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1.
Manavalan Arulmani Kalaichelvan Aparanjini Kalyanasundaram Vasanthi Perumal Arumugam Manavalan Arivuchelvi P. Thangavelu Kalaichelvan 《World journal of microbiology & biotechnology》2007,23(4):475-481
An extracellular thermostable alkaline protease isolated from Bacillus
laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified
protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the
molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed
a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme
had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific
serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced
by the metal ions Ca2+ and Mg2+ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain
completely when used with Wheel detergent. 相似文献
2.
《Bioscience, biotechnology, and biochemistry》2013,77(2):246-250
Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu2+ and Hg2+. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl2, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca2+ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH2-terminal amino acid was deleted. 相似文献
3.
M. Vidyasagar S. Prakash S. K. Jayalakshmi K. Sreeramulu 《World journal of microbiology & biotechnology》2007,23(5):655-662
An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease.
Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme
displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was
thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial
pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM
concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl2 concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best
nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease
secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease. 相似文献
4.
C. R. Tremacoldi R. Monti H. S. Selistre-De-Araújo E. C. Carmona 《World journal of microbiology & biotechnology》2007,23(2):295-299
An extracellular alkaline serine protease has been purified from a strain of Aspergillus clavatus, to apparent homogeneity, by ammonium sulfate precipitation and chromatography on Sephadex G-75. Its molar mass, estimated
by SDS-PAGE, was 35 kDa. Maximum protease activity was observed at pH 9.5 and 40°C. The enzyme was active between pH 6.0 and
11.0 and was found to be unstable up to 50°C. Calcium at 5 mM increased its thermal stability. The protease was strongly inhibited
by PMSF and chymostatin as well as by SDS, Tween 80 and carbonate ion. Substrate specificity was observed with N-p-Tos-Gly-Pro-Arg-p-nitroanilide and N-Suc-Ala-Ala-Ala-p-nitroanilide being active substates. Parts of the amino acid sequence were up to 81% homologous with those of several fungal
alkaline serine proteases. 相似文献
5.
Kemel Jellouli Ali Bougatef Laila Manni Rym Agrebi Rayda Siala Islem Younes Moncef Nasri 《Journal of industrial microbiology & biotechnology》2009,36(7):939-948
A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases
when it was grown at 30°C in media containing casein as carbon source (14,000 U ml−1). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular
weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence
of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide
range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity
of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic
constants K
m and K
cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 105 min−1, respectively. The catalytic efficiency (K
cat
/K
m) was 7.23 × 108 min−1 M−1. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability
and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that
of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme. 相似文献
6.
Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about
1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold
purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified
enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active
at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C
up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn2+, Co2+ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up
to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating
the potential role of this enzyme for detergent application. 相似文献
7.
Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42 总被引:1,自引:0,他引:1
Kazan D Denizci AA Oner MN Erarslan A 《Journal of industrial microbiology & biotechnology》2005,32(8):335-344
An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease
activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate
by DEAE-cellulose chromatography followed by (NH4)2SO4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum
temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca2+ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal
pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses
of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis
by the purified enzyme was 10.59 kcal mol−1 (44.30 kJ mol−1). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h
at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was
not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k
cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K
m and k
cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×103 min−1, respectively. 相似文献
8.
Studies on the <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> CCT-4518 laccase purified by hydrophobic interaction chromatography 总被引:1,自引:0,他引:1
A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield
of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by
12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K
m values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 μM, respectively. The enzyme’s pH optimum
for syringaldazine was 4.2 and optimal activity was 50°C. The enzyme showed to be thermostable because when kept at 50°C for
24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by l-cysteine, β-mercaptoethanol, NaN3, NaF, and HgCl2. 相似文献
9.
Michelin M Ruller R Ward RJ Moraes LA Jorge JA Terenzi HF Polizeli Mde L 《Journal of industrial microbiology & biotechnology》2008,35(1):17-25
An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated
as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and
pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C,
with a t
50 of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated
by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose,
p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis,
analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix.
The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase). 相似文献
10.
Karbalaei-Heidari HR Amoozegar MA Hajighasemi M Ziaee AA Ventosa A 《Journal of industrial microbiology & biotechnology》2009,36(1):21-27
The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary
growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed
that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations
of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with
68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography.
The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic
activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still
remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at
pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc
SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the
protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view. 相似文献
11.
Bin Wu Licheng Wu Daijie Chen Zhijun Yang Minyu Luo 《Journal of industrial microbiology & biotechnology》2009,36(3):451-459
A novel fibrinolytic enzyme from Fusarium sp. CPCC 480097, named Fu-P, was purified to electrophoretic homogeneity using ammonium sulfate precipitation and ion exchange
and gel filtration chromatography. Fu-P, a single protein had a molecular weight of 28 kDa, which was determined by SDS-PAGE
and gel filtration chromatography. The isoelectric point of Fu-P determined by isoelectric focusing electrophoresis (IEF)
was 8.1, and the optimum temperature and pH value were 45°C and 8.5, respectively. Fu-P cleaved the α-chain of fibrin (ogen)
with high efficiency, and the β-chain and γ-γ (γ-)-chain with lower efficiency. Fu-P activity was inhibited by EDTA and PMSF,
and the enzyme exhibited a high specificity for the chymotrypsin substrate S-2586. Fu-P was therefore identified as a chymotrypsin-like
serine metalloprotease. The first 15 amino acids of the N-terminal sequence of Fu-P were Q-A-S–S-G-T-P-A-T-I-R-V-L-V–V and
showed no homology with that of other known fibrinolytic enzymes. This protease may have potential applications in thrombolytic
therapy and in thrombosis prevention. 相似文献
12.
The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing
agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the
organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed
by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level
of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however
the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml
at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg2+, while Mn2+ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex
chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE
revealed a molecular weight of approximately 22.5 kDa. The enzyme had K
m and V
max values of 2.5 and 26 μmol/mg per minute, respectively. 相似文献
13.
Ping Yu Xinxin Wang Xingxing Huang Qian Ren Tingting Yan 《Preparative biochemistry & biotechnology》2013,43(7):718-726
AbstractA propanol-tolerant neutral protease was purified and characterized from Bacillus sp. ZG20 in this study. This protease was purified to homogeneity with a specific activity of 26,655?U/mg. The recovery rate and purification fold of the protease were 13.7% and 31.5, respectively. The SDS-PAGE results showed that the molecular weight of the protease was about 29?kDa. The optimal temperature and pH of the protease were 45?°C and 7.0, respectively. The protease exhibited a good thermal- and pH stability, and was tolerant to 50% propanol. Mg2+, Zn2+, K+, Na+ and Tween-80 could improve its activity. The calculated Km and Vmax values of the protease towards α-casein were 12.74?mg/mL and 28.57?µg/(min mL), respectively. This study lays a good foundation for the future use of the neutral protease from Bacillus sp. ZG20. 相似文献
14.
A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory.
It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions
for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin,
0.05% urea and NaCl, 0.03% K2HPO4, 0.04% KH2PO4, and 0.01% MgCl2·6H2O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase
production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography
on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction
were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme
activity was significantly inhibited by EDTA, Zn2+ and Hg2+. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing
applications. 相似文献
15.
A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain
was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate
and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol,
benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme
production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident
with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol;
the enzyme was highly active at 60–80°C and displayed optimum activity at 70°C. The protease was significantly stable and
catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis
of the enzyme adversely. Crude enzyme preparation was more stable at 37°C in solvents as compared to partially purified and
purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information
on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant
protease from salt-tolerant alkaliphilic actinomycetes. 相似文献
16.
W. Chungool W. Thongkam P. Raweesri A. Thamchaipenet P. Pinphanichakarn 《World journal of microbiology & biotechnology》2008,24(4):549-556
Acetyl esterase was produced by Streptomyces sp. PC22 at comparable levels of about 0.3 U ml−1 using either 1.0% (w/v) birchwood xylan or 1.5% (w/v) corn husks as a carbon source and cultivating at 45 °C, at pH 9 for
3 or 2 days, respectively. The enzyme was purified from culture filtrate to about 54-fold purity by ammonium sulfate precipitation,
followed by consecutive chromatography using a Macro-Prep DEAE, t-butyl hydrophobic interaction and hydroxyapatite, respectively. The approximate molecular weight of the purified enzyme was
155 kDa as analyzed by gel filtration, and it contained four identical 34 kDa subunits, as assessed by SDS-PAGE. It had K
m and V
max values for p-nitrophenyl acetate of 0.43 mM and 70.78 U mg−1 and 7.8 mM and 1,027 U mg−1 for α-naphthyl acetate, respectively. Its optimal pH and temperature were 6.5–7.0 and 50 °C, respectively. It was stable for 30 min
at a broad range of pH values, from 5.0 to 9.0, and at temperatures up to 60 °C. The purified enzyme had no other xylanolytic
activities. It showed cooperative action on birchwood xylan degradation, when used in combination with xylanase from the same
strain and β-xylosidase from Streptomyces sp. CH7. Enhancement was 1.4-fold, compared to the expected amount of individual enzymes alone. This indicates that the enzyme
has potential industrial applications, especially for utilizing renewable hemicelluloses containing acetyl xylan for the production
of biofuels or other fermentation products. 相似文献
17.
Alfredo E. Pérez-Guzmán Teresa Cruz y Victoria Ramón Cruz-Camarillo Humberto Hernández-Sánchez 《World journal of microbiology & biotechnology》2006,22(9):953-958
Summary An X-prolyl-dipeptidylaminopep tidase (Pep-XP) was purified from the crude intracellular extract of Lactococcus lactis subsp. cremoris NRRL 634 by ion exchange and gel filtration chromatographies. The enzyme was purified 80-fold with a recovery of 6%, and appeared as a single band with a molecular weight of about 80 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE). The peptidase showed its maximal activity on arginyl-proline-p-nitroanilide at pH 7.0 and at a temperature of 45 °C, although there was a good activity of Pep-XP in the pH range of 5.5–7.0 and temperatures between 40 and 50 °C. The Michaelis constant (K
m) and the maximum reaction velocity (V
max) values were 0.92 mM and 7.9 U/mg protein min, respectively. The activity of Pep-XP was completely inhibited by phenylmethanesulphonyl fluoride, an inhibitor of serine peptidases, and metal chelators had little effect on enzyme activity. The purified enzyme hydrolyzed synthetic substrates whose structure is X-Pro-Y like Lys-Pro-pNA, but did not hydrolyse Pro-pNA or azocasein, showing that the enzyme did not have aminopeptidase or endopeptidase activities. 相似文献
18.
I Saba PH Qazi SA Rather RA Dar QA Qadri N Ahmad S Johri SC Taneja S Shawl 《World journal of microbiology & biotechnology》2012,28(3):1071-1079
A Psychrotolerant alkaline protease producing bacterium IIIM-ST045 was isolated from a soil sample collected from the Thajiwas
glacier of Kashmir, India and identified as Stenotrophomonas sp. on the basis of its biochemical properties and 16S ribosomal gene sequencing. The strain could grow well within a temperature
range of 4–37°C however, showed optimum growth at 15°C. The strain was found to over-produce proteases when it was grown in
media containing lactose as carbon source (157.50 U mg−1). The maximum specific enzyme activity (398 U mg−1) was obtained using soya oil as nitrogen source, however, the inorganic nitrogen sources urea, ammonium chloride and ammonium
sulphate showed the lowest production of 38.9, 62.2 and 57.9 U mg−1. The enzyme was purified to 18.45 folds and the molecular weight of the partially purified protease was estimated to be ~55 kDa
by SDS-PAGE analysis. The protease activity increased as the increase in enzyme concentration while as the optimum enzyme
activity was found when casein (1% w/v) was used as substrate. The enzyme was highly active over a wide range of pH from 6.5
to 12.0 showing optimum activity at pH 10.0. The optimum temperature for the enzyme was 15°C. Proteolytic activity reduced
gradually with higher temperatures with a decrease of 56% at 40°C. The purified enzyme was checked for the removal of protein
containing tea stains using a silk cloth within a temperature range of 10–60°C. The best washing efficiency results obtained
at low temperatures indicate that the enzyme may be used for cold washing purposes of delicate fabrics that otherwise are
vulnerable to high temperatures. 相似文献
19.
The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid
supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The
molecular mass determined by SDS-PAGE was 31 kDa. The K
m and k
cat values were found to be 1 mg/ml and 76 sec−1, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when
exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when
exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea.
Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992. 相似文献
20.
Nack-Shick Choi Jae Jun Song Dong-Min Chung Young Jae Kim Pil Jae Maeng Seung-Ho Kim 《Journal of industrial microbiology & biotechnology》2009,36(3):417-426
A novel fibrinolytic enzyme (AJ) was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography. Based on a 2D gel, AJ
was found to consist of three active isoforms (pI 5.5–6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5–3.0 and 85°C, respectively.
AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K
m) and K
cat values of AJ towards α-casein were 0.38 mM and 19.73 s−1, respectively. AJ cleaved the Aα-chain of fibrinogen but did not affect the Bβ- and γ-chains, indicating that it is an α-fibrinogenase.
The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly,
AJ was very stable at acidic condition, SDS, and heat (100°C), whereas it was easily degraded at neutral and alkaline conditions.
In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of
acid and heat stability has not yet been reported for other fibrinolytic enzymes. 相似文献