DNA technology,directed evolution and metagenomic approaches have proved tobe very useful for enzyme quality improvement and discovery ofnovel enzymes.75 Quantitative enhancement, i.e. increasing theenzyme productivity, can also be achieved through improving thestrain, for which techniques such as site-directed mutagenesis,recombinant DNA technology and growth media standardisationcan be adopted.75 As the genes are directly related to enzymeproduction in organisms, the strain improvement can be achievedby implementing changes at the gene level. Directed evolutionand metagenomic approach has emerged as a powerful tool forbiocatalyst engineering,76 in order to develop enzymes with novelproperties, even without requiring knowledge of the enzymestructure and catalytic mechanisms. This approach uses thegenetic diversity of the microorganisms in a certain environment,

as a whole, to encounter new or improved genes and geneproducts for biotechnological purposes. Recent developmentshave focused on using a combination of rational protein design anddirected evolution procedures for making smaller libraries.77 Basedon this approach, saturation mutagenesis has been extensivelyused in recent enzyme improvement protocols. In this approachrandomisation of all amino acids is done at one or more thanone positions in an enzyme.78,79 In this case the modifiedsequence space is smaller and therefore, faster to screen. Usingthis concept, iterative saturation mutagenesis was introducedas a new and more efficient method for directed evolutionof functional enzymes.79,80 Both rational protein design anddirected evolution can be repeated or combined to generatethe desired property of an enzyme. So, these protein engineeringstrategies have been established as efficient tools to successfullyimprove biotechnologically relevant properties of enzymes.79,81,82

Moreover, in recent years, computational protein design is gainingmore and more attention as a novel strategy to predict the effectsof mutations on protein structure, function or stability of librariesof enzyme variants generated by means of in silico approaches.83

Thermal stability is a major requirement for commercial enzymes,being critical for industrial applications, as thermal denaturationleads to enzyme inactivation.84 Several approaches can be appliedto improve thermal stability: directed evolution with randommutagenesis based on error-prone polymerase chain reaction(PCR), DNA shuffling, or the recent iterative saturation mutagenesisguided by rational design. Factors generally known to enhancethermal stability of protein are the hydrophobicity profile, the



Table 6. Characterisation of some thermostable alkaline lipases

Microorganism

Mol. wt.

(kDa)

Optimum

temperature (◦C)

Optimum

pH

Stable

temperature (◦)

range

Stable

pH range Reference

Acinetobacter sp. EH28 NM 50 10.0 30–60 8.0–11.0 30

Aneurinibacillusthermoaerophilus HZ

50 65 7.0 50–70 4.0–10.0 31

Bacillus cereus C7 NM 60 8.0 50–65 7.6–9.0 32

Burkholderia cepacia ATCC25416

33 60 11 50–70 9.0–11.5 33

Bacillus sp. 33 50 10.0 40–60 8.0–9.0 34

Bacillus sp. LBN-4 NM 65 8.0 45–75 7.0–8.5 35

Bacillus sp. 45 55 7.0 45–55 3.0–9.0 36

Bacillus subtilis EH 37 NM 60 8.0 50–60 7.0–9.0 37

Bacillus licheniformes 35 45 9.0 37–60 6.0–11.0 38

Bacillus alcalophilus B-M20 NM 60 10.6 NM NM 39

Bacillus smithi BTMS11 45 50 8.0 30–80 7.0–9.0 40

Bacillus coagulans BTS-3 31 55 8.5 50–60 7.0–9.0 41

Bacillus stearothermophilus NM 70 6.0 30–70 7.0–9.0 42


11 60 9.0 and 10.0 30–60 5.0–11.0 43


57.8 55 8.0 40–60 7.0–9.0 28

Caldanaerobactersubterraneus DSM 15242

34.2 75 7.0 60–80 6.5–9.0 21

Staphylococcus aureus 49 55 8.0 10–65 5.0–11.0 44

Streptomycesthermocarboxydus ME168

21 50 8.5 45–65 4.0–10.0 45


32.1 75 8.0 60–80 6.5–9.0 21

NM, not mentioned.

number of hydrogen bonds, amino acid composition and theirdistribution or interactions in the protein.85 Minimal changes in theprotein structure are sufficient for thermostability improvement,especially when changes take place on the protein surface whichwas demonstrated by Ahmad and co-workers.86 Error-prone PCRwas used by Sharma and collaborators to generate a highlythermostable mutant lipase starting from a metagenomic library:the improved mutant showed a 144-fold enhanced thermostabilityat 60 ◦C.87 The lipCCR11 gene for thermostable lipase (CCR11)from Geobacillus thermoleovorans was successfully expressed inEscherichia coli using pET-3b, PinPoint Xa, pET-28a (+), pGEM-Tplasmid vectors where expression was induced by Isopropylβ-D-1-thiogalactopyranoside (IPTG).76 The thermal stability andactivity of the lipase was enhanced by directed evolution applyingisoleucine to threonine mutation where the mutagenesis wasperformed by error-prone PCR and the library created was screenedfor thermostable lipase.88 By using recombinant DNA technology,Antibiotic resistance Marker (ARM) lipase gene from a Geobacillussp. was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host and there was increasedlevel of production and purification yield of the ARM lipase.89

Similarly, two thermostable lipase producing genes isolatedfrom two bacterial strains, viz. Caldanaerobacter subterraneusDSM 15242 and Thermoanaerobacter thermohydrosulfuricus DSM7021, were successfully cloned in Escherichia coli TOP10 host andlipase with improved thermostability and stability at alkaline pHcompared with that of wild type.21

POTENTIAL APPLICATIONS IN INDUSTRYLipases form an integral part of the industries ranging fromfood, dairy, pharmaceuticals, agrochemical and detergentsto oleo-chemicals, tea industries, cosmetics, leather and inseveral bioremediation processes.90 Following proteases andcarbohydrases, lipases are considered to be the third largestgroup of commercial enzymes based on total sales volume. Thecommercial use of lipases is a billion-dollar business that comprisesa wide variety of different applications.91 Some of the applicationsof lipase are summarised in Table 7. Lipases are valued biocatalystsbecause they are highly stable in organic solvents, show broadsubstrate specificity, and usually show high stereo-selectivity incatalysis.92 Most of the industrial processes in which lipases areused function at temperatures exceeding 45 ◦C. The enzymes,thus, are required to exhibit an optimum temperature of around50 ◦C.93 So, lipases which are alkaline and thermostable are themore sought after enzymes for industrial applications because oftheir ability to retain activity in harsh conditions. Because of theincreasing industrial applications, it is necessary to screen newermicrobes that produce thermostable and alkaline lipases. In thisreview the applications of only thermostable and alkaline bacteriallipases are being discussed.

Applications in the oleo-chemical industryLipases are hydrolases that act on carboxylic ester bonds. Thephysiological role of lipases is to hydrolyse triglycerides intodiglycerides, monoglycerides, fatty acids and glycerol. In addition



Table 7. Some applications of lipase90

Industry Action Product application

Dairy food Hydrolysis of milk, fat, cheese ripening, modificationof butter fat

Development of flavouring agent in milk cheese andbutter

Bakery food Flavour improvement Shelf life propagation

Beverages Improved aroma Alcoholic beverages, e.g. sake wine

Food dressings Quality improvement Maysoine dressing and whippings

Health food Trans esterification Health food

Meat and fish Flavour development Meat and fish product fat removal

Fats and oils Trans esterification and hydrolysis Cocoa butter, margarine fatty acids, glycerol mono- anddiglycerides

Laundry Reducing biodegradable stains Cleaning cloths

Cosmetics Esterification Skin and sun-tan creams, bath oil etc.

Surfactants Replaces phospholipases in the production oflysophospholipids

Polyglycerol and carbohydrates fatty acid esters used asindustrial detergents and as emulsifiers in foodformulation such as sauces and ice creams

Agrochemicals Esterification Herbicides such as phenoxypropionate

Pharmaceuticals Hydrolysis of epoxyester alcohols Produce various intermediates used in the manufactureof medicine

Fuel industries Trans esterification Biodiesel production

Pollution control Hydrolysis and trans esterification of oils and grease To remove hard stains, and hydrolyse oils and greases

to their natural function of hydrolysing carboxylic ester bonds,lipases can catalyse esterification, inter-esterification and trans-esterification reactions in non-aqueous media.94 In the modernoleo-chemical industry the use of immobilised lipases to initiatevarious reactions (hydrolysis, alcoholysis and glycerolysis) theuse of mixed substrates is rapidly increasing. Thus, the useof immobilised enzyme ensures high productivity as well ascontinuous running of the processes. This offers the greatest hopefor successful fat splitting and modification without substantialinvestment in expensive equipment as well as in expenditure oflarge amounts of thermal energy.95 The scope for the application oflipases in the oleo-chemical industry is enormous as it saves energyand minimises thermal degradation during hydrolysis, glycerolysisand alcoholysis.96 Immobilised lipase from Bacillus subtilis EH37strain was used to produce ethyl caprylate,30 a very useful foodand beverage flavouring agent with sweet fruity–flowery smell.97

Lipase from Streptomyces thermocarboxydus ME168 strain wassuccessfully reported to produce sugar esters from a wide rangeof substrates such as glucose, fructose and various vinyl esters.45

Applications in the detergent industryThe use of various enzymes in detergents is now very commonworldwide; more than half of all commercial detergents presentlyavailable contain enzymes. Such detergents are becoming moreand more popular because of their ability to impart softness, theirresiliency to fabrics, easy dissolution in water, and they are mildto eyes and skins. Worldwide demand for detergents has beengrowing continuously for the past decade There has been also atremendous increase in the significance of the biotechnologicalapplications of lipases during the last two decades as they displayamazing versatility in catalytic behaviour. The most commerciallyimportant application of lipases is their addition to detergents.98

Lipases were generally added to the detergents primarily incombination with proteases and cellulases.1 The lipases are addedto the detergent formulations because of their ability to removefat stains and oil or fatty deposits on clothes. To be a suitableadditive in detergents, lipases should be both thermostable andalkaline besides demonstrating catalytic activity in the presence of

various components of detergent formulations.99 Several lipase-producing organisms and their lipases are characterised as potentdetergent additives.20,30,100,101 Addition of bacterial thermostablealkaline lipases to some commercially available detergents hasincreased the ability of detergents to remove the oil or fatty stainsfrom clothes,102 proving that lipases are potential additives fordetergents.

Applications in the paper industryThe pulp and paper industry processes huge quantities ofbiomass rich in lignocelluloses every year. The technology for pulpmanufacturing is highly diverse and there lies opportunities forusing microbial enzymes for increasing and improving production.The enzymatic pitch control method using lipases has been in usein large-scale paper-making processes as a routine operation sincethe early 1990s.103 ‘Pitch’, or the hydrophobic components ofwood (mainly triglycerides and waxes), causes severe problemsin pulp and paper manufacture. Lipases are used to removethe pitch from the pulp produced for paper making.104 Theaddition of lipase from Pseudomonas sp. KWI-56 to a de-inking composition for ethylene oxide–propylene oxide adductstearate improved whiteness of paper and reduced residual inkspots.105

Applications in the leather industryNowadays lipases have found a great application in leather indus-try, mainly in fat removal or for degreasing purposes. Degreasingis an essential stage in the processing of skins from small animalsand hides from cattle.94 Lipase enzymes can remove fats andgrease from skins and hides, particularly those with a moderate fatcontent. The main advantages of using lipases over the conven-tional method are that uniformity in colour and a cleaner finishof the product along with improved hydrophobic (waterproof)properties.94 Many Bacillus sp. strains, which grow successfullyunder highly alkaline conditions, were found to be useful inleather processing.106 Thermostable alkaline lipases from bacteriaisolated from slaughter-house wastewater have shown potential



to be used in leather industry.48 NovoLime, a protease/lipaseblend for enzyme-assisted liming of hides and skins, and NovoCorAD, an acid lipase for degreasing of hides and skins, are somecommercially available lipases for leather industry.94

Applications in bioremediationApplication of lipases in bioremediation processes is a newaspect in lipase biotechnology. The wastes of lipid-processingfactories, restaurants, dairies etc. can be cleaned using lipasesfrom different origins. Lipase-producing bacterial strains playa key role in the enzymatic remediation of polluted soils.107

Lipase activity is an indicator parameter for testing hydrocarbondegradation in soil.108 Lipases from thermophilic and alkaliphilicbacteria have been shown to hydrolyse fat particles in slaughter-house wastewater48 and are therefore potential candidates for usein bioremediation. Bacterial consortia of three lipase-producingbacterial strains (P. aeruginosa, B. subtilis and Acinetobactercalocoaceticus) were shown to have potential for bioremediationof lipid-rich wastewater.109

Applications in the production of biodieselThe resources of fossil fuels are constantly diminishing and, as aresult, the price of crude oil is increasing drastically. Such burningproblems have led researchers to explore the use of vegetableoils as alternative fuels.110 The biodiesel from vegetable oil doesnot produce sulfur oxide and the amount of soot particulatesis one-third in comparison with the amount from conventionaldiesel. Because of these environmental advantages, biodiesel fuelcan be expected as a substitute for conventional diesel fuel.111

A lipase from Thermomyces lanuginosus (TL 100 L) was used inthe hydrolysis of soybean oil by a hydroesterification process andit was reported that the lipase hydrolysed 89% of the oil after48 h of reaction.112 Burkholderia multivorans PSU-AH130 producesan extracellular thermostable lipase which can produce biodieselfrom palm oil.28

FUTURE PERSPECTIVESMost of the thermostable and alkaline enzymes are characterisedby their high catalytic efficiency at high temperature and highpH range at which mesophilic enzymes lose their activity. Dueto this characteristic, thermostable and alkaline enzymes areuseful in biotechnology-based industries in order to shortenproduction time, minimise energy consumption and preventundesired chemical transformations and the loss of volatilecompounds. However, more study is required to overcomeseveral bottlenecks, such as high enzyme production cost, lowstability and the low biodiversity of extremophile microbesexplored so far. The introduction and development of the latestrecombinant DNA technology and protein engineering have hada positive impact in increasing the production of recombinantenzymes. Such efforts may increase the amount of enzymesproduced and lower the cost of production. Therefore, effortsshould be made to achieve economic benefits by production ofthermostable and alkaline lipases in heterologous hosts and thetailoring of their properties as desired by protein engineering.As enzyme technology is a clean and green technology incomparison to the chemical-based technology, in future theuse of enzymes in various fields, such as food agriculture,pharmaceuticals, bioremediation etc. will take a more positiveturn.

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