research article production of thermoalkaliphilic lipase...

Research ArticleProduction of Thermoalkaliphilic Lipase from Geobacillusthermoleovorans DA2 and Application in Leather Industry

Deyaa M Abol Fotouh1 Reda A Bayoumi23 and Mohamed A Hassan4

1Electronic Materials Research Department Advanced Technology and NewMaterials Institute (ATNMRI) City of Scientific Researchand Technological Applications (SRTA-City) New Borg El-Arab City PO Box 21934 Alexandria Egypt2Biology Department Faculty of Science and Education Taif University Khormah Branch PO Box 21974 Taif Saudi Arabia3Botany and Microbiology Department Faculty of Science (Boys) Al-Azhar University PO Box 11884 Cairo Egypt4Protein Research Department Genetic Engineering and Biotechnology Research Institute (GEBRI) City of Scientific Research andTechnological Applications (SRTA-City) New Borg El-Arab City PO Box 21934 Alexandria Egypt

Correspondence should be addressed to Deyaa M Abol Fotouh dabolfotouhgmailcomand Mohamed A Hassan m adelmicroyahoocom

Received 30 September 2015 Revised 30 November 2015 Accepted 3 December 2015

Academic Editor Hartmut Kuhn

Copyright copy 2016 Deyaa M Abol Fotouh et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Thermophilic and alkaliphilic lipases are meeting a growing global attention as their increased importance in several industrialfields Over 23 bacterial strains novel strainwith high lipolytic activity was isolated from Southern Sinai Egypt and it was identifiedas Geobacillus thermoleovorans DA2 using 16S rRNA as well as morphological and biochemical features The lipase was producedin presence of fatty restaurant wastes as an inducing substrate The optimized conditions for lipase production were recorded tobe temperature 60∘C pH 10 and incubation time for 48 hrs Enzymatic production increased when the organism was grown in amedium containing galactose as carbon source and ammonium phosphate as nitrogen source at concentrations of 1 and 05 (wv)respectively Moreover the optimum conditions for lipase production such as substrate concentration inoculum size and agitationrate were found to be 10 (wv) 4 (vv) and 120 rpm respectivelyThe TA lipase with Triton X-100 had the best degreasing agentby lowering the total lipid content to 26 as compared to kerosene (75) or the sole crude enzyme (89) It can be concluded thatthe chemical leather process can be substituted with TA lipase for boosting the quality of leather and reducing the environmentalhazards

1 Introduction

Lipases (triacylglycerol acylhydrolases EC 3113) are ubiq-uitous enzymes of considerable physiological significanceand industrial potential [1] Lipases catalyze the hydrolysis oftriacylglycerols to glycerol and free fatty acids Today lipasesare the choice of biocatalyst as they show unique chemo-regio- enantioselectivities which enable the production ofnovel drugs agrochemicals and fine products [2]

Due to the ability of many lipases to perform both hydro-lytic and synthetic reactions they find immense applicationsin industries like foods detergents pharmaceuticals leathercosmetics textile dairy and even biodiesel [3 4]

Lipases are widely present in plants and animals butalmost all the commercially available lipases are usually

obtained from microorganisms that produce a wide varietyof extracellular lipases [5]

Cost of lipase production process was considered as amajor obstacle in the industries Therefore many efforts arebeing made to use wastes as raw materials for lipase produc-tion Agricultural residues for lipase production as well asother value added products would hold a prominent positionin future biotechnologies mainly because of its ecofriend-liness and flexibility to both developing and developedcountries Several residues such as oil cakes fibrous residuesand industrial effluent have increasing attention as abundantand cheap renewable feedstock [5 6] Enzymes from ther-mophiles and alkaliphiles have become the subject of specialinterest for biotechnological applications due to their highstability at adverse operational andor storage conditions [7]

Hindawi Publishing CorporationEnzyme ResearchVolume 2016 Article ID 9034364 9 pageshttpdxdoiorg10115520169034364

2 Enzyme Research

Many advantages were earned for carrying out biotech-nological and industrial processes in high temperatureshigh solubility of substrates (in particular for poorly solubleor polymeric molecules) resulting in higher product yieldhigher reaction rates increased availability of substratesdecreased risk of microbial contamination and lower vis-cosity of reaction mixtures which in turn reduces the costsrelated to pumping filtration and centrifugation and savinga great power cost would be exploited for cooling [8 9]

In the present study a detailed description of isolationidentification and optimization of TA lipase production con-ditions from G thermoleovorans DA2 will be demonstratedAn attempt to utilize restaurant fatty wastes as the mainsubstrate for lipase production was carried out which mayraise the lipolytic activity and decrease the overall cost of theproduction process In addition this approach has a greatenvironmental endeavor through minimizing the ecologicalhazards accompanied with the accumulation of wastes

The application of TA lipase from G thermoleovoransDA2 in leather tanning process as a degreasing agent replacedthe commonly utilized organic solvent (Kerosene) Kanagarajet al reported that it is fundamental to add hydrolyticenzymes such as lipases and proteases in the soaking step forhelping the fat degradation and raise the leather quality [10]

Substitution of the traditional chemical tanning process-ing with more ecofriendly treating procedures for examplethe enzymatic steps became a necessity because of therecorded environmental hazards resulting from the pollutionof water careless disposal of solid wastes and gaseous emis-sions [11]

2 Materials and Methods

21 Bacterial Strain Awide variety of samples were collectedfrom many localities in Egypt including desert and hotsprings of Southern Sinai Wadi El-Natron swamps desert ofQina and Suez governorates and the soil of El-Basateenslaughter house

All samples were suspended in sterilized saline solution(085wv) which were cultured on plates of nutrient agarmedium with pH 9 and incubated at 65∘C for 48 hrs Theseparated colonies had undergone a series of (agar streakmethod) for purification and the morphological character-istics of each isolate were investigated The purpose of thismethod was to isolate the thermoalkalophilic microorgan-isms

22 Screening

221 Qualitative Assay All strains were screened for inves-tigating their lipase activity on agar plates containing Rho-damine B 0001 (wv) nutrient broth 08 (wv)NaCl 04(wv) agar 1 (wv) and olive oil 3 in distilled water andthe pH was adjusted to be 9 [12]

Plates were incubated at 65∘C for 18 hrs and the lipaseactivity was identified as an orange halo zone around coloniesunder UV light at 350 nm

222 Quantitative Assay To detect the most potent ther-moalkaliphilic lipase producing bacteria all bacterial isolateswere grown on medium composed of yeast extract 1 g oliveoil 10mL gum Arabic 10 g CaCl

21 g and mineral salt solu-

tion 1mL per liter The medium pH is initially adjusted at 9by using 6N NaOH and incubated at 65∘C and 100 rpm for18 hrs The most potent thermoalkaliphilic lipase producingisolate was purified by ldquoagar streak methodrdquo and underwentbiochemical investigations and it was identified by 16S rRNAtechnique

223 Bacterial Identification The bacterial isolate was iden-tified using 16S rRNA sequence The genomic DNA wasextracted by the following method which was described bySambrook et al [13]

The PCR amplification was carried out according toHassan et al and Abdou and Hassan The reaction was per-formed using forwarded 16S rRNA primer (51015840-AAATGG-AGGAAGGTGGGGAT-31015840) and reverse 16S rRNA primer(51015840-AGGAGGTGATCCAACCGCA-31015840) The PCR machine(TECHNE TC-3000 FTC302) was programmed as follows3min denaturation at 95∘C followed by 35 cycles that con-sisted of 1min at 95∘C 1min at 58∘C and 1min at 72∘C andthe final extension was 10min at 72∘C [14 15]

The PCR product was cleaned up for sequencing usingQiagen kit for DNA purification from aqueous PCR DNAsequencingmethod which was developed by Sanger et al wascarried out using 3130X DNA Sequencer (Genetic AnalyzerApplied Biosystems Hitachi Japan) [16]

224 Sequence Analysis and Phylogenetic Tree ConstructionSimilarity of the obtained nucleotide sequence was per-formed by basic local alignment search tool (BLAST) againstreference sequences available in National Center for Biotech-nology Information GenBank (NCBI GenBank) The refer-ence sequences were collected from GenBank and the align-ments using Clastal W were performed for constructing thephylogenetic tree using MEGA 5 software version 51 [17]

23 Lipase Production G thermoleovorans DA2 was grownon a liquid production medium containing ( wv) yeastextract 01 g NaNO

3 02 g KH

2PO4 01 g MgSO

4sdot7H2O

005 g KCl 005 g and CaCl2 01 g supplemented with 1 g of

fatty restaurant wastesThe medium was adjusted at pH 9 (pH-Meter Model-

420A Orion Co USA) and 100mL of medium in 500mLErlenmeyer flasks was inoculated with G thermoleovoransDA2 and incubated at 65∘C and 100 rpm for 18 hrs on a rotaryshaker (Innova J-25 New Brunswick scientific USA)

24 Assay of Thermoalkaliphilic Lipase Activity Lipase activ-ity was detected by a spectrophotometric assay using p-nitro-phenyl laurate (pNPL) as a substrate according to Castro-Ochoa et al and Amara et al with slight modifications Inbrief the reaction mixture consisted of 01mL enzyme ext-ract 08mL 01M phosphate buffer (pH 8) and 01mL 001MpNPL in isopropanolThe hydrolytic reaction was carried outat 60∘C for 30min and then terminated by 025mL of 01M

Enzyme Research 3

Na2CO3added The mixture was centrifuged at 10000 rpm

for 15min and the absorbance was determined at 410 nmusing spectrophotometer (spectrophotometer Lambda EZ201 Perkin Elmer USA) One unit of lipase activity wasdefined as the amount of enzyme that caused the releaseof 1 120583mol of p-nitrophenol (molar absorption coefficient46mMminus1 cmminus1) from pNP-laurate in 30min under test con-ditions [18 19]

25 Determination of Protein Content Protein content wasdetermined according to Lowry Method [20]

26 Optimization of Various Production Conditions of TALipase from G thermoleovorans DA2 Several experimentswere conducted to study the effect of physical and nutrientson culture conditions for TA lipase production by G ther-moleovorans DA2 All the previously mentioned productionconditions were investigated throughout the following exper-iments

261 Effect of Incubation Temperature on Production of TALipase from G thermoleovorans DA2 Most favorable pro-duction temperature was studied by incubating the inocu-lated productionmedium at varying temperatures (50 55 6065 70 75 80 85 and 90∘C) The culture filtrate was used forthe lipase activity

262 Effect of pH of the Medium on the Production of TALipase from G thermoleovorans DA2 For optimization ofproduction pH the production medium of different pHnamely 6 7 8 9 10 11 and 12 was inoculatedwith culture andincubated in shaker for 18 hrs at 65∘C and lipase activity ofculture filtrate was determined

263 Effect of Incubation Time on Production of TA Lipasefrom G thermoleovorans DA2 To optimize incubation timefor the maximum production of lipase the productionmedium was incubated at 60∘C in the shaker for 12 18 2436 48 56 and 72 hrs

264 Effect of Carbon Source on Production of Lipase from Gthermoleovorans DA2 Various carbon sources such as glu-cose galactose xylose ribose rhamnosemelezitose sucroselactose maltose cellobiose sorbitol mannitol and inulinwere used in the production medium at the concentration of1 (wv) to check the effect of carbon source on lipase pro-duction The medium without carbon source served as con-trol and the culture filtrate was assayed for enzyme activity

265 Effect of Nitrogen Source on Production of TA LipasefromG thermoleovorans DA2 To study the effect of nitrogensource various nitrogen sources such as ammonium chlo-ride ammonium sulphate ammonium nitrate ammoniummolybdate ammonium phosphate sodium nitrite urea cal-cium nitrate potassium nitrate and peptone were conductedin the productionmedium at the concentration of 05 (wv)The main medium included sodium nitrate only served ascontrol The culture filtrate was assayed for enzyme activity

266 Effect of Substrate Concentration on Production ofLipase from G thermoleovorans DA2 The effect of substrateconcentration on the production of TA lipasewas determinedby varying the concentration of fatty restaurantwastes that is025 05 1 2 5 10 20 and 40 (wv)

267 Effect of Inoculum Size on Production of Lipase fromG thermoleovorans DA2 Heavy cell suspension of G ther-moleovorans DA2 was prepared by growing bacterial iso-late on nutrient broth and limiting growth level at 3 times107 CFUsdotmLminus1 Different inocula sizes of culture including025 05 1 2 4 5 and 10 (vv) were applied

268 Effect of Agitation Rate on Production of TA Lipasefrom G thermoleovorans DA2 To optimize the agitation ratefor maximum lipase production the inoculated productionmedium was agitated at different rotations per minute (rpm)such as 40 80 120 and 150 rpmThe culture filtrate was usedto check the enzyme activity

27 Application of TA Lipase from G thermoleovorans DA2 inDegreasing of Leather The crude TA lipase from G thermo-leovoransDA2was used as degreaser agent in leather industryas compared to traditional methods which depends uponsolvents and surfactants

271 Preparation of Skin Samples A piece of sheep skinwas obtained after it had undergone the common processingsteps that is liming dehairing and bating Eight skin piecesabout 5 times 5 cm dimension were cut and divided into 4 groups(each of 2 pieces)

Group (A) the skin was soaked in 50mL (Kerosene)and was incubated at 25∘C as a traditional methodGroup (B) the skin was immersed in 50mL of 10 ofthe crude TA lipase and was incubated at 60∘CGroup (C) the skin was treated with a mixture ofcrude TA lipase (10) +Kerosene in 4 1 ratio andwasincubated at 60∘CGroup (D) the skin was bathed in a mixture of crudeTA lipase (10) + Triton X-100 in 10 1 ratios and wasincubated at 60∘C

All groups of skin were treated for 2 hrs then the skin wascollected and dried [21]

272 Total Lipid Measurement Technique Total lipid of thetreated skin pieces were measured relying on Soxhlet appa-ratus [22] The obtained values were then compared with thelipid content of the control

3 Results

31 Bacterial Strain Selection and Identification Out of sevenisolates the most efficient lipase producer was isolated fromdesert of southern Sinai based on enzyme assay method andit was identified using 16S rRNA and selected for furtherstudies

4 Enzyme Research

17

99

1312

38

82

50

26

21

26

29

90

38

41

2

Geobacillus thermodenitrificans NG80-2 (DQ2437881)

Geobacillus vulcani BGSC 97A1 (AY6089401)

Geobacillus stearothermophilus BGSC 9A19 (AY6089411)

Geobacillus stearothermophilus mb-5 (EU2146311)

Geobacillus thermoleovorans BGSC 96A1 (NR 1152861)

Geobacillus sp N60 (DQ6420881)

Geobacillus thermoleovorans NP54 (JN8715951)

Geobacillus thermoparaffinivorans it-12 (EU2146151)

Bacillus thermoleovorans CCR11 (AJ5365991)

Geobacillus thermoparaffinivorans ZLG (FJ7960731)

Geobacillus thermoleovorans LEH-1 (NR 0369851)

Geobacillus kaustophilus HTA426 (NR 0749891)

Geobacillus thermoleovorans DA2 (KR338989)

Geobacillus sp SBS-4S (AB3065191)

Geobacillus sp sbs4s2 (AB3065211)

Geobacillus sp sbs4L (AB3065201)

Geobacillus thermodenitrificans NG80-2 (NR 0749761)

Geobacillus thermoleovorans R-32498 (FN4286371)

Geobacillus kaustophilus mt-12 (EU6520831)

Geobacillus sp R-32630 (FN4286711)



Figure 1 Phylogenetic position of Geobacillus thermoleovorans DA2 within the genus Geobacillus The branching pattern was generated byneighbor-joining tree method and the GenBank accession numbers of the 16S rRNA nucleotide sequences are indicated in brackets The barindicates a Jukes-Cantor distance of 2

32 Bacterial Identification by 16S rRNA The amplified 16SrRNA gene fragment was investigated using DNA ladder(Gene ruler 50 bpndash1031 bp DNA ladder) and it was 380 bp

The BLAST algorithm was used to retrieve for homol-ogous sequences in GenBank to the obtained 16S rRNAsequence The bacterial isolate revealed 99 identity to fullgenome of Geobacillus thermoleovorans Geobacillus stearo-thermophilus G thermoparaffinivorans G thermodenitrifi-cans and G kaustophilus Based on the morphological bio-chemical and molecular characteristics the isolate was iden-tified and released in NCBI GenBank as Geobacillus ther-moleovoransDA2 under the accession numbers (KR338990)and the branching pattern was analyzed by 500 bootstrapreplicates as in Figure 1 G thermoleovorans DA2 is an aero-bic spore-forming nonmotile rod able to grow at high tem-peratures (50ndash80∘C) with an optimum growth at 65∘C

33 Optimization of Production Conditions of TA Lipase fromG thermoleovorans DA2

331 Effect of IncubationTemperature onProductionofTALip-ase fromG thermoleovorans DA2 Most suitable temperature

for maximum production of TA lipase (14685UmL) fromG thermoleovorans DA2 was found to be 60∘C as shown inFigure 2(a)

332 Effect of pH of the Medium on Production of TA Lipasefrom G thermoleovorans DA2 The maximum productionof TA lipase from G thermoleovorans DA2 was observedat pH 10 (15715UmL) and after that the lipase activitywas decreased with increasing the pH values as shown inFigure 2(b)

333 Optimization of Incubation Time for Production of TALipase from G thermoleovorans DA2 The maximum pro-duction of lipase was observed at 48 hrs (24811 UmL) Opti-mal incubation time was found to be 48 hrs corresponding tomaximum enzyme activity and after this decline in enzymeactivity was observed (Figure 2(c))

334 Effect of Carbon Source on Production of TA Lipase fromG thermoleovorans DA2 Maximum production of TA lipasewas obtained from the medium which was supplemented

Enzyme Research 5

50 55 60 65 70 75 80 85 90 95 10045Temperature (∘C)

0

25

50

75

100

125

150

175

200Li

pase

activ

ity (U

mL)

(a)

0

25

50

75

100

125

150

175

200

Lipa

se ac

tivity

(Um

L)

6 7 8 9 10 11 12 135pH

(b)

0

50

100

150

200

250

300

Lipa

se ac

tivity

(Um

L)

10 20 30 40 50 60 70 80 900Incubation time (hr)

(c)

Figure 2 (a) Effect of incubation temperature on production of TA lipase from G thermoleovorans DA2 (b) Effect of pH on production ofTA lipase from G thermoleovorans DA2 (c) Effect of incubation time on production of TA lipase from G thermoleovorans DA2

with galactose (1) as carbon source giving enzyme activityof 70186UmL (Figure 3(a))

335 Effect ofNitrogen Source onProduction of TALipase fromG thermoleovorans DA2 The lipase production was highest(84304UmL) inmedium containing ammoniumphosphateat a concentration (05) as nitrogen source (Figure 3(b))

336 Effect of Substrate Concentration on Production of TALipase from G thermoleovorans DA2 Maximum enzymeactivity (89243UmL) was observed with 10 (wv) fattyrestaurant wastes (Figure 3(c))

337 Optimization of Inoculum Size for Production of TALipase from G thermoleovorans DA2 Figure 3(d) revealsthat the inoculum size (4 vv) gave the maximum lipaseproduction with an activity of 91723UmL

338 Effect of Agitation Rate on Production of TA Lipasefrom G thermoleovorans DA2 The maximum lipase activ-ity (102191 UmL) was observed at 120 rpm as shown inFigure 3(e)

34 Application of TA Lipase in Degreasing of Leather Thethermoalkaliphilic lipase from G thermoleovorans DA2 plusTriton X-100 was the most efficient leather degreasing agentwhere the total lipid content decreased from 175 to 26 asshown in Table 1

Themixture of TA lipase plus kerosene decreased the totallipid content to 57 so it came in second level

4 Discussion

The applications of lipases are constantly increased in indus-trial and biotechnological fields therefore that should besupported by discovering novel lipase types with improvedcharacters The common about enzymes is their sensitivityto adverse conditions such as high temperature extremeacidity andor alkalinity drought and high salinity but extre-mozymes enzymes derived from extremophilic microorgan-isms are an attractive alternative to tuning a given biocatalystfor a specific industrial application They are capable ofcatalyzing their respective reactions in nonaqueous environ-ments watersolvent mixtures at extremely high pressuresacidic and alkaline pH at temperatures up to 140∘C or near

6 Enzyme Research

Carbon source

Inul

inM

anni

tol

Sorb

itol

Sucr

ose

Cel

lobi

ose

Mal

tose

Lact

ose

Glu

cose

Mel

ezito

seRh

amno

seRi

bose

Xylo

seG

alac

tose

0

100

200

300

400

500

600

700

800Li

pase

activ

ity (U

mL)

(a)

Nitrogen source

Sodi

um n

itrat

e

Pept

one

Pot

nitr

ate

Calc

ium

nitr

ate

Ure

a

Sodi

um n

itrite

Am

m p

hosp

hate

Am

m m

olyb

date

Am

m n

itrat

e

Am

m s

ulph

ate

Am

m c

hlor

ide

0100200300400500600700800900

Lipa

se ac

tivity

(Um

L)(b)

0

100

200

300

400

500

600

700

800

900

1000

Lipa

se ac

tivity

(Um

L)

5 10 15 20 25 30 35 40 45 500Substrate concentration ( wv)

(c)

0

100

200

300

400

500

600

700

800

900

1000Li

pase

activ

ity (U

mL)

1 2 3 4 5 6 7 8 9 10 11 120Inoculum size ( vv)

(d)

400

600

800

1000

1200

Lipa

se ac

tivity

(Um

L)

Agitation rate (rpm)40 80 120 160 2000

(e)

Figure 3 (a) Effect of carbon source on production of lipase from G thermoleovorans DA2 (b) Effect of nitrogen source on productionof lipase from G thermoleovorans DA2 (c) Effect of substrate concentration on production of TA lipase from G thermoleovorans DA2 (d)Effect of inoculum size on production of TA lipase from G thermoleovorans DA2 (e) Effect of agitation rate on production of lipase fromGeobacillus thermoleovorans DA2

Enzyme Research 7

Table 1 Effect of using the TA lipase from G thermoleovorans DA2 as a degreasing agent and the total lipid content of leather samples

Group of leather samples Type of leather treatment Total lipid content ()Control (without treatment) 1750

Group (A) Organic solvent (Kerosene) 750Group (B) 10 crude lipase produced by G thermoleovorans DA2 890Group (C) 10 crude lipase produced by G thermoleovorans DA2 + Kerosene 570Group (D) 10 crude lipase produced by G thermoleovorans DA2 + Triton X-100 260

the freezing point of water [23] The present investigation isan attempt to discover novel bacterial strainswith the capabil-ity of producing lipases able to tolerate high temperature andhigh alkalinity as well Seven of twenty three strains showeddifferent lipolytic activities in incubation temperature 65∘Cand pH 9 the most potent producing strain is identified asGthermoleovorans DA2 selected to further studies as the mostpromising source of thermoalkaliphilic lipase

Optimum temperature in the present investigation camein correspondence with the optimum temperature for lipaseproduced byG stearothermophilus strain-5 [24] On the otherhand the temperature for the highest production of lipase byG thermodenitrificans AZ1 was 55∘C [25]

Optimum pH value for the present study suggests morealkaliphilic behavior comparing to lipases produced by Geo-bacillus thermoleovorans CCR11 and the alkaliphilic Bacillussp (KS4) which was found to give their highest lipase pro-ductions at pH 8 [26 27] Berekaa et al reported that theoptimum pH for lipase production by G stearothermophilusstrain-5 was 7 [28]

G thermoleovorans DA2 was found to consume 48 hrsto give its maximum production of TA lipase comparing toBacillus thermoleovoransCCR11 which is recorded to producethe highest lipase rate after 44 hrs while the highest produc-tion of lipase by the thermophilic strain Bacillus sp strain 42was recorded after 72 hrs [26 29]

Galactose was found to be the most supportive carbonsource for the TA lipase production by G thermoleovoransDA2 suggesting high ability of the strain to uptake and utilizethe monosaccharide galactose in the metabolic processes oflipase production as the lactose disaccharide was the secondoptimum carbon source Glycerol was found to be the mostinducing carbon source for lipase production by G stearo-thermophilus strain-5 while it is the highest lipase productionby B subtilis ZR-1 supported by the sole olive oil [12 28]

Peptone was the most supportive nitrogen source forlipase production by Bacillus sp LBN2 [30] In contrast mixof yeast extract and peptone was the best for the productionof thermoalkaliphilic lipase by B coagulans BTS-3 [31]Moreover sole yeast extract was the best for lipase producedby B licheniformisMTCC-10498 [32]

Abd El Rahman found that 125 of the slaughter housewaste was the optimum substrate concentration for lipaseproduction by B stearothermophilus B-78 while Gayathri etal have reported that 2 of palm oil was the optimum con-centration for lipase production by B stearothermophilus [3334]

Ten percent (10 vv) was the optimum inoculum forlipase production by B licheniformis MTCC-10498 [32]

However Bacillus flexus XJU-1 gave the best production oflipase depending on 2 (vv) inoculum size while 6 (vv)was the optimum inoculum size for the best lipase productionby Pseudomonas gessardii [35 36]

The optimum revolution per minute for the maximumproduction of TA lipase by G thermoleovorans DA2 wasfound to be 120 rpm which came in complete accordancewith the optimum agitation rate for highest lipase productionby B licheniformisMTCC-10498 [32] while Veerapagu et alreported that the optimized value for greatest lipase produc-tion by Pseudomonas gessardii was 160 rpm [36]

Once samples of sheep leather were treatedwith TA lipaseproduced by the strain G thermoleovorans DA2 mixed withTriton X-100 fatty content of the leather depleted from 175to 25This may be due to the elevated processing tempera-ture which takes a hand in destroying thewalls of skin fat cellsand increase solubility of the fat content very well MoreoverTriton X-100 had supportive effect in reducing the surfacetension of the lipids and facilitated the enzyme efficacy Saranet al recorded degreasing rate more than 95 by 5ndash10mesophilic lipase enzyme produced by Bacillus subtilis solelyafter treating for 12 hrs [11]

Moreover their interpretations came in complete accor-dance with our observations about the higher quality appear-ance and smoothness levels of the enzyme-treated leathercompared to chemically processed skin The obtained resultsare suggesting that advanced efforts to replace the ecohaz-ardous traditional chemical tanning process with the greenenzymatic techniques are worthy to be achieved

5 Conclusion

In the present study thermoalkaliphilic lipase was producedfrom the new bacterial isolate which was identified using thesequence of 16S rRNAgene and biochemical tests asGeobacil-lus thermoleovorans DA2 Wide ranges of growth factorswere studied for maximizing TA lipase enzyme The produc-tion of TA lipase with high amount using cheap substrate(fatty restaurant wastes) made the production process cost-effectiveThe results exhibited the potential application of theTA lipase in leather industry as a degreaser suggesting moreecofriendly tanning processes in the future

Conflict of Interests

The authors have no conflict of interests to declare

8 Enzyme Research

References

[1] R Sharma Y Chisti and U C Banerjee ldquoProduction purifica-tion characterization and applications of lipasesrdquo Biotechnol-ogy Advances vol 19 no 8 pp 627ndash662 2001

[2] R Kaushik S Saran J Isar and R K Saxena ldquoStatisticaloptimization of medium components and growth conditionsby response surface methodology to enhance lipase productionby Aspergillus carneusrdquo Journal of Molecular Catalysis B Enzy-matic vol 40 no 3-4 pp 121ndash126 2006

[3] F Hasan A A Shah and A Hameed ldquoIndustrial applicationsof microbial lipasesrdquo Enzyme and Microbial Technology vol 39no 2 pp 235ndash251 2006

[4] A Robles-Medina P A Gonzalez-Moreno L Esteban-Cerdanand E Molina-Grima ldquoBiocatalysis towards ever greenerbiodiesel productionrdquo Biotechnology Advances vol 27 no 4 pp398ndash408 2009

[5] A Salihu M Z Alam M I AbdulKarim and H M SallehldquoLipase production an insight in the utilization of renewableagricultural residuesrdquo Resources Conservation and Recyclingvol 58 pp 36ndash44 2012

[6] J Sahasrabudhe S Palshikar A Goja and C Kulkarni ldquoUseof ghee residue as a substrate for microbial lipase productionrdquoInternational Journal of Scientific amp Technology Research vol 1no 10 pp 61ndash64 2012

[7] B P Golaki S Aminzadeh A A Karkhane et al ldquoCloningexpression purification and characterization of lipase 3646from thermophilic indigenous Cohnella sp A01rdquo ProteinExpression and Purification vol 109 pp 120ndash126 2015

[8] A Trincone ldquoMarine biocatalysts enzymatic features andapplicationsrdquoMarine Drugs vol 9 no 4 pp 478ndash499 2011

[9] S P S Bisht and S Panda ldquoIsolation and identification of newlipolytic thermophilic bacteria from an Indian hot springrdquoInternational Journal of PharmaandBio Sciences vol 2 pp 229ndash235 2011

[10] J Kanagaraj T Senthilvelan R C Panda and S Kavitha ldquoEco-friendly waste management strategies for greener environmenttowards sustainable development in leather industry a compre-hensive reviewrdquo Journal of Cleaner Production vol 89 pp 1ndash172015

[11] S Saran R V Mahajan R Kaushik J Isar and R K SaxenaldquoEnzyme mediated beam house operations of leather industrya needed step towards greener technologyrdquo Journal of CleanerProduction vol 54 pp 315ndash322 2013

[12] M Rabbani M R Bagherinejad HM Sadeghi et al ldquoIsolationand characterization of novel thermophilic lipase-secretingbacteriardquo Brazilian Journal of Microbiology vol 44 no 4 pp1113ndash1119 2013

[13] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Cold Spring Harbor Laboratory ColdSpring Harbor NY USA 2nd edition 1989

[14] M A Hassan B M Haroun A A Amara and E A SerourldquoProduction and characterization of keratinolytic protease fromnew wool-degrading Bacillus species isolated from Egyptianecosystemrdquo BioMed Research International vol 2013 Article ID175012 14 pages 2013

[15] H M Abdou and M A Hassan ldquoProtective role of omega-3polyunsaturated fatty acid against lead acetate-induced toxicityin liver and kidney of female ratsrdquo BioMed Research Interna-tional vol 2014 Article ID 435857 11 pages 2014

[16] F Sanger S Nicklen and A R Coulson ldquoDNA sequencingwith chain-terminating inhibitorsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 74 no12 pp 5463ndash5467 1977

[17] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[18] L D Castro-Ochoa C Rodrıguez-Gomez G Valerio-Alfaroand R O Ros ldquoScreening purification and characterizationof the thermoalkalophilic lipase produced by Bacillus ther-moleovorans CCR11rdquo Enzyme and Microbial Technology vol 37no 6 pp 648ndash654 2005

[19] A A AmaraM A Hassan A T Abulhamd and BMHarounldquoNon-mucoid P aeruginosa aiming to a safe production ofprotease and lipaserdquo International Science and InvestigationJournal vol 2 no 5 pp 103ndash113 2013

[20] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[21] A Afsar and F Cetinkaya ldquoStudies on the degreasing of skinby using enzyme in liming processrdquo Indian Journal of ChemicalTechnology vol 15 no 5 pp 507ndash510 2008

[22] F Shahidi ldquoExtraction and measurement of total lipidsrdquo inCurrent Protocols in Food Analytical Chemistry John Wiley ampSons 2003

[23] S Elleuche C Schroder K Sahm and G Antranikian ldquoExtre-mozymes-biocatalysts with unique properties from extremo-philic microorganismsrdquo Current Opinion in Biotechnology vol29 no 1 pp 116ndash123 2014

[24] M Sifour T I Zaghloul H M Saeed M M Berekaa andY R Abdel-Fattah ldquoEnhanced production of lipase by thethermophilic Geobacillus stearothermophilus strain-5 using sta-tistical experimental designsrdquo New Biotechnology vol 27 no 4pp 330ndash336 2010

[25] Y R Abdel-Fattah N A Soliman S M Yousef and E REl-Helow ldquoApplication of experimental designs to optimizemedium composition for production of thermostable lipaseesterase by Geobacillus thermodenitrificans AZ1rdquo Journal ofGenetic Engineering and Biotechnology vol 10 no 2 pp 193ndash200 2012

[26] M G Sanchez-Otero I I Ruiz-Lopez D E Avila-Nietoand R M Oliart-Ros ldquoSignificant improvement of Geobacillusthermoleovorans CCR11 thermoalkalophilic lipase productionusing Response Surface Methodologyrdquo New Biotechnology vol28 no 6 pp 761ndash766 2011

[27] D Sharma B K Kumbhar A K Verma and L TewarildquoOptimization of critical growth parameters for enhancingextracellular lipase production by alkalophilic Bacillus sprdquo Bio-catalysis and Agricultural Biotechnology vol 3 no 4 pp 205ndash211 2014

[28] M M Berekaa T I Zaghloul Y R Abdel-Fattah H MSaeed and M Sifour ldquoProduction of a novel glycerol-induciblelipase from thermophilicGeobacillus stearothermophilus strain-5rdquoWorld Journal of Microbiology amp Biotechnology vol 25 no 2pp 287ndash294 2009

[29] M A Eltaweel R N Z R A Rahman A B Salleh and MBasri ldquoAn organic solvent-stable lipase from Bacillus sp strain42rdquo Annals of Microbiology vol 55 no 3 pp 187ndash192 2005

[30] L Bora and M Bora ldquoOptimization of extracellular ther-mophilic highly alkaline lipase from thermophilic Bacillus spisolated from hot spring of Arunachal Pradesh Indiardquo BrazilianJournal of Microbiology vol 43 pp 30ndash42 2012

Enzyme Research 9

[31] S Kumar K Kikon A Upadhyay S S Kanwar and R GuptaldquoProduction purification and characterization of lipase fromthermophilic and alkaliphilic Bacillus coagulans BTS-3rdquo ProteinExpression and Purification vol 41 no 1 pp 38ndash44 2005

[32] C K Sharma P K Sharma and S S Kanwar ldquoOptimization ofproduction conditions of lipase from B licheniformis MTCC-10498rdquo Research Journal of Recent Sciences vol 1 no 7 pp 25ndash32 2012

[33] M A Abd El Rahman Production of thermostable microbialenzymes for application in bio-detergent technology [MS disser-tation] Faculty of Science Al-Azhar University Cairo Egypt2006

[34] V R Gayathri P Perumal L P Mathew and B PrakashldquoScreening and molecular characterization of extracellularlipase producing Bacillus species from coconut oil mill soilrdquoInternational Journal of Science and Technology vol 2 no 7 pp502ndash509 2013

[35] F N Niyonzima and S S More ldquoConcomitant production ofdetergent compatible enzymes by Bacillus flexus XJU-1rdquo Brazil-ian Journal of Microbiology vol 45 no 3 pp 903ndash910 2014

[36] M Veerapagu A S Narayanan K Ponmurgan and K R JeyaldquoScreening selection identification production and optimiza-tion of bacterial lipase from oil spilled soilrdquo Asian Journal ofPharmaceutical and Clinical Research vol 6 supplement 3 pp62ndash67 2013

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

2 Enzyme Research

Many advantages were earned for carrying out biotech-nological and industrial processes in high temperatureshigh solubility of substrates (in particular for poorly solubleor polymeric molecules) resulting in higher product yieldhigher reaction rates increased availability of substratesdecreased risk of microbial contamination and lower vis-cosity of reaction mixtures which in turn reduces the costsrelated to pumping filtration and centrifugation and savinga great power cost would be exploited for cooling [8 9]

In the present study a detailed description of isolationidentification and optimization of TA lipase production con-ditions from G thermoleovorans DA2 will be demonstratedAn attempt to utilize restaurant fatty wastes as the mainsubstrate for lipase production was carried out which mayraise the lipolytic activity and decrease the overall cost of theproduction process In addition this approach has a greatenvironmental endeavor through minimizing the ecologicalhazards accompanied with the accumulation of wastes

The application of TA lipase from G thermoleovoransDA2 in leather tanning process as a degreasing agent replacedthe commonly utilized organic solvent (Kerosene) Kanagarajet al reported that it is fundamental to add hydrolyticenzymes such as lipases and proteases in the soaking step forhelping the fat degradation and raise the leather quality [10]

Substitution of the traditional chemical tanning process-ing with more ecofriendly treating procedures for examplethe enzymatic steps became a necessity because of therecorded environmental hazards resulting from the pollutionof water careless disposal of solid wastes and gaseous emis-sions [11]

2 Materials and Methods

21 Bacterial Strain Awide variety of samples were collectedfrom many localities in Egypt including desert and hotsprings of Southern Sinai Wadi El-Natron swamps desert ofQina and Suez governorates and the soil of El-Basateenslaughter house

All samples were suspended in sterilized saline solution(085wv) which were cultured on plates of nutrient agarmedium with pH 9 and incubated at 65∘C for 48 hrs Theseparated colonies had undergone a series of (agar streakmethod) for purification and the morphological character-istics of each isolate were investigated The purpose of thismethod was to isolate the thermoalkalophilic microorgan-isms

22 Screening

221 Qualitative Assay All strains were screened for inves-tigating their lipase activity on agar plates containing Rho-damine B 0001 (wv) nutrient broth 08 (wv)NaCl 04(wv) agar 1 (wv) and olive oil 3 in distilled water andthe pH was adjusted to be 9 [12]

Plates were incubated at 65∘C for 18 hrs and the lipaseactivity was identified as an orange halo zone around coloniesunder UV light at 350 nm

222 Quantitative Assay To detect the most potent ther-moalkaliphilic lipase producing bacteria all bacterial isolateswere grown on medium composed of yeast extract 1 g oliveoil 10mL gum Arabic 10 g CaCl

21 g and mineral salt solu-

tion 1mL per liter The medium pH is initially adjusted at 9by using 6N NaOH and incubated at 65∘C and 100 rpm for18 hrs The most potent thermoalkaliphilic lipase producingisolate was purified by ldquoagar streak methodrdquo and underwentbiochemical investigations and it was identified by 16S rRNAtechnique

223 Bacterial Identification The bacterial isolate was iden-tified using 16S rRNA sequence The genomic DNA wasextracted by the following method which was described bySambrook et al [13]

The PCR amplification was carried out according toHassan et al and Abdou and Hassan The reaction was per-formed using forwarded 16S rRNA primer (51015840-AAATGG-AGGAAGGTGGGGAT-31015840) and reverse 16S rRNA primer(51015840-AGGAGGTGATCCAACCGCA-31015840) The PCR machine(TECHNE TC-3000 FTC302) was programmed as follows3min denaturation at 95∘C followed by 35 cycles that con-sisted of 1min at 95∘C 1min at 58∘C and 1min at 72∘C andthe final extension was 10min at 72∘C [14 15]

The PCR product was cleaned up for sequencing usingQiagen kit for DNA purification from aqueous PCR DNAsequencingmethod which was developed by Sanger et al wascarried out using 3130X DNA Sequencer (Genetic AnalyzerApplied Biosystems Hitachi Japan) [16]

224 Sequence Analysis and Phylogenetic Tree ConstructionSimilarity of the obtained nucleotide sequence was per-formed by basic local alignment search tool (BLAST) againstreference sequences available in National Center for Biotech-nology Information GenBank (NCBI GenBank) The refer-ence sequences were collected from GenBank and the align-ments using Clastal W were performed for constructing thephylogenetic tree using MEGA 5 software version 51 [17]

23 Lipase Production G thermoleovorans DA2 was grownon a liquid production medium containing ( wv) yeastextract 01 g NaNO

3 02 g KH

2PO4 01 g MgSO

4sdot7H2O

005 g KCl 005 g and CaCl2 01 g supplemented with 1 g of

fatty restaurant wastesThe medium was adjusted at pH 9 (pH-Meter Model-

420A Orion Co USA) and 100mL of medium in 500mLErlenmeyer flasks was inoculated with G thermoleovoransDA2 and incubated at 65∘C and 100 rpm for 18 hrs on a rotaryshaker (Innova J-25 New Brunswick scientific USA)

24 Assay of Thermoalkaliphilic Lipase Activity Lipase activ-ity was detected by a spectrophotometric assay using p-nitro-phenyl laurate (pNPL) as a substrate according to Castro-Ochoa et al and Amara et al with slight modifications Inbrief the reaction mixture consisted of 01mL enzyme ext-ract 08mL 01M phosphate buffer (pH 8) and 01mL 001MpNPL in isopropanolThe hydrolytic reaction was carried outat 60∘C for 30min and then terminated by 025mL of 01M

Enzyme Research 3


















3 Results


4 Enzyme Research

17

99

1312

38

82

50

26

21

26

29

90

38

41

2
































Enzyme Research 5

50 55 60 65 70 75 80 85 90 95 10045Temperature (∘C)

0

25

50

75

100

125

150

175

200Li

pase

activ

ity (U

mL)

(a)

0

25

50

75

100

125

150

175

200

Lipa

se ac

tivity

(Um

L)

6 7 8 9 10 11 12 135pH

(b)

0

50

100

150

200

250

300

Lipa

se ac

tivity

(Um

L)


(c)









4 Discussion


6 Enzyme Research

Carbon source

Inul

inM

anni

tol

Sorb

itol

Sucr

ose

Cel

lobi

ose

Mal

tose

Lact

ose

Glu

cose

Mel

ezito

seRh

amno

seRi

bose

Xylo

seG

alac

tose

0

100

200

300

400

500

600

700

800Li

pase

activ

ity (U

mL)

(a)

Nitrogen source

Sodi

um n

itrat

e

Pept

one

Pot

nitr

ate

Calc

ium

nitr

ate

Ure

a

Sodi

um n

itrite

Am

m p

hosp

hate

Am

m m

olyb

date

Am

m n

itrat

e

Am

m s

ulph

ate

Am

m c

hlor

ide

0100200300400500600700800900

Lipa

se ac

tivity

(Um

L)(b)

0

100

200

300

400

500

600

700

800

900

1000

Lipa

se ac

tivity

(Um

L)


(c)

0

100

200

300

400

500

600

700

800

900

1000Li

pase

activ

ity (U

mL)

1 2 3 4 5 6 7 8 9 10 11 120Inoculum size ( vv)

(d)

400

600

800

1000

1200

Lipa

se ac

tivity

(Um

L)


(e)


Enzyme Research 7
















5 Conclusion




8 Enzyme Research

References
































Enzyme Research 9














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Enzyme Research 3


















3 Results


4 Enzyme Research

17

99

1312

38

82

50

26

21

26

29

90

38

41

2
































Enzyme Research 5

50 55 60 65 70 75 80 85 90 95 10045Temperature (∘C)

0

25

50

75

100

125

150

175

200Li

pase

activ

ity (U

mL)

(a)

0

25

50

75

100

125

150

175

200

Lipa

se ac

tivity

(Um

L)

6 7 8 9 10 11 12 135pH

(b)

0

50

100

150

200

250

300

Lipa

se ac

tivity

(Um

L)


(c)









4 Discussion


6 Enzyme Research

Carbon source

Inul

inM

anni

tol

Sorb

itol

Sucr

ose

Cel

lobi

ose

Mal

tose

Lact

ose

Glu

cose

Mel

ezito

seRh

amno

seRi

bose

Xylo

seG

alac

tose

0

100

200

300

400

500

600

700

800Li

pase

activ

ity (U

mL)

(a)

Nitrogen source

Sodi

um n

itrat

e

Pept

one

Pot

nitr

ate

Calc

ium

nitr

ate

Ure

a

Sodi

um n

itrite

Am

m p

hosp

hate

Am

m m

olyb

date

Am

m n

itrat

e

Am

m s

ulph

ate

Am

m c

hlor

ide

0100200300400500600700800900

Lipa

se ac

tivity

(Um

L)(b)

0

100

200

300

400

500

600

700

800

900

1000

Lipa

se ac

tivity

(Um

L)


(c)

0

100

200

300

400

500

600

700

800

900

1000Li

pase

activ

ity (U

mL)

1 2 3 4 5 6 7 8 9 10 11 120Inoculum size ( vv)

(d)

400

600

800

1000

1200

Lipa

se ac

tivity

(Um

L)


(e)


Enzyme Research 7
















5 Conclusion




8 Enzyme Research

References
































Enzyme Research 9














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Enzyme Research

17

99

1312

38

82

50

26

21

26

29

90

38

41

2
































Enzyme Research 5

50 55 60 65 70 75 80 85 90 95 10045Temperature (∘C)

0

25

50

75

100

125

150

175

200Li

pase

activ

ity (U

mL)

(a)

0

25

50

75

100

125

150

175

200

Lipa

se ac

tivity

(Um

L)

6 7 8 9 10 11 12 135pH

(b)

0

50

100

150

200

250

300

Lipa

se ac

tivity

(Um

L)


(c)









4 Discussion


6 Enzyme Research

Carbon source

Inul

inM

anni

tol

Sorb

itol

Sucr

ose

Cel

lobi

ose

Mal

tose

Lact

ose

Glu

cose

Mel

ezito

seRh

amno

seRi

bose

Xylo

seG

alac

tose

0

100

200

300

400

500

600

700

800Li

pase

activ

ity (U

mL)

(a)

Nitrogen source

Sodi

um n

itrat

e

Pept

one

Pot

nitr

ate

Calc

ium

nitr

ate

Ure

a

Sodi

um n

itrite

Am

m p

hosp

hate

Am

m m

olyb

date

Am

m n

itrat

e

Am

m s

ulph

ate

Am

m c

hlor

ide

0100200300400500600700800900

Lipa

se ac

tivity

(Um

L)(b)

0

100

200

300

400

500

600

700

800

900

1000

Lipa

se ac

tivity

(Um

L)


(c)

0

100

200

300

400

500

600

700

800

900

1000Li

pase

activ

ity (U

mL)

1 2 3 4 5 6 7 8 9 10 11 120Inoculum size ( vv)

(d)

400

600

800

1000

1200

Lipa

se ac

tivity

(Um

L)


(e)


Enzyme Research 7
















5 Conclusion




8 Enzyme Research

References
































Enzyme Research 9














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Enzyme Research 5

50 55 60 65 70 75 80 85 90 95 10045Temperature (∘C)

0

25

50

75

100

125

150

175

200Li

pase

activ

ity (U

mL)

(a)

0

25

50

75

100

125

150

175

200

Lipa

se ac

tivity

(Um

L)

6 7 8 9 10 11 12 135pH

(b)

0

50

100

150

200

250

300

Lipa

se ac

tivity

(Um

L)


(c)









4 Discussion


6 Enzyme Research

Carbon source

Inul

inM

anni

tol

Sorb

itol

Sucr

ose

Cel

lobi

ose

Mal

tose

Lact

ose

Glu

cose

Mel

ezito

seRh

amno

seRi

bose

Xylo

seG

alac

tose

0

100

200

300

400

500

600

700

800Li

pase

activ

ity (U

mL)

(a)

Nitrogen source

Sodi

um n

itrat

e

Pept

one

Pot

nitr

ate

Calc

ium

nitr

ate

Ure

a

Sodi

um n

itrite

Am

m p

hosp

hate

Am

m m

olyb

date

Am

m n

itrat

e

Am

m s

ulph

ate

Am

m c

hlor

ide

0100200300400500600700800900

Lipa

se ac

tivity

(Um

L)(b)

0

100

200

300

400

500

600

700

800

900

1000

Lipa

se ac

tivity

(Um

L)


(c)

0

100

200

300

400

500

600

700

800

900

1000Li

pase

activ

ity (U

mL)

1 2 3 4 5 6 7 8 9 10 11 120Inoculum size ( vv)

(d)

400

600

800

1000

1200

Lipa

se ac

tivity

(Um

L)


(e)


Enzyme Research 7
















5 Conclusion




8 Enzyme Research

References
































Enzyme Research 9














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Enzyme Research

Carbon source

Inul

inM

anni

tol

Sorb

itol

Sucr

ose

Cel

lobi

ose

Mal

tose

Lact

ose

Glu

cose

Mel

ezito

seRh

amno

seRi

bose

Xylo

seG

alac

tose

0

100

200

300

400

500

600

700

800Li

pase

activ

ity (U

mL)

(a)

Nitrogen source

Sodi

um n

itrat

e

Pept

one

Pot

nitr

ate

Calc

ium

nitr

ate

Ure

a

Sodi

um n

itrite

Am

m p

hosp

hate

Am

m m

olyb

date

Am

m n

itrat

e

Am

m s

ulph

ate

Am

m c

hlor

ide

0100200300400500600700800900

Lipa

se ac

tivity

(Um

L)(b)

0

100

200

300

400

500

600

700

800

900

1000

Lipa

se ac

tivity

(Um

L)


(c)

0

100

200

300

400

500

600

700

800

900

1000Li

pase

activ

ity (U

mL)

1 2 3 4 5 6 7 8 9 10 11 120Inoculum size ( vv)

(d)

400

600

800

1000

1200

Lipa

se ac

tivity

(Um

L)


(e)


Enzyme Research 7
















5 Conclusion




8 Enzyme Research

References
































Enzyme Research 9














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Enzyme Research 7
















5 Conclusion




8 Enzyme Research

References
































Enzyme Research 9














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 Enzyme Research

References
































Enzyme Research 9














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Enzyme Research 9














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article production of thermoalkaliphilic lipase...

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