Purification and characterization of an extracellular lipa from a thermophilic Rhizopus oryzae strain isolated from palm fruit
Abel Hiol,a Marie D.Jonzo,a Nathalie Rugani,b Danielle Druet,a Louis Sarda,c
Louis Claude Comeau a,*
a
Universite D’Aix-Marille III.Institut de Nutrition,Laboratoire de Chimie Biologique Applique ´e,Faculte ´des Sciences de Saint–Je ´ro ˆme 13397
Marille Cedex 20,France
b
Laboratoire de Microbiologie,Faculte ´des Sciences de Saint–Je ´ro ˆme 13397Marille Cedex 20,France
c
Laboratoire de Biochimie,Faculte ´des Sciences Saint Charles,Universite ´de Provence 13331Marille Cedex 3,France
Received 7July 1999;received in revid form 4November 1999;accepted 8November 1999
Abstract
We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae .Culture conditions were optimized and
highest lipa production amounting to 120U/ml was achieved after 4days of cultivation.The extracellular lipa was purified 1200-fold by ammonium sulfate precipitation,sulphopropyl-Sepharo chromatography,Sephadex G 75gel filtration and a cond sulphopropyl-Sepharo chromatography.The specific activity of the purified enzyme was 8800U/mg.The lipolytic enzyme has a molecular mass of 32kDa by SDS-polyacrylamide gel electrophoresis and gel filtration.The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6.Analysis of Rhizopus oryzae lipa by RP-HPLC confirmed the homogeneity of the enzyme preparation.Determination of the N-terminal quence over 19amino acid residues showed a high homology with lipas of the same genus.The optimum pH for enzyme activity was 7.5.Lipa was s
table in the pH range from 4.5to 7.5.The optimum temperature for lipa activity was 35°C and about 65%of its activity was retained after incubation at 45°C for 30min.The lipolytic enzyme was inhibited by Triton X100,SDS,and metal ions such as Fe 3ϩ,Cu 2ϩ,Hg 2ϩand Fe 2ϩ.Lipa activity against triolein was enhanced by sodium cholate or taurocholate.The purified lipa had a preference for the hydrolysis of saturated fatty acid chains (C 8–C 18)and a 1,3-position specificity.It showed a good stability in organic solvents and especially in long chain-fatty alcohol.The enzyme poorly hydrolyzed triacylglycerols containing n-3polyunsaturated fatty acids,and appeared as a suitable biocatalyst for lective esterification of sardine free fatty acids with hexanol as substrate.About 76%of sardine free fatty acids were esterified after 30h reaction whereas 90%of docosahexaenoic acid (DHA)was recovered in the unesterified fatty acids.©2000Elvier Science Inc.All rights rerved.
Keywords:Palm oil acidification;Palm fruit;Rhizopus oryzae ;Extracellular lipa;Purification;Enzyme characterization;Docosahexaenoic acid
1.Introduction
长城的诗句Lipas reprent a group of enzymes having the ability to hydrolyze triacylglycerols at lipid-water interface.Lipo-lytic activity is highly incread upon enzyme binding to the lipid surface,a phenomeno
n known as interfacial activation [1,2].Lipas can also transterify triacylglycerols or syn-thesize ester bonds in non aqueous media [3,4].Lipas have been widely ud for biotechnological applications in dairy industry,oil processing,production of surfactants and
preparation of enantiomerically pure pharmaceuticals [5].Many studies have been carried out to understand the struc-ture-function relationships of lipas in view to improve their properties and to extend their industrial applications.The tridimensional (3D)structures of lipas from fungi Rhizomucor miehei and Geotrichum candidum ,and from human pancreas lipa have been first determined [6–8].Since then,3D structures of at least eleven lipas of mi-crobial origin have been solved [9].The enzymes display variable biochemical properties.Although the quence ho-mology among them is limited,the 3D structures of lipas share a common motif known as ␣/hydrola fold.Elu-cidation of the 3D structures of lipas provides an elegant explanation for interfacial activation.Actually the active
*Corresponding author.Tel.:ϩ33-4-91-28-87-17;fax:ϩ33-4-91-28-87-78.
E-mail address:Louis Comeau@lcba.u-3mrs.fr (L.C.
Comeau)Enzyme and Microbial Technology 26(2000)421–430
0141-0229/00/$–e front matter ©2000Elvier Science Inc.All rights rerved.PII:S0141-0229(99)00173-8
site of the enzyme is covered by an amphiphilic loop(lid or flap),which prevents access of substrate.Upon binding of lipa to interface,the active site becomes accessible and a hydrophobic area is expod by structural rearrangement which includes movement of the lid[10].
诗词佳句Many studies on microbial lipas have investigated their effects on food spoilage and/orflavour changes,particularly in the dairy industry[11].The main criterion recognized for asssing the quality of palm oil is the level of free fatty acids.Several lipolytic fungi,predominantly Mucor spp., Humicola lanuginosa,and Rhizopus spp.have been isolated from palm fruit from various origins[12,13].Although the deterioration of palm oil wasfirst attributed to fungal lipas,only few studies concerning the isolation and bio-chemical characterization of the lipolytic enzymes respon-sible for the relea of fatty acids have been carried out to definitely asss their involvement in the biodeterioration process.We have recently reported the purification and characterization of a new lipa from Mucor hiemalis f. hiemalis isolated from palm fruit[14]together with the Rhizopus oryzae strain studied here.The production of lipolytic enzymes from ae strain,isolated from the effluent treatment pond of a palm oil mill has been already described[15]but no further purification an
d characteriza-tion of the enzyme were reported.Since then,the taxonomic diversity of the Rhizopus genus was reinvestigated and veral species were combined ae[16].In this paper we describe an optimized fermentation process for the production of an extracellular lipa from a thermophilic R. oryzae strain isolated from Cameroonian palm fruit.The enzyme has been purified to homogeneity and its biochem-ical and molecular properties have been studied and com-pared to tho of lipas from other fungi of the Rhizopus genus.
2.Materials and methods
Palm fruit were obtained from the CIRAD(Centre de Coope´ration Internationale en Recherche Agronomique pour le De´veloppement,Montpellier,France).
2.1.Microorganisms
Peptone from cain pancreatically digested free from sulfonamide antagonists and Sabouraud-4%malt agar were obtained from Merck.Q-Sepharo,Sephadex G75,Sul-phopropyl-Sepharo,and
reagents for electrophoresis were from Pharmacia Biotech(Orsay,France).Hydrophilic ion exchange resins(Duolite A561,A568and Amberlite IRC50)were a gift of Chauny,Rhom,and Haas(France). Polyvinylidene difluoride(PVDF)membrane and pI stan-dards were obtained from Bio–Rad(Richmond,CA,USA). Corn steep liquid was a gift of Roquette(Lille,France).
2.2.Strain conrvation
ae strain isolated from palm fruit was main-tained in Sabouraud-4%malt agar with0.1%palm oil at 4°C.For inoculum preparation,the tubes were incubated at 30°C for4days.After dilution in0.1%peptone solution,the spore suspension(about106spores liter)was kept at Ϫ20°C.
2.3.Culture conditions
The organism was cultivated in2l(CMF100Basic,B. Braun,France)fermentor in a medium containing4%(w/v) corn steep liquor,1%peptone, 1.4%KH2PO4,0.24% K2HPO4,and0.04%MgSO4.The initial pH of culture medium was adjusted to5.6with0.5M NaOH.A metallic grill,ud as growth support,was placed in the fermentor which was autoclaved at120°C for30min.After inocula-tion with10ml of a106spores liter suspension,the culture wasflushed with oxygen during2min,with shaking main-tained at100rev./min.Growth temperature was maintained at3
5°C during thefirst day of cultivation and then was reduced to28°C for the remainder of the fermentation period.Samples were withdrawn at different time intervals. The pH and lipa activity in the culture broth were deter-mined.Growth studies were performed in batch cultivation as previously described[17].
2.4.Purification
All purification steps were carried out at4°C except when stated otherwi.At the end of the cultivation period, mycelia were removed byfiltration(Whatman paper).The filtrate was centrifuged at10000ϫg for25min and the resulting supernatant was ud as starting material for lipa purification.Benzamidine,a protea inhibitor,and sodium azide were added to the supernatant to afinal concentration of2mM and0.02%,respectively,to prevent proteolytic degradation and microbial growth.Proteins from the culture medium were precipitated by two-step procedure.Thefirst step consisted in the addition of ammonium sulfate up to 30%saturation at0°C followed by centrifugation at 12000ϫg for45min.The precipitate proteins were discarded.Ammonium sulfate was then added to the super-natant to thefinal concentration of75%saturation and the suspension was kept at0°C for4h under gentle stirring.The precipitated was collected by centrifugation at10000ϫg for15min and further dissolved in30ml of20mM Tris-HCl buffer pH6.8containing0.02%sodium azide and 2mM Benzamidin
e(buffer A).The enzyme solution was dialyzed overnight against the same buffer and the insoluble material were discarded by centrifugation.The enzyme so-lution was applied to a Sulphopropyl-Sepharo column
422 A.Hiol et al./Enzyme and Microbial Technology26(2000)421–430
(2.6cmϫ10cm)previously equilibrated in buffer A.The column was washed to remove unbound proteins and the enzyme was eluted by a0to0.5M NaCl linear gradient in the same buffer at aflow rate of0.8ml/min.The peak of lipa activity was eluted with NaCl at concentration around 0.15M.Active fractions from the Sulphopropyl-Sepharo column were pooled and the supernatant was dialyzed against buffer A.The enzyme was partially purified batch-wi by mixing the dialyzed solution with75ml of Q-Sepharo equilibrated in buffer ae lipa was not adsorbed on Q-Sepharo.The enzyme solution was then concentrated to10ml by ultrafiltration with an Amicon cell using a PM10membrane.The concentrated lipa solution (10ml)was loaded onto a Sephadex G75column(2.6ϫ10 cm)previously equilibrated with20mM Tris-HCl pH6.8, containing0.15M NaCl and elution was performed with the same buffer at aflow rate of0.4ml/min.Fractions contain-ing lipa were pooled and dialyzed against20mM Tris-HCl pH6.8.The sample was then applied to a Sulphopropyl column(2.6ϫ10cm)equilibrated with20mM Tris-HCl pH6.8.The column was washed wit
h0.1M NaCl in the same Tris buffer and the lipa was eluted with0.15M NaCl in the Tris buffer.The pooled active fractions were concentrated by ultrafiltration and stored atϪ20°C for v-eral months without loss of lipa activity.
2.5.Determination of lipa activity
Lipa activity was determined titrimetrically as de-scribed previously[18],using a VIT90Video titrator(Ra-diometer,Copenhagen,Denmark)and0.05N NaOH as titrant.The reaction medium contained500l trioctanoyl-glycerol as substrate disperd by stirring in20ml of10 mM Tris-HCl pH7with0.1M NaCl and10mM CaCl2. Hydrolysis was measured at pH7.0and30°C under nitro-gen atmosphere.The reaction was initiated by addition of enzyme.Controls were carried out as above but the enzyme solution was inactivated by boiling for15min.Kinetics of hydrolysis were zero-order within thefirstfive min of the reaction.Activity against long-chain triacylglycerol plant oils(palm oil,olive oil,rapeed oil,sunflower oil,and corn oil)was determined with20ml of substrate emulsion pre-pared in a blender(Sofraca.Turmix,Vitrolles,France)by mixing40ml of oil in400ml of a2%solution of gum acacia prepared in distilled water.One lipa unit corre-sponded to the relea onemole of fatty acid per min under assay conditions.The specific activity is the number of lipa units per mg protein.
2.6.Rapid detection of lipa on Rhodamine B–olive oil agar plates
The detection of lipa was adapted from Samad et al [19].Lipa activity was detected upon UV irradiation as orange-redfluorescent halos.2.7.Assay of estera activity
Estera activity was assayed spectrophotometrically us-ing p-nitrophenylacetate(p-NPA)as substrate according to Erlanson[20].The substrate solution was prepared by dis-solving18mg of p-NPA in1ml of methanol.This solution was added with a submerged pipette to99ml of0.05N acetate buffer pH5.0.Assays were performed with1ml of the aqueous solution of p-NPA added with enzyme(100to 200ml)and0.5M Tris-HCl buffer pH7.4infinal volume of3ml.The incubations were performed at room temper-ature and the relea of p-nitrophenol was measured during 5min at400nm against a blank prepared with heat-inacti-vated enzyme in a Ultraspec III spectrophotometer(Phar-macia Biotech computed with Kinetic software).
2.8.Effect of temperature and pH on lipa activity
The optimal temperature for the hydrolysis of trioctanoin by lipa was determined by measuring the rate of the reaction at temperatures ranging from20°C to50°C under standard assay conditions,as described above.Controls were performed with boiled enzyme.For studying thermal stability,the enzy
me was incubated at temperatures ranging from20°C to50°C for30min and residual activity was determined.The optimal pH enzyme activity was deter-mined by measuring trioctanoin hydrolysis at pH from5to 9in various buffers.The effect of pH on lipa stability was studied by measuring residual activity at pH7.0after1h incubation at pH ranging from5.5to9.0.
2.9.Protein content
Protein was estimated colorimetrically according to Lowry et al[21]using bovine rum albumin as standard.In chromatography experiments,protein concentration in frac-tions was routinely estimated by measuring the absorbance at280nm.
2.10.Analysis of lipolysis products by high-performance liquid chromatography(HPLC)
The lipolysis system(final volume4ml)was prepared with200mM triolein,diolein or monoolein suspended in20 mM Tris-HCl buffer,pH7.0.This system was added with 30units of purifiae lipa and stirred for30min with12mm stirring bar at350rev./min.Samples(40l) were withdrawn everyfive min,diluted with800l of acetone andfiltered through0.2HV Milliporefilter.The reaction products were analyzed by reverd-pha HPLC (Shimadzu,Kyoto,Japan)on a C18Beckman column(5m internal diameterϫ15cm)using acetone/acetonitrile (1:1v/v)as the mobile pha.
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2.11.Analytical gel electrophoresis and isoelectric focusing
Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis(SDS-PAGE)and isoelectric focusing were run on a Bio–Rad Mini-Protean II DUAL SLAB cell according to the manufacturer’s instructions.SDS-PAGE was performed as described by Laemmli et al.[22]in a12%polyacryl-amide gel slab in the prence of0.1%SDS.For the esti-mation of the molecular mass of the enzyme,the following proteins were ud as markers:phosphoryla(94kDa), rum albumin(67kDa),ovalbumin(43kDa),carbonic anhydra(30kDa),soy bean trypsin inhibitor(20kDa), and lysozyme(14.4kDa).
2.12.Electrotransfer on PVDF membrane
After SDS-PAGE,the purified lipa was electrotrans-fered onto a PVDF membrane with the Mini Trans-Blot electrophoretic transfer cell from Bio–Rad according to the manufacturer’s instruction.The PVDF membrane was rind with distilled water and stained with Coomassie Bril-lant Blue R-250.The stained component was ud for N-terminal quencing.
2.1
3.Amino acid analysis and N-terminal quence determination
Amino acid analysis was performed in duplicate with a Beckman6300quencer equipped with a PTH amino acid analyzer(System Gold,Beckman).The protein was hydro-lyd in6N HCl for24h(110°C)and derivatized with PITC before being injected on a30cm picotac column. Amino acids were eluted by using an acetonitrile linear concentration gradient in50mM sodium acetate buffer. N-terminal quence analysis was performed by automated Edman degradation using instrumentation protocols from Applied Biosystems.A model470A gas-pha quencer equipped with online120A PTH-analyr and a model 900A control/data analysis module were ud[23].
2.14.Analytical RP-HPLC of purified lipa
The purified lipa preparation was analyzed for homo-geneity by RP-HPLC under conditions previously described [24].Elution was achieved with a linear gradient(total volume:70ml)from2to72%acetonitrile in0.1%triflu-oroacetic acid in water at a rateflow of1ml per min.Protein was detected at230nm.
2.15.Determination of molecular mass by gelfiltration
Purified lipa(100g)was loaded in one ml onto a Sephadex G75(2.6ϫ30cm)column.Serum albumin(67 kDa),ovalbumin(43kDa),chymotrypsinogen A(25kDa) and cytochrome C(12.5kDa)were ud as protein standards.2.16.Carbohydrate content
The phenol-sulfuric acid method[25]was ud to deter-mine the degree of glycosylation of the purified lipa,using manno as reference sugar.A deglycosylated sample of purified lipa was prepared by incubating the purified en-zyme(10g)with60mU of endoglycosida H at37°C for 1h.
2.17.Effect of metal ions and enzyme inhibitors
The purified lipa(15U)was incubated with various compounds at30°C for1h and at different concentrations of effectors and residual activity was measured against trioctanoin under standard assay conditions.
2.18.Water content of enzyme preparation
The water content in lipa preparation was determined using a DL18Mettler autotitrator according to the Karl Fisher’s method.The water activity of immobilized lipa was measured at30°C using a Novasima hygrometric n-sor calibrated with three saturated salt solution of known a w, namely LiC
l.H2O(a wϭ0.113),Mg(NO3)2,6H2O(a wϭ0.514),and KNO3(a wϭ0.914)as previously described [26].牙齿旅行记
2.19.Lipa immobilization
Immobilization ae lipa on a weak cation-exchange resin(Amberlite IRC50)was performed as fol-lows:the enzyme solution(8800U/mg)was mixed at4°C with3g of Amberlite IRC50previously equilibrated in50 mM acetate buffer pH 6.0by shaking at150rev./min overnight.Non-adsorbed lipa activity and protein were determined.The sample of ae lipa (IROL)was washed with acetate buffer.The IROL was collected byfiltration,dried over P2O5and stored at4°C. The enzyme theorecal loading was defined as the number of lipa units adsorbed from the enzyme solution per gram of the Amberlite support.
Immobilization of Mucor hiemalis f.hiemalis lipa on a weak anion-exchange resin was achieved as described above except that Duolite A568was ud as the carrier,and that immobilization was performed in50mM phosphate buffer pH7.0.
2.20.Preparation of free fatty acids from sardine oil
Purified Sardine triacylglycerols(40g)from industrial wastes[27]were mixed in120ml of1N NaOH in90
% aqueous ethanol and placed in a250ml stopperedflask.The temperature of the mixture was brought to50°C under nitrogen atmosphere and stirring for90min.The fatty acids were extracted3ϫwith cold hexane.The composition of
创业路径424 A.Hiol et al./Enzyme and Microbial Technology26(2000)421–430
the resulting free fatty acids mixture(Sardine-FFA)was analyzed by gas-liquid chromatography(GLC).
2.21.Esterification of Sardine-FFA
Experiments were carried out to determine the optimal conditions(type of alcohol,enzyme concentration,alcohol/ free fatty acids ratio water content of the enzyme prepara-tion,optimum temperature,and time cour of the reaction). For esterification of hexanol with Sardine-FFA,reactions mixtures containing1.8mM of hexanol and3.6mM of fatty acids were prepared in a50ml stoppered testflasks and maintained under shaking at30°C.After20min,the im-mobilized lipa(500U)was added to start the reaction. The acid values before and after the reaction were measured by titrating with1N NaOH and the extent of esterification was calculated on the ba of the amount of fatty acids
consumed during the reaction.All samples were tested in triplicate.After30h reaction,unesterified fatty acids were extracted three times with cold hexane and their methyl esters were analyzed by a Shimadzu CR4A gas chromato-graph,fitted with aflame-ionization detector(260°C).The column ud was a BPX70(0.22mmϫ25m from SGE, France).
2.22.Stability in organic solvent
The enzyme was incubated in the prence of various solvents as described by Sztajer et al.[28]and residual activity was measured by using p-NPA as substrate.
All experiments were performed in triplicate.Mean val-ues and standard deviations are reported.Statistic analys were computed with SAS Software(SAS Institute,Inc., Cary,NC,USA).
3.Results and discussion
3.1.Production and purification ae lipa
ae strain ud in this study is classified as thermophilic becau it can grow at50°C.However,no lipolytic activity was detected in the culture broth at tem-perature above35°C.As shown in Fig.1,the production of extracellular lipa incread steadily with cultivation time. Maximal
enzyme concentration was reached after four days of culture(120U/ml)at the late logarithmic growth.At this stage,it could be obrved that the mass of mycelium decread,probably following cell lysis.Therefore,it can-not be excluded that the relea of lipa in the culture broth might originate from cell lysis instead of true enzyme -cretion.Proteolysis and loss of lipa activity was prevented by adding of benzamidine at2mM during the enzyme purification.A summary ae lipa purification is prented in Table1.Lipa was purified about1200-fold over the culturefiltrate with a22%yield.The specific activity of lipa determined against trioctanoin was8800 U/mg.This value is comparable to tho previously re-ported for lipas from other Rhizopus spp.such as R. delemar[29],R.niveus II[30],ae853ATCC [31].
3.2.Biochemical characterization
As shown in Fig.2,upon analytical SDS-PAGE,the purified enzyme,appeared as a single band with molecular mass around32kDa.Upon gelfiltration,a single peak of lipa activity was eluted and corresponded to a protein of molecular mass around30kDa(data not shown).The results indicate that the enzyme is a monomer.The isoelec-tric point of the enzyme was determined to be7.6by isoelectric focusing(data not shown).Homogeneity of the sample ae lipa was definitely confirmed by analytical reverd-pha HPLC(data not shown).Becau the purified lipa was elut
ed with12%acetonitrile,one can suggest that this lipa exhibited a weak affinity for the hydrophobic pha(C18)ud in RP-HPLC.Under the same chromatography conditions,lipa from Mucor hi-emalis f.hiemalis[14]was eluted with9%acetonitrile while triacylglycerol lipa I from Penicillium cyclopium lipa is eluted with55%acetonitrile[32].The purified lipa con-tained very low amount of carbohydrate as estimated by the method of Dubois.Actually no significant modification in the molecular mass was obrved by SDS-PAGE analysis after treatment of the enzyme with endoglycosyda H(Fig.
2).The amino acid composition ae lipa isolated in this study is comparable to tho of lipas from other Rhizopus spp.(Table2).Taken together,the total percent-age of the hydrophobic residues(Ile,Leu,Val,Met,Tyr, Phe)varies from28%to33%.This percentage is in the same range as that lopium triacylglycerol lipa. The N-terminal quence ae lipa was deter-mined over thefirst nineteen amino acid residues and es-tablished as follows:STNSASAGGKVVAATTQI.
带月的诗
文控管理
Com-Fig.1.Time cour production of Rhizopus oryzae lipa.Cultivation was performed with(w/v)4%Corn Steep Liquor and1%peptone at30°C. Lipa activity was determined against trioctanoin as substrate.
小金丸的作用
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A.Hiol et al./Enzyme and Microbial Technology26(2000)421–430
parison with the N-terminal quence of lipas from other strains ae previously characterized and lipas from various Rhizopus spp.(R.delemar and R.niveus ),allows veral obrvations (Table 3).It ems that five additional amino acid residues (STNSA)are prent at the N terminus of purified lipa ae as compared to R.delemar and R.niveus lipas who N-terminal -quences are SDGGKVVAATTQI.In addition,aspartic acid at position 2in the later proteins was changed to alanine.Interesting is the obrvation that,in addition to R.delemar lipa originally purified and characterized by Haas et al.
[29]having the SDGGKVVAATTQI N-terminal quence,two forms of the recombinant enzyme have been isolated from culture supernatants.One form carries the additional NSA tripeptide at its N terminus [33]and,in the other,an additional peptide of 28amino acids is attached to the N terminus [35].This peptide corresponds to the C-terminal region of the proquence of ae lipa which probably plays important roles in folding and transport of the precursor protein in the cretory process.The pentapep-tide STNSA found at the N terminus of the enzyme isolated here actually corr
esponds to the C-terminus of the pro-quence of the ae lipa expresd and produced by Saccharomyces cerevisiae [35].The prence of the additional dipeptide Ser-Thr at the N terminus of the lipa produced by ae strain might originate from a difference of proteolytic cleavage during processing of the mature enzyme due to amino acid substitutions in the pro-quence,the nature of the proteas,or both.
Table 1
Purification of lipa from Rhizopus oryzae Step
Total activity a U Total protein (mg)Specific activity (U/mg)Purification fold Yield (%)Filtrate
2160003060071100Ammonium sulfate fractionation 1728008811962880Sulphopropyl-Sepharo 9330071131418843Sephadex G75
7128013.1544077733Sulfopropyl-Sepharo
47520
5.4
8800
1260
22
a
Activity was determined against
trioctanoin.
Fig.2.Polyacrylamide gel electrophoresis of purified Rhizopus oryzae lipa.Electrophoresis was performed in 12%polyacrylamide gel in the prence of 0.1%SDS.Line 1,purified lipa;line 2,purified lipa treated with endoglycosida H;line 3,molecular mass marker proteins.
Table 2
Amino acid composition of the lipas from Rhizopus spp.Amino acid Rhizopus oryzae lipa a Rh.delemar lipa b Rh.niveus lipa c Asx 98.98.3Glx 8.38.59Ser 99.38.7Gly 10.88.18.3His 2.1 2.6 2.8Arg 3.2 3.4 3.5Thr 10.18.57.6Ala 6.9 5.7 5.3Pro 7.9 5.7 6.5Tyr 3.5 4.6 4.3Val 5.310.111.2Met 0.30.30.3Ileu 5.4 6.7 6.2Leu 7.6 6.2 6.5Phe 6.1 5.7 5.7Lys
4.6
5.7
5.8
Amino acid composition is expresd as mole percent of total amino acids.Residues of cysteine and tryptophane are not taken in account in the calculation.a
长征是什么this study,b Ref [29],c Ref [30].
426 A.Hiol et al./Enzyme and Microbial Technology 26(2000)421–430
