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1.
Laila Manni Olfa Ghorbel-Bellaaj Kemel Jellouli Islem Younes Moncef Nasri 《Applied biochemistry and biotechnology》2010,162(2):345-357
Chitin is a polysaccharide found in abundance in the shell of crustaceans. In this study, the protease from Bacillus cereus SV1 was applied for chitin extraction from shrimp waste material of Metapenaeus monoceros. A high level of deproteinization 88.8% ± 0.4 was recorded with an E/S ratio of 20. The demineralization was completely achieved
within 6 h at room temperature in HCl 1.25 M, and the residual content of calcium in chitin was below 0.01%. 13C CP/MAS-NMR spectral analysis of chitin prepared by the enzymatic deproteinization of shrimp wastes was found to be similar
to that obtained by alkaline treatment and to the commercial α-chitin. The degree of N-acetylation, calculated from the spectrum,
was 89.5%. Chitin obtained by treatment with crude protease from B. cereus was converted to chitosan by N-deacetylation, and the antibacterial activity of chitosan solution against different bacteria
was investigated. Results showed that chitosan solution at 50 mg/mL markedly inhibited the growth of most Gram-negative and
Gram-positive bacteria tested. Furthermore, the antioxidant potential of the protein hydrolysates obtained during enzymatic
isolation of chitin was evaluated using various in vitro assays. All the samples exerted remarkable antioxidant activities. These results suggest that enzymatic deproteinization
of the shrimp shell wastes, using B. cereus SV1 protease, could be applicable to the chitin production process. 相似文献
2.
Laila Manni Kemel Jellouli Olfa Ghorbel-Bellaaj Rym Agrebi Anissa Haddar Alya Sellami-Kamoun Moncef Nasri 《Applied biochemistry and biotechnology》2010,160(8):2308-2321
The current increase in amount of shrimp wastes produced by the shrimp industry has led to the need in finding new methods
for shrimp wastes disposal. In this study, an extracellular organic solvent- and oxidant-stable metalloprotease was produced
by Bacillus cereus SV1. Maximum protease activity (5,900 U/mL) was obtained when the strain was grown in medium containing 40 g/L shrimp wastes
powder as a sole carbon source. The optimum pH, optimum temperature, pH stability, and thermal stability of the crude enzyme
preparation were pH 8.0, 60 °C, pH 6–9.5, and <55 °C, respectively. The crude protease was extremely stable toward several
organic solvents. No loss of activity was observed even after 60 days of incubation at 30 °C in the presence of 50% (v/v) dimethyl sulfoxide and ethyl ether; the enzyme retained more than 70% of its original activity in the presence of ethanol
and N,N-dimethylformamide. The protease showed high stability toward anionic (SDS) and non-ionic (Tween 80, Tween 20, and Triton
X-100) surfactants. Interestingly, the activity of the enzyme was significantly enhanced by oxidizing agents. In addition,
the enzyme showed excellent compatibility with some commercial liquid detergents. The protease of B. cereus SV1, produced under the optimal culture conditions, was tested for shrimp waste deproteinization in the preparation of chitin.
The protein removal with a ratio E/S of 20 was about 88%. The novelties of the SV1 protease include its high stability to organic solvents and surfactants. These
unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition,
the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin. 相似文献
3.
Ali Nel-H Hmidet N Ghorbel-Bellaaj O Fakhfakh-Zouari N Bougatef A Nasri M 《Applied biochemistry and biotechnology》2011,164(7):1096-1110
Alkaline proteases from the viscera of the striped seabream (Lithognathus mormyrus) were extracted and characterized. Interestingly, the crude enzyme was active over a wide range of pH from 6.0 to 11.0, with
an optimum pH at the range of 8.0–10.0. In addition, the crude protease was stable over a broad pH range (5.0–12.0). The optimum
temperature for enzyme activity was 50 °C. The crude alkaline proteases showed stability towards various surfactants and bleach
agents and compatibility with some commercial detergents. It was stable towards several organic solvents and retained more
than 50% of its original activity after 30 days of incubation at 30 °C in the presence of 25% (v/v) dimethyl sulfoxide, N,N-dimethylformamide, diethyl ether, and hexane. The crude enzyme extract was also tested for shrimp waste deproteinization
in the preparation of chitin. The protein removal with a ratio enzyme/substrate of 10 was about 79%. 相似文献
4.
Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified
enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima
at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from
40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0
for 24 h. The apparent K
m and V
max of Chi72 for colloidal chitin were 0.23 mg ml−1 and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the
recycling of chitin waste. 相似文献
5.
Byproducts generated in high levels by marine processes have been recognized for their value as recyclable or reclaimable waste. Among marine byproducts, shrimp shells, crab shells, and squid pens have the highest chitin content. The chemical treatments of these chitin-containing byproducts for preparing chitin and chitosan create waste disposal problems because neutralization and detoxification of the discharged wastewater are necessary. Therefore, the cost of chitin and chitosan preparations was far higher than those of their raw materials, marine chitin-containing byproducts. Chitin and chitosan have been widely used as the major carbon source of bacteria for producing chitinolytic enzymes. In 1997, the bifunctional chitinase/lysozymes from Pseudomonas aeruginosa K-187 using shrimp and crab shells as the sole carbon/nitrogen (C/N) source was first reported. Thereafter, the use of squid pens as the only C/N source for producing enzymes and bioactive materials had also been studied. The use of shellfish chitin waste as the sole C/N source not only solves environmental problems, it decreases the production costs for microbial conversion. This review summarizes our recent research of microbial reclamation of these marine byproducts for producing enzymes and bioactive materials; the characterization and applications of these products were also studied. 相似文献
6.
An exoinulinase has been isolated, purified and characterised from a commercially available broth of Aspergillus ficuum. The enzyme was purified 4.2-fold in a 21% yield with a specific activity of 12,300 U mg−1(protein) after dialysis, ammonium sulphate fractionation and Sephacryl S-200 size exclusion and ion exchange chromatography.
The molecular weight of this enzyme was estimated to be 63 kDa by SDS-PAGE. It exhibited a pH and temperature optima of 5.4
and 50 °C respectively and under such conditions the enzyme remained stable with 96% and 63.8% residual activity after incubation
for 12 h and 72 h respectively. The respective K
m and V
max values were 4.75 mM and 833.3 μmol min−1 ml−1, respectively. Response surface methodological statistical analysis was evaluated for the maximal production of fructose
from the hydrolysis of pure commercial chicory inulin. Incubation of the dialyzed crude exoinulinase (100 U/ml, 48 h, 50 °C,
150% inulin, pH 5.0) produced the highest amount of fructose (106.4 mg/ml) under static batch conditions. The purified exoinulinase
was evaluated for fructose production and the highest amount (98 mg/ml) was produced after 12 h incubation at 50 °C, 150%
inulin pH 5.0. The use of a crude exoinulinase preparation is economically desirable and the industrial production of fructose
from inulin hydrolysis is biotechnologically feasible. 相似文献
7.
Yun Wang Jin-Zhu Song Qian Yang Zhi-Hua Liu Xiao-Mei Huang Yan Chen 《Applied biochemistry and biotechnology》2010,162(3):843-854
A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans. Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum. The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The
chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated
an activity of 0.77 U ml−1 for the glycol chitin substrates, and its specific activity was 4.17 U mg−1 for it. The optimal temperature and pH of the purified enzyme were 50 °C and 8.0, respectively. When glycol chitin was used
as the substrate, K
m was 4.92 mg ml−1, and K
cat showed 6.25 s−1, thus the ratio of K
cat and K
m was 1.27 ml s−1 mg−1. The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid. 相似文献
8.
Nahed Fakhfakh-Zouari Noomen Hmidet Anissa Haddar Safia Kanoun Moncef Nasri 《Applied biochemistry and biotechnology》2010,162(2):329-344
A keratinolytic enzyme (KerA1) secreted by a newly isolated Bacillus pumilus strain A1 cultivated in medium containing chicken feather meal was purified and characterized, and the gene was isolated
and sequenced. The molecular mass of the purified enzyme was estimated to be 34,000 Da by sodium dodecyl sulfate (SDS) polyacrylamide
gel electrophoresis and gel filtration. The optimum pH and temperature for the purified keratinase were 9.0 and 60 °C, respectively,
using keratin as a substrate. KerA1 showed a high stability towards nonionic surfactants. It was found to be relatively stable
toward the strong anionic surfactant (SDS). The deduced amino acid sequence of the keratinase KerA1 differs from both the
organic solvent tolerant protease of B. pumilus 115b and the dehairing protease of B. pumilus UN-31-C-42 by one and nine amino acids, respectively. These results suggest that this keratinase may be a useful alternative
and ecofriendly route for handling the abundant amount of waste feathers and for applications in detergent formulations. 相似文献
9.
Horticultural waste in wood chips form collected from a landscape company in Singapore was utilized as the substrate for the
production of cellulase and hemicellulase under solid-state fermentation by Trichoderma reesei RUT-C30. The effects of substrate pretreatment methods, substrate particle size, incubation temperature and time, initial
medium pH value, and moisture content on cellulase and hemicellulase production were investigated. Enzyme complex was obtained
at the optimal conditions. This enzyme mixture contained FPase (15.0 U/g substrate dry matter, SDM), CMCase (90.5 U/g SDM),
β-glucosidase (61.6 U/g SDM), xylanase (52.1 U/g SDM), and β-xylosidase (10.4 U/g SDM). The soluble protein concentration
in the enzyme complex was 26.1 mg/g SDM. The potential of the crude enzyme complex produced was demonstrated by the hydrolysis
of wood chips, wood dust, palm oil fiber, and waste newspaper. The performance of the crude enzyme complex was better than
the commercial enzyme blend. 相似文献
10.
An-hua Li Mei-ying Xu Wei Sun Guo-ping Sun 《Applied biochemistry and biotechnology》2011,163(5):600-611
A rhamnolipid production strain newly isolated from oil-contaminated soil was identified as Pseudomonas aeruginosa GIM32 by its morphology and 16S rDNA sequence analysis. The effect of carbon source and carbon to nitrogen (C/N) ratio on
rhamnolipids production was investigated. Palm oil was favorable as a carbon source for rhamnolipid production. The maximum
biomass and rhamnolipid concentration were 8.24 g/L and 30.4 g/L, respectively, with an optimization medium containing 50 g/L
palm oil and 5 g/L sodium nitrate. Molasses distillery wastewater as an unconventional substrate for rhamnolipid production
was investigated. It was found that 2.6 g/L of rhamnolipids was produced; this amount was higher than that of past reports
using wastewater as a substrate. In addition, 44% of the chemical oxygen demand of wastewater was removed at the same time
under the optimization condition. Eleven kinds of different molecular weight rhamnolipid homologues were identified in the
rhamnolipids obtained from molasses distillery wastewater by P. aeruginosa GIM32 by LC–MS analysis. 相似文献
11.
Background
The alkaline protease from Pseudomonas aeruginosa(AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region. 相似文献12.
Fatemeh Moradian Khosro Khajeh Hossein Naderi-Manesh Majid Sadeghizadeh 《Applied biochemistry and biotechnology》2009,159(1):33-45
Bacillus sp. HR-08 screened from soil samples of Iran, is capable of producing proteolytic enzymes. 16S rDNA analysis showed that
this strain is closely related to Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, and Bacillus atrophaeus. The zymogram analysis of the crude extract revealed the presence of five extracellular proteases. One of the proteases was
purified in three steps procedure involving ammonium sulfate precipitation, DEAE-Sepharose ionic exchange and Sephacryl S-200
gel filtration chromatography. The molecular mass of the enzyme on SDS-PAGE was estimated to be 29 kDa. The protease exhibited
maximum activity at pH 10.0 and 60 °C and was inhibited by PMSF but it was not affected by cysteine inhibitors, suggesting
that the enzyme is a serine alkaline protease. Irreversible thermoinactivation of enzyme was examined at 50, 60, and 70 °C
in the presence of 10 mM CaCl2. Results showed that the protease activity retains more than 80% and 50% of its initial activity after incubation for 30 min
at 60 and 70 °C, respectively. This enzyme had good stability in the presence of H2O2, nonionic surfactant, and local detergents and its activity was enhanced in the presence of 20% of dimethyl sulfoxide (DMSO),
dimethyl formamide (DMF) and isopropanol. The enzyme retained more than 90% of its initial activity after pre-incubation 1 h
at room temperature in the presence of 20% of these solvents. Also, activation can be seen for the enzyme at high concentration
(50%, v/v) of DMF and DMSO. 相似文献
13.
Wanwisa Moon-ai Ploypat Niyomploy Ruethairat Boonsombat Polkit Sangvanich Aphichart Karnchanatat 《Applied biochemistry and biotechnology》2012,166(8):2138-2155
Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of
the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has
long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested
for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate
precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall
SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa,
as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C.
With nitroblue tetrazolium and riboflavin as substrates, the K
m values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V
max values of 333.7 ± 0.034 and 254.1 ± 0.022 μmol min−1 mg protein−1. This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen
peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn2+and Fe2+ ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production
in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC50 = 14.36 ± 0.15 μg protein/ml). 相似文献
14.
Chuan-Lu Wang Cheng-Jung Chen Anh Dzung Nguyen Tzu-Wen Liang Yawo-Kuo Twu Shin-Yi Huang San-Lang Wang 《Research on Chemical Intermediates》2014,40(6):2363-2369
We describe the use of chitinous materials as adsorbents for purification of protease and chitosanase from bromelain solution and chitosanase from culture supernatants of three bacterial strains: Serratia marcescens TKU011, Bacillus cereus TKU022 and Acinetobacter calcoaceticus TKU024. The best adsorption results were observed when crude shrimp shell chitin (CSSC) and 750-nm chitin nanoparticles (CNP) were used. The optimum temperatures for protease adsorption from bromelain solution (22.9 mg/mL) by CSSC (0.1 g) and by 750-nm CNP (0.1 g) were 4 and 25 °C, respectively. The purification folds of bromelain by CSSC and 750-nm CNP were 5.2 and 4.5, respectively. For purification of protease from culture supernatants of TKU011, 750-nm CNP was 4.0-fold better than CSSC. However, CSSC exhibited purification folds of 2.9 and 3.3 for the chitosanases from TKU022 and TKU024, respectively. The adsorbed chitinolytic enzymes TKU015 chitinase (30 kDa) and TKU024 chitosanase (27 kDa) exhibited high purity by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Thus, CSSC and 750-nm CNP indicate potential for use as tools for one-step purification of bacterial chitinolytic enzymes from culture supernatants. 相似文献
15.
Aran H-Kittikun Poonsuk Prasertsan Wolfgang Zimmermann Phisit Seesuriyachan Thanongsak Chaiyaso 《Applied biochemistry and biotechnology》2012,166(8):1969-1982
The extracellular lipase from Streptomyces thermocarboxydus ME168 was purified to 9.5-fold with 20% yield, following concentration by acetone precipitation, ion exchange chromatography
(Resource Q) and gel filtration chromatography (Superdex 200), respectively. The purified enzyme had an apparent molecular
mass of 21 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of the lipase
was ASDFDDQILG and was different from most other reported lipase. The enzyme showed maximum activity at 50 °C with the half-life
of 180 min at 65 °C. It showed high stability at a broad pH range of 5.5–9.5 and was thermostable at the temperature range
of 25–60 °C. The K
m
and V
max were 0.28 mM and 1,428 U/mg, respectively, using p-nitrophenyl palmitate as substrate. It was active toward p-nitrophenyl ester with medium to long acyl chain (C8–C16). Lipase activity was inhibited by Zn2+, dithiothreitol (DTT), EDTA and some organic solvents, e.g., ethanol, acetone, dioxane, acetronitrile, tert-butanol and pyridine. Immobilized crude lipase of S. thermocarboxydus ME168 on celite could be used to synthesize sugar esters from glucose and vinyl acetate, vinyl butyrate or vinyl caproate
in tert-butanol:pyridine (55:45 v/v) at 45 °C with conversion yields of 93, 67 and 55%, respectively. 相似文献
16.
Rym Agrebi Noomen Hmidet Mohamed Hajji Nawrez Ktari Anissa Haddar Nahed Fakhfakh-Zouari Moncef Nasri 《Applied biochemistry and biotechnology》2010,162(1):75-88
In this study, Mirabilis jalapa tuber powder (MJTP) was used as a new complex organic substrate for the growth and production of fibrinolytic enzymes by
a newly isolated Bacillus amyloliquefaciens An6. Maximum protease activity (1,057 U/ml) with casein as a substrate was obtained when the strain was grown in medium containing
(grams per liter) MJTP 30, yeast extract 6, CaCl2 1, K2HPO4 0.1, and K2HPO4 0.1. The strain was also found to grow and produce extracellular proteases in a medium containing only MJTP, indicating that
it can obtain its carbon, nitrogen, and salts requirements directly from MJTP. The B. amyloliquefaciens An6 fibrinase (BAF1) was partially purified, and fibrinolytic activity was assayed in a test tube with an artificial fibrin
clot. The molecular weight of the partially purified BAF1 fibrinolytic protease was estimated to be 30 kDa by sodium dodecyl
sulfate polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for the caseinolytic activity
were 60 °C and 9.0, respectively. The enzyme was highly stable from pH 6.0 to 11.0 and retained 62% of its initial activity
after 1 h incubation at 50 °C. However, the enzyme was inactivated at higher temperatures. The activity of the enzyme was
totally lost in the presence of phenylmethylsulfonyl fluoride, suggesting that BAF1 is a serine protease. 相似文献
17.
Horticultural waste collected from a landscape company in Singapore was utilized as the substrate for the production of laccase
under solid-state fermentation by Trametes versicolor. The effects of substrate particle size, types of inducers, incubation temperature and time, initial medium pH value, and
moisture content on laccase production were investigated. The optimum productivity of laccase (8.6 U/g substrate) was achieved
by employing horticultural waste of particle size greater than 500 μm and using veratryl alcohol as the inducer. The culture
was at 30 °C for 7 days at moisture content of solid substrate of 85% and initial pH 7.0. The decolorization was also investigated
in order to assess the degrading capability of the ligninolytic laccase obtained in the above-mentioned cultures. The decolorization
degree of a model dye, phenol red, was around 41.79% in 72 h of incubation. By far, this is the first report on the optimization
of laccase production by T. versicolor under solid-state fermentation using horticultural waste as the substrate. 相似文献
18.
Silva MF Freire DM de Castro AM Di Luccio M Mazutti MA Oliveira JV Treichel H de Oliveira D 《Applied biochemistry and biotechnology》2011,164(6):755-766
One relevant limitation hindering the industrial application of microbial lipases has been attributed to their production
cost, which is determined by the production yield, enzyme stability among other. The objective of this work was to evaluate
the concentration and immobilization of lipase extracts from Penicillium brevicompactum obtained by solid-state fermentation of babassu cake and castor bean cake. The precipitation with ammonium sulfate 60% of
saturation of crude extract obtained with babassu cake as raw material showed an enhancement in hydrolytic and esterification
activities from 31.82 to 227.57 U/g and from 170.92 to 207.40 U/g, respectively. Concentrated lipase extracts showed preference
to medium-chain triglycerides and fatty acids. It is shown that the enzyme activity is maintained during storage at low temperatures
(4 and −10°C) for up to 30 days. Higher esterification activities were achieved when the lipase extract was immobilized in
sodium alginate and activated coal. 相似文献
19.
Daniela de Araújo Viana Carolina de Albuquerque Lima Rejane Pereira Neves Cristina Souza Mota Keila Aparecida Moreira José Luiz de Lima-Filho Maria Taciana Holanda Cavalcanti Attilio Converti Ana Lúcia Figueiredo Porto 《Applied biochemistry and biotechnology》2010,162(3):830-842
Cow raw milk from dairy cooperatives was examined for its microbial composition. Among the isolates identified, 17.6% were
yeasts. The most frequent genus was Candida, although members belonging to the genera Brettanomyces, Dekkera, and Geotricum were also identified. Although qualitative and quantitative tests for extracellular proteolytic activity were positive for
all the species isolated, Candida buinensis showed the highest response (23.5 U/mg); therefore, it was selected for subsequent investigation. The results of fermentations
carried out at variable temperature, pH, and soybean flour concentration, according to a 23 full factorial design, demonstrated that this yeast ensured the highest production of extracellular proteases (573 U/mL)
when cultivated at 35 °C, pH 6.5, and using soybean flour concentrations in the range 0.1–0.5% (w/v). The cell-free supernatants showed the highest activity at 25 °C and pH 7.0, and satisfactory stability in the ranges 25–30 °C
and pH 7–9. The first-order rate constants of protease inactivation in the cell-free supernatants were calculated at different
temperatures from semi-log plots of the residual activity versus time and then used in Arrhenius and Eyring plots to estimate
the main thermodynamic parameters of thermoinactivation (E* = 40.0 kJ/mol; ΔH* = 37.3 kJ/mol; ΔS* = −197.5 J/mol K; ΔG* = 101 kJ/mol). 相似文献
20.
Yao Guo Fengxia Lu Haizhen Zhao Yanchong Tang Zhaoxin Lu 《Applied biochemistry and biotechnology》2010,162(2):498-509
A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of
605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the
AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around
94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions
after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum
temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below
40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting
revealed that rGOD exhibited a K
m value of 16.95 mM and a K
cat value of 484.26 s−1. 相似文献