Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ibab20 Download by: [197.133.37.118] Date: 18 April 2017, At: 08:30 Biocatalysis and Biotransformation ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20 Biochemical and biotechnological studies on a novel purified bacillus cholesterol oxidase tolerant to solvent and thermal stress Fathy N ElBaz, Rawia F Gamal, Ashraf F ElBaz, Nasser E Ibrahim & Ahmed ElMekawy To cite this article: Fathy N ElBaz, Rawia F Gamal, Ashraf F ElBaz, Nasser E Ibrahim & Ahmed ElMekawy (2017): Biochemical and biotechnological studies on a novel purified bacillus cholesterol oxidase tolerant to solvent and thermal stress, Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2017.1306742 To link to this article: http://dx.doi.org/10.1080/10242422.2017.1306742 View supplementary material Published online: 18 Apr 2017. Submit your article to this journal View related articles View Crossmark data
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Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=ibab20
Download by: [197.133.37.118] Date: 18 April 2017, At: 08:30
Biochemical and biotechnological studies on anovel purified bacillus cholesterol oxidase tolerantto solvent and thermal stress
Fathy N ElBaz, Rawia F Gamal, Ashraf F ElBaz, Nasser E Ibrahim & AhmedElMekawy
To cite this article: Fathy N ElBaz, Rawia F Gamal, Ashraf F ElBaz, Nasser E Ibrahim &Ahmed ElMekawy (2017): Biochemical and biotechnological studies on a novel purified bacilluscholesterol oxidase tolerant to solvent and thermal stress, Biocatalysis and Biotransformation, DOI:10.1080/10242422.2017.1306742
To link to this article: http://dx.doi.org/10.1080/10242422.2017.1306742
Biochemical and biotechnological studies on a novel purified bacilluscholesterol oxidase tolerant to solvent and thermal stress
Fathy N ElBaza, Rawia F Gamalb, Ashraf F ElBaza, Nasser E Ibrahimc and Ahmed ElMekawya,d
aDepartment of Industrial Biotechnology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City (USC),Sadat City, Egypt; bDepartment of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt; cDepartmentof Bioinformatics, Genetic Engineering and Biotechnology Research Institute, University of Sadat City (USC), Sadat City, Egypt;dFaculty of Engineering, Computer and Mathematical Sciences, School of Chemical Engineering, University of Adelaide, Adelaide,Australia
ABSTRACTA novel bacterial strain was isolated and identified as Bacillus pumilus, with the capability to pro-duce cholesterol oxidase enzyme (55 kDa). The production of the enzyme was optimized viatwo-step statistical approach. Out of eight factors screened in Plackett–Burman, only four hadsignificant effects on enzyme activity. The optimization process of these four variables byBox–Behnken revealed that the maximum enzyme activity (90U/mL) was significantly obtainedafter 6 days of fermentation with 0.3%, 1% and 0.2% of NH4NO3, yeast extract and Tween 80,respectively. The purified enzyme showed optimum activity at pH 7.5 and temperature of 40 �C.The enzyme retained 100% of its activity after storage at 40 �C for 60min. The enzyme alsoexhibited enhanced stability in the presence of Tween 80, methanol and isopropanol. This solv-ent and thermal stress tolerant enzyme, produced by B. pumilus, may provide a practical optionfor industrial and analytical applications.
ARTICLE HISTORYReceived 27 November 2016Revised 25 February 2017Accepted 25 February 2017
Cholesterol (5-cholesten-3-ol) is a type of steroid,mainly located in the membranes of animal cells(Hassanein et al. 2012). It has been related to coloncancer development and the strong toxic effects of anumber of cholesterol oxides suggested a possiblerole for them in the occurrence of cardiovascular dis-eases, attributed to the high levels of blood choles-terol (Kim et al. 2002). Due to the importance ofmonitoring the two types of cholesterol (low-densitylipoprotein (LDL) and high-density lipoprotein (HDL))in serum for the diagnosis of atherosclerotic or hyper-lipaemia diseases, some methods have been devel-oped for the separation of HDL and LDL cholesterolsusing different types of detergents (Praveen et al.2011). These efforts focused on developing sensitive invitro analysing methods for monitoring and measuringcholesterol in different biological samples (Saxena andGoswami 2010; Saxena et al. 2011). The enzymatictechnique, utilizing cholesterol oxidase (ChO; EC1.1.3.6) enzyme, is a common method among choles-terol assessment techniques (Kasabe et al. 2015). Somebacterial species are capable of degrading cholesterol
by ChO, so that cholesterol is oxidized to 4-cholesten-3-one, and oxygen is reduced to hydrogen peroxide asshown in Figure 1 (Kim et al. 2002).
This enzyme has a wide range of clinical and indus-trial applications, for example, food, agricultural andpharmaceutical fields, leading to a considerableincreased demand for it (Niwas et al. 2013). Severalbacterial genera can produce ChO with the ability toassimilate cholesterol as a single carbon source, suchas Streptomyces (Praveen et al. 2011; Niwas et al.2013), Rhodococcus (Yazdi et al. 2008; Ahmad andGoswami 2014; Kasabe et al. 2015), Bardetella (Linet al. 2010), Chromobacterium (Doukyu et al. 2008),Enterobacter (Ye et al. 2008), Pseudomonas (Ghosh andKhare 2016), Mycobacterium (Yao et al. 2013) andBifidobacterium (Park et al. 2008). Stable and sensitiveChO is a current crucial need in order to get the bene-fit of this multipurpose enzyme. These needs might beachieved by enhancing the current ChO producerstrains by employing the contemporary recombinantbiotechnologies, although they entail some drawbackslike the development of inclusion bodies, heterologousprotein degradation and improper protein folding
(Amid and Hassan 2015). An alternative approach is toselect some novel microbial producers with the abilityto produce ChO with superior stability and stress toler-ance for improved applicability, for example, toenhance the differential assay technique for measuringLDL and HDL cholesterols in serum.
The objective of our study is the identification andisolation of new Bacillus strains, from the oily waste ofships that have the potential to produce tolerant andstable ChO under different conditions in order to beapplied in a wide range of medical fields. In the courseof the study, the taxonomic classification of a novelenzyme producer bacterial strain is described. Moreover,the fermentative conditions of the enzyme productionare statistically optimized through a two-step approachusing Plackett–Burman and Box–Behnken experimentaldesigns. The enzyme is purified and its pH tolerance,thermal stability and solvents tolerance are assessed.
Materials and methods
Materials and samples
The oily waste samples were kindly provided byDepartment of Industrial Biotechnology in University ofSadat City located in Egypt. Cholesterol and 4-choles-ten-3-one were purchased from Sigma Chemicals (St.
Louis, MO). Yeast extract and agar were supplied byOxoid (Hampshire, UK). Gel filtration column andSephadex G-100 were purchased from Pharmacia,Uppsala, Sweden. Protein marker was brought fromAffymetrix USBVR (Cleveland, OH). Bradford protein assaykit was purchased from Bio-Rad Laboratories, Richmond,CA. All other chemical reagents, solvents and salts wereof analytical grade and purchased from local suppliers.
Isolation and ChO screening of bacterial cells
The samples (5% v/v) were inoculated into cholesterolenrichment medium (1 g NH4NO3, 0.25 g K2HPO4,0.25 g MgSO4, 0.001 g FeSO4, 5 g yeast extract, 1 gcholesterol (dissolved in 0.1% Tween 80) and distilledwater up to 1 L; pH 7) and incubated under shakingcondition at 37 �C for 7 days. Enriched cultures wereserially diluted with saline solution and plated onmodified cholesterol agar (Praveen et al. 2011).Colonies were examined for halos and positive oneswere isolated by frequent single-colony isolation onthe same medium. Pure colonies were streaked onChO indicator agar medium plates (1 g cholesterol, 1 gTriton X-100, 0.1 g o-dianisidine, 1000U of peroxidase,15 g agar and distilled water up to 1 L) and incubatedfor 2–4 days at 37 �C (Niwas et al. 2013). The growingcolonies were examined for the formation of brownpigments surrounding them and positive ones wereisolated as ChO-producing bacteria and maintained at4 �C for further study. The ChO production by differentisolates was verified in Erlenmeyer flasks (250mL),each containing 100mL modified cholesterol growthmedium. The flasks were inoculated with 1% of cellssuspension and incubated at 37 �C on a rotary shaker(150 rpm) for 7 days, after which the enzyme activitywas assayed.
Taxonomic studies of the ChO producer strain
The highest ChO-producing bacterial culture wasdetermined and identified through 16S rRNA hom-ology technique. Isolation of DNA, amplification bypolymerase chain reaction (PCR), and sequencing ofthe amplified product was performed according to theearlier reported procedures (Praveen and Tripathi2009). The pairwise sequence alignment was carriedout via BLAST (http://www.ncbi.nlm.nih.gov/) tool andmultiple sequence alignment by CLUSTALW. Thephylogenetic tree was created by neighbor-joiningapproaches using MEGA 7 software (State College, PA,USA). The resulting nucleotide sequence was depos-ited in the GenBank database under accession numberKX685666.
Figure 1. Reaction steps catalysed by ChO, which catalysesthe oxidation of the cholesterol, in the presence of O2, to cho-lest-5-en-3-one which isomerized to cholest-4-en-3-one withthe reduction of prosthetic group FAD to FADH2 which thenregenerated again by reduction of the free O2 to H2O2.
The activities of extracellular and membrane-boundenzymes were measured according to the techniqueof Inouye et al. (Yehia et al. 2015). The total assay mix-ture was 0.525mL, containing 0.1mL of enzyme solu-tion in 0.4mL Tris-HCl buffer (125mM, pH 7.5),incubated for 3min at 37 �C, and 25 lL of cholesterol(12mM) dissolved in isopropanol. Absolute ethanol(2.5mL) was added after 30min, and the 4-cholesten-3-one degradation product amount was determinedby measuring its absorbance at 240 nm. Authentic 4-cholesten-3-one was used as analytical comparativestandard (10–100 lg/mL dissolved in isopropanol). Oneunit of the ChO activity was defined as the amount ofenzyme which releases 1lmol of 4-cholestene-3-onefrom cholesterol per minute. The total proteins wereassessed using Bradford’s method applying bovineserum albumin (BSA) as the standard (Bradford 1976).
Fermentative optimization of ChO production
The production of ChO enzyme was optimizedemploying modified cholesterol medium with differentexamined variables, with ChO activity measured as thedependent response. Eight variables were screened,via the application of Plackett–Burman design (PBD),based on the impact percentage of each variable(Abdel-Monem et al. 2012; ElBaz et al. 2016). Thetested variables include cholesterol concentration, pH,harvesting time, NH4NO3 concentration, yeast extractconcentration, aeration (medium volume/flask volume),cholesterol addition time and Tween 80 concentration.Two levels were assigned for each variable; low (�1)and high (þ1) levels (Table 1). The matrix of the testedvariables resulted in 12 experiments in which the lev-els of the eight factors were diverged.
Based on the results of PBD, the optimization pro-cess was expanded to a Box–Behnken design (BBD).The significant factors recognized from PBD; harvestingtime (X3), NH4NO3 concentration (X4), yeast extract
concentration (X5) and Tween 80 concentration (X8),were selected, with a broader range of levels, to bethe main independent variables for BBD (Table 1).Three levels were assigned for each factor; low (�1),medium (0) and high (þ1) levels. The matrix of thetested variables resulted in 27 experiments. To predictthe optimum point, a second-order polynomial equa-tion was fitted for the correlation of the relationshipbetween the ChO activity and the independent varia-bles (ElMekawy et al. 2013).
Purification of the ChO enzyme
All the purification steps were performed at 5 �C. Thegrowing culture broth was centrifuged at 104�g for10min and the cell-free supernatant was exploited asan extracellular crude ChO source. To obtain the mem-brane-bound enzyme, the collected cells were washedtwice using ethyl acetate and then frozen at �18 �C.The extraction of membrane-bound ChO from frozencells was carried out in 1mM phosphate buffer (pH 7),containing 0.7% Triton X-100 (v/v), for 18 h at 5 �Cunder continuous stirring. The extracted enzyme wassubsequently centrifuged and the cell-free supernatantwas used as a crude membrane-bound enzyme source.
The ChO enzyme precipitation by ammonium sul-phate was optimized, using different saturation con-centrations (45, 50, 60, 70 and 75%) under differentpH values (5.5, 6, 7, 8 and 8.5), through 10 trials.Ammonium sulphate was gradually added to thecrude ChO solution with continuous stirring till it wascompletely solubilized at the defined pH value. Theammonium sulphate traces in the precipitated pelletwere removed by dialysis against distilled water in acellophane bag (MWCO 10 kDa) for 3 h, followed bydialysis against phosphate buffer (pH 7.5). Theobtained concentrate was then completely loaded in aSephadex G-100 gel filtration column with dimensionsof 2 cm� 60 cm. The column was equilibratedand eluted with 0.1 M phosphate buffer (pH 7.5) at20mL/h. Three millilitres of each fraction was sampled
Table 1. Independent variables and their verified levels for the Plackett–Burman and Box–Behnken experimental designs.Design type Independent variable Symbol Actual values Coded levels
and analysed for protein content and ChO activity. Thepurified ChO was assessed for the existence of its FADcofactor by measuring its absorption at 360 and450 nm to determine the presence of FADH2 (reducedstate) and FAD (oxidized state), respectively (Pandiniet al. 2010).
Enzyme molecular weight determination
The molecular weight of the purified enzyme wasdetermined by Na dodecyl sulphate polyacrylamidegel electrophoresis (SDS-PAGE) (Laemmli 1970) in aMini Protean II vertical tank apparatus (Bio-Rad,Munich, Germany) applying acrylamide (10%) separat-ing gel along with acrylamide (5%) stacking gel includ-ing 0.1% SDS. A mixture of the purified enzyme andreducing buffer was prepared, then heated at 90 �C for4min, and introduced into separate lanes. Standardproteins with medium molecular weight range wereloaded onto the first lane and the gel was stainedwith Coomassie Brilliant Blue R-250.
Biochemical characterization of purified ChO
Effect of pH on ChO activity and stability
The optimum pH of the ChO activity was determinedby measuring absorbance at 240 nm, attributable tothe formation of 4-cholesten-3-one, and the residualenzyme activity was then calculated. The optimumenzyme activity was detected within a pH range of5–8.5 at 37 �C for 5min via 100mM sodium acetatebuffer (pH 5–5.5), 100mM phosphate buffer (pH6–7.5), 50mM Tris-HCl buffer (pH 8) and 100mM gly-cine buffer (8.5). The pH stability of ChO was alsomeasured after the enzyme was incubated at 37 �C for60min in buffers with different pH values (6.5, 7.5 or8.5) and the residual enzyme activity was determinedevery 10min.
Effect of temperature on ChO activity and stability
The optimum temperature for ChO enzyme activitywas verified, using 100mM phosphate buffer (pH 7.5),through its exposure to a range of different tempera-tures from 20–70 �C with increments of 5 �C.Furthermore, the thermal stability was examined byincubating the ChO enzyme at pH 7.5 at a tempera-ture range of 40–60 �C for 0–60min with increment of10min. The effect of the enzyme storage in refriger-ator (4 �C) on its stability was also studied. The ChOenzyme, in 0.1 M phosphate buffer (pH 7), was storedfor 30 days in refrigerator and the residual activity wasassayed every five days. The effect of temperature on
enzymatic reaction was expressed in terms of the tem-perature coefficient Q10, which is a measurement forthe reaction velocity upon increasing the temperatureby 10 �C.
Effect of different cholesterol solubilizers
Different solvents are often used as solubilizers ofcholesterol, for that reason the stability of ChOenzyme was examined in the presence of various solu-bilizers. Cholesterol (0.1 g) was completely dissolved at60 �C in 1mL of methanol, ethanol, isopropanol orTween 80 and the dissolved cholesterol was added to1mL of enzyme solution. A combination of two solu-bilizers; methanol and Tween 80, was also examinedvia two methods. In the first method, cholesterol(0.1 g) was completely dissolved at 60 �C in 1mLmethanol followed by the addition of Tween 80(0.4mL), while in the second one, cholesterol wascompletely dissolved at 60 �C in Tween 80 followed bythe addition of methanol using the same amounts asthe first method. The relative activities were measuredafter the incubation with all solubilizers.
Results and discussion
Isolation and identification of ChO-producingbacterium
Bacterial colonies were isolated from the oily samplesto be screened for their capability to produce ChOenzyme. After the isolation of colonies, 20 bacterialisolates formed halos around their growing colonieson the agar medium, showing the ability to grow oncholesterol medium with cholesterol as a single-carbonsource. Cholesterol is converted to 4-cholesten-3-oneand hydrogen peroxide by ChO, resulting in the for-mation of azo compound and the colour of the ChOindicator medium becomes dark brown. Out of 20 bac-terial isolates, only 16 were able to form brown pig-ment around them. Quantitative determination of theChO activity of the selected isolates was performedthrough fermentation in cholesterol medium. The bac-terial strain with highest ChO activity (16.8 U/mL) wasselected for advanced studies. The selected isolate wasidentified with 16S rRNA homology analysis. Thephylogenetic tree was obtained by means of someneighbours that possess the maximum homologousgene sequences (Figure S1). Sequence of the isolatedstrain, with highest ChO activity, was highly similar(98%) to the 16S rDNA sequence of Bacillus pumilusstrain MS5-14 (GenBank accession no. KX685666).
The B. pumilus ChO had not been investigated so far,and as a result, its characteristics were further studied.
ChO production optimization
The PBD was statistically employed for the screeningof different variables that are significantly affecting theChO production by B. pumilus (Table S1). The ChOactivity values widely ranged from 0.06 to 9U/mL,which highlights the importance of screening the sig-nificant effects of growth conditions and mediumcomponents on the enzyme activity (Elbaz et al. 2015).The lowest ChO activity was noticed in the 5th run,while the highest one was obtained in the 1st run.This improvement in the enzyme activity was obtainedunder pH 8 after 5 days, with medium/flask ratio of 0.35 and the concentrations of cholesterol, NH4NO3,yeast extract and Tween 80 were 0.2, 0.05, 0.3 and 0.4%, respectively.
These significant factors were further optimized viaBBD which involves regression equation, connectingthe response to the coded levels of the independentvariables. The significant variables, namely the harvest-ing time (X3), NH4NO3 (X4), yeast extract (X5) andTween 80 (X8) concentrations, were matrixed in theBBD with three levels for each of them. The non-sig-nificant variables were stabilized at their low (�1)/high(þ1) levels depending on their negative/positive effectvalues, respectively. The results showed that harvest-ing time, yeast extract and Tween 80 concentrationssignificantly increased the B. pumilus ChO activity(Table S2). The maximum ChO activity (90U/mL) wasobtained in the 8th run after 6 days upon using con-centrations of 0.3, 1 and 0.2% for NH4NO3, yeastextract and Tween 80, respectively. This activity wastenfold more than that obtained from the PBD screen-ing experiment.
The amount and type of carbon and nitrogen sub-strates have been the focus of several industrial inves-tigations in order to obtain cost-effective growthmedia components. This type of research is particularlyimportant when the optimum growth conditions ofthe applied microbial strains are unknown. These nutri-tional factors, in addition to growth conditions, forexample, harvesting time, pH and aeration, have beennoticed to determine the optimum growth and theability of bacterial cells to produce cellular products(Tari et al. 2007). Accordingly, one aim of this studywas to optimize growth medium components usingdifferent concentrations of NH4NO3, yeast extract andTween 80, resulting in maximum ChO production of B.pumilus and recognizing the other essential factorsthat would also bring about high ChO production.
Generally, the bacterial ChO production has been opti-mized in very few studies (Moradpour et al. 2014).After comparing the current results with the previouslyconducted studies on ChO production from variousbacterial isolates, it was evidently observed that B.pumilus enzyme activity surpassed those results. Intwo studies performed to optimize the ChO produc-tion by Streptomyces sp. using different carbon/nitro-gen sources and cholesterol inducer concentration, thehighest activity obtained was 6U/mL and 20U/mg(Praveen et al. 2011; Niwas et al. 2013). Similarly, theBordetella ChO production was optimized, and themaximum activity was 1.7 U/mL (Lin et al. 2010).Moreover, the ChO produced by Enterobacter andRhodococcus, without optimization, showed activity of0.43 and 0.398U/mL, respectively (Ye et al. 2008;Kasabe et al. 2015).
Purification and identification of bacterial ChO
The purification of the crude ChO was performedthrough two consecutive stages, starting with ammo-nium sulphate precipitation, then gel filtration chroma-tography using Sephadex G-100 column. The ChOprecipitation was optimized using different concentra-tions of ammonium sulphate under different pH val-ues. The optimum conditions for the precipitationprocess was 60% saturation with ammonium sulphateunder pH 7, which resulted in the highest specificactivity (0.14U/mg protein) (Table 2). After applyingthese conditions, the fraction precipitated with ammo-nium sulphate displayed a total enzyme activity of17U, corresponding to 68% of the recovered activitywith a total protein amount of 118mg. Although 45fractions were collected from the Sephadex G-100 col-umn, the maximum enzymatic activity was onlydetected in fractions 28–35 (Figure 2). Being a flavo-protein, ChO shows a maximum absorption at 450 and360 nm in its oxidized and reduced states, respectively(Kasabe et al. 2015). The majority of the collected frac-tions (40) showed absorptions at 360 and 450 nm, but
Table 2. Optimization of the first step purification of ChO pre-cipitation by ammonium sulphate.Ammoniumsulphate conc. (%) pH ChO (U/mL)
80% of these fractions had shown very low ChO activ-ity. Eight fractions exhibiting the maximum recoveredactivity, were pooled leading to a total ChO activity of4.8 U and specific activity of 1.37 U/mg. The purifica-tion process totally resulted in an escalation in specificactivity from 0.13U/mg in crude broth to 1.37U/mgafter the gel filtration chromatography step (Table 3).
The molecular weight of the purified ChO, accord-ing to SDS-PAGE analysis, was 55 kDa in the form ofone protein band stained on the gel, which empha-sizes the purified enzyme homogeneity (Figure 3). Anumber of studies investigated the production andpurification of ChO from different bacterial isolates.They reported a molecular weight in the range of55–62 kDa (Praveen et al. 2011, Niwas et al. 2013),which coincided with the molecular weight of the B.pumilus purified ChO enzyme.
pH stability
Generally, every enzyme is active in a certain range ofpH, and hence, a particular optimum pH has to bedetected. The effect of different pH values on theactivity and stability of the purified ChO are shown inFigure 4. After the examination of the effect of a pHrange of 5–8.5 on the enzyme activity, the optimum
pH was observed to be 7.5 (Figure 4(A)). The enzymeconsiderably lost its activity at a pH value less than6.5. On the other side, the enzyme retained more than80% of its activity at pH values of 7–8. The pH stability
Figure 2. Elution profile of the Sephadex G-100 gel filtration column to purify ChO from Bacillus pumilus showing a single peak ofenzyme activity.
Table 3. Purification of the Cho enzyme produced by Bacilluspumilus.
Figure 3. SDS-PAGE of ChO enzyme after gel filtration purifica-tion step; Right lane: Molecular marker and left lane: PurifiedChO fraction.
6 F. N. ELBAZ ET AL.
was also examined by incubating the enzyme underpH values of 6.5, 7.5 or 8.5 for 60min (Figure 4(B)).The enzyme reserved more than 80% of its activityafter being incubated under tested pH values for60min. Nevertheless, only 10% of the enzyme activityvanished after 30min under all pH values. Theseresults revealed the high stability of the enzyme at pHrange of 6.5–8.5. Comparable results were obtainedwith ChOs purified from other bacterial species, whichkept most of their activities within pH range from 7(Doukyu et al. 2008; Ye et al. 2008; Lin et al. 2010;Praveen et al. 2011; Niwas et al. 2013) to 8 (Kasabeet al. 2015). The role of pH is well known in changing
the ionization status of the enzyme substrate complex,which affects the ionic bonds that help to determinethe 3D structure of the enzyme, leading to enzymeinactivation (Illanes 2008).
Thermal stability
The thermal stability of the ChO enzyme has beenwell verified in this study. The ChO enzyme activitywas examined under a temperature range of 20–70 �C.The optimum temperature was observed to be 40 �C,while the enzyme activity sharply declined at tempera-tures higher than 45 �C (Figure 5(A)). Moreover, theenzyme activity after incubation at various tempera-tures for 60min was evaluated. The enzyme sustained100% of its activity after 60min when stored at 40 �C,whereas the same pattern of activity was observedwhen the enzyme was incubated at 50 �C with slightdecline after 30min till it reached 83% by the end ofthe incubation period (Figure 5(B)). On the other side,when the enzyme was incubated at 60 �C, its activitysharply declined till it reached 13% after 40min, andthe enzyme was totally inactivated by the end of incu-bation period. The enzyme stability was furtherchecked at a lower temperature (4 �C) for elongatedperiod of incubation (30 days). About 20% reductionin the activity was observed after 10 days, then theactivity gradually declined until it reached 40% after30 days (Figure 5(C)). The effect of temperature on theenzymatic reaction was determined in terms of thetemperature coefficient (Q10). The maximum obtainedQ10 (2.13) was observed when the temperature wasraised from 30 �C to 40 �C (Figure 5(D)), which is inagreement with the results of the optimum tempera-ture value for the enzyme activity and stability.
Figure 4. Effect of different pH values on the activity (A) andthe stability (B) of ChO.
Figure 5. Thermal stability profile of ChO enzyme. (A): Effect of different temperatures on the enzyme activity, (B): Stability ofChO, (C): Enzyme activity at 4 �C and (D): Temperature coefficients.
BIOCATALYSIS AND BIOTRANSFORMATION 7
The optimum temperature (40 �C) was within therange of the optimum temperature of the related bac-terial ChO enzymes, which had values in the range of25–65 �C (Doukyu et al. 2008; Ye et al. 2008).
Tolerance to solubilizers
Organic solvents and detergents are regularly used tosolubilize cholesterol. Stability of ChO was inspected inthe presence of different organic solvents and deter-gent (Figure 6). ChO displayed an outstanding stabilitywhen solubilized in several organic solvents and deter-gent. The highest enzyme activity was obtained whenmethanol was used as a solvent (265%). Furthermore,the enzyme activity was enhanced (>100%) with alltested solubilizers except ethanol. The effect of thetwo solubilizers with highest enzyme activity (metha-nol and Tween 80) were further studied when com-bined with each other via two different methods.Solubilizing cholesterol in hot methanol (60 �C) fol-lowed by the addition of Tween 80 was observed tobe a suitable solvating and emulsifying method forhigher ChO activity, which accounts for approximately70% improvement compared to that of each singlesolubilizer.
The effect of different solvents and detergents onseveral bacterial ChO enzymes has been investigatedin several studies. B. pumilus ChO activity was
compared to other studies which applied the samesolvents and detergents (Figure 7). The relative activ-ities of different purified bacterial enzymes greatlydiverged in the range of 4–250%. The minimum rela-tive activity (4%) was obtained when the BordetellaChO was subjected to methanol (Lin et al. 2010), whilethe maximum one (250%) was obtained upon solubi-lizing of Streptomyces parvus ChO in isopropanol(Praveen et al. 2011). It was clear that methanol exhib-ited an unprecedented qualitative and quantitativeenhancement in the relative activity of the B. pumilusChO, which accounts for more than twofold, comparedto the maximum improvement of enzyme activity(115%) ever obtained in previous studies (Niwas et al.2013). This enhancement also extended to Tween 80,which displayed a resultant relative activity (171%)superior to the maximum activity obtained after itsapplication with Streptomyces parvus purified enzyme(150%) (Praveen et al. 2011). Furthermore, the applica-tion of isopropanol solvent with B. pumilus ChOresulted in an intermediate relative activity (131%)compared to other studies that gave effects rangingfrom 115% to 250% (Praveen et al. 2011; Kasabe et al.2015). Accordingly, ChO from the isolated strain hasshown improved stability in the presence of Tween 80,methanol and isopropanol.
Moreover, commercial enzymes from Nocardia sp.,Pseudomonas sp., Streptomyces sp. and Cellulomonassp. were reported to be inactivated upon the employ-ment of methanol, ethanol and isopropanol (Niwaset al. 2013). The B. pumilus ChO enzyme under studywas more stable in detergent/organic solvents com-pared to commercial ones and therefore can beapplied in different chemical reactions, for example,bioconversion of several 3b-hydroxysteroids and syn-thesis of steroid hormones (Niwas et al. 2013). It hasbeen known that alcohols generally create structuralvariations in proteins and peptides. Methanolimproved the enzymatic activity possibly by acting asa molecular lubricant that enhances the enzyme’s
Figure 6. Effects of different solubilizers on the stability ofChO enzyme produced by Bacillus pumilus.
Figure 7. Comparison between the relative activities of the current purified ChO and previous related studies in terms of the fourtested solubilizers; ethanol, methanol, isopropanol and Tween 80 (1 (Niwas et al. 2013), 2 (Doukyu et al. 2008), 3 (Ye et al. 2008),4 (Praveen et al. 2011), 5 (Lin et al. 2010), 6 (Kasabe et al. 2015) and 7 this study).
8 F. N. ELBAZ ET AL.
conformational flexibility to facilitate its catalytic activ-ity (Wiggers et al. 2007).
Conclusions
The purification and optimization of growth conditionsand medium components for the B. pumilus ChOenzyme were exclusively performed in this study.Besides detecting the optimum growth conditions ofthis bacterial isolate, this study also worked as a para-digm for the application of statistical approaches withthe bacterial systems to give the operator enoughsuppleness to select the optimum factors in terms ofthe enzyme production response via the polynomialmodel. After two sequential steps of optimizationcourse, the highest ChO enzyme activity was obtainedafter 6 days upon the usage of NH4NO3, as a nitrogensource, at 0.3%. Moreover, the purification of B. pumi-lus ChO was performed by gel filtration chromatog-raphy and confirmed by using SDS-PAGE analysis inwhich single band was obtained and the enzymemolecular weight was determined to be 55 kDa. Thepurified bacterial ChO had thermal stability and toler-ance for organic solvents, which are considered asbeneficial features, rendering the newly isolated strain,B. pumilus, as a potential source of ChO which couldbe employed in clinical and various research purposes.
Disclosure statement
The authors report no conflicts of interest. The authors aloneare responsible for the content and writing of this article.
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