共查询到20条相似文献,搜索用时 78 毫秒
1.
Lignin peroxidase was purified (72-fold) from Acinetobacter calcoaceticus NCIM 2890. The purified lignin peroxidase (55–65 kDa) showed dimeric nature. The maximum enzyme activity was observed at
pH 1.0, between a broad temperature range of 50 and 70°C, at H2O2 concentration (40 mM) and the substrate concentration (n-propanol, 100 mM). Purified lignin peroxidase was able to oxidize a variety of substrates including Mn2+, tryptophan, mimosine, l-Dopa, hydroquinone, xylidine, n-propanol, veratryl alcohol, and ten textile dyes of various groups indicating as a versatile peroxidase. Most of the dyes
decolorized up to 90%. Tryptophan stabilizes the lignin peroxidase activity during decolorization of dyes. 相似文献
2.
Production of chitinolytic enzymes with Trichoderma longibrachiatum IMI 92027 in solid substrate fermentation 总被引:1,自引:0,他引:1
Kovacs K Szakacs G Pusztahelyi T Pandey A 《Applied biochemistry and biotechnology》2004,118(1-3):189-204
Thirty Trichoderma strains representing 15 species within the genus were screened for extracellular production of chitinolytic enzymes in solid
substrate fermentation. Trichoderma longibrachiatum IMI 92027 (ATCC 36838) gave the highest yield (5.0 IU/g of dry matter of substrate) after 3 d of fermentation on wheat bran-crude
chitin (9:1 mixture) medium. The optimal moisture content (66.7%), chitin content (20%), initial pH of the medium (2.0–5.0),
and time course (5 d) of solid substrate fermentation were determined for strain IMI 92027. Cellulase, xylanase, α-amylase,
and β-xylosidase activities were also detected. The pH and temperature optima of the chitinase complex of T. longibrachiatum IMI 92027 were 4.5 and 55°C, respectively. The enzyme totally lost its activity at 70°C in 5 min in the absence of the substrate
but retained about 15% of its initial activity even at 70°C after a 60-min incubation in the presence of solid substrate fermentation
solids. Purification of protein extract from the solid substrate fermentation material revealed high chitinolytic activities
between pI 5.9 and 4.8, where N-acetyl-β-d-hexosaminidase and chitinase peaks have been found in the same pI range. Two chitinases of 43.5 and 30 kDa were purified at acidic pI. 相似文献
3.
A 56.56-kDa extracellular chitinase from Paenibacillus sp. D1 was purified to 52.3-fold by ion exchange chromatography using SP Sepharose. Maximum enzyme activity was recorded
at pH 5.0 and 50 °C. MALDI-LC-MS/MS analysis identified the purified enzyme as chitinase with 60% similarity to chitinase
Chi55 of Paenibacillus ehimensis. The activation energy (E
a) for chitin hydrolysis and temperature quotient (Q
10) at optimum temperature was found to be 19.14 kJ/mol and 1.25, respectively. Determination of kinetic constants k
m, V
max, k
cat, and k
cat/k
m and thermodynamic parameters ΔH*, ΔS*, ΔG*, ΔG*E–S, and ΔG*E–T revealed high affinity of the enzyme for chitin. The enzyme exhibited higher stability in presence of commonly used protectant
fungicides Captan, Carbendazim, and Mancozeb compared to control as reflected from the t
1/2 values suggesting its applicability in integrated pest management for control of soil-borne fungal phytopathogens. The order
of stability of chitinase in presence of fungicides at 80 °C as revealed from t
1/2 values and thermodynamic parameters E
a(d) (activation energy for irreversible deactivation), ΔH*, ΔG*, and ΔS* was: Captan > Carbendazim > Mancozeb > control. The present study is the first report on thermodynamic and kinetic characterization
of chitinase from Paenibacillus sp. D1. 相似文献
4.
New Thermostable Amylase from <Emphasis Type="Italic">Bacillus cohnii</Emphasis> US147 with a Broad pH Applicability 总被引:1,自引:0,他引:1
Ghorbel RE Maktouf S Massoud EB Bejar S Chaabouni SE 《Applied biochemistry and biotechnology》2009,157(1):50-60
A new thermophilic bacterial strain identified as Bacillus cohnii US147 was isolated from the southern Tunisian soil. The identification was based on physiological tests and molecular techniques
related to the 16S ribosomal ribonucleic acid. The isolated strain produced amylase, which was purified. This amylase had
an apparent molecular mass of 30 kDa as estimated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Amylase
US147 showed K
m and V
max values of 0.7 mg/ml and 2.2 U/ml, respectively, with starch as the substrate. The enzyme was active in acid and basic pH
and had a maximal activity on starch at pH 9 and 70 °C. The enzyme was stable at pH 9 for 72 h and retained half of its activity
after incubation at 70 °C for 150 min. A partially inhibition (15%, 25%, 23%, 20%, and 22%) was obtained with 1 mM SDS, 1 mM
NaBO3, 1 mM H2O2, 1 mM Zn+2, and 5 mM ethylenediamine tetraacetic acid (EDTA), respectively. The amylase recovered its original activity by the addition
of 10 mM Ca 2+ to the 5 mM EDTA. These properties indicated a possible use of this amylase in starch saccharification, in detergent, and
in other industrial applications. 相似文献
5.
An extracellular thermostable α-galactosidase producing Aspergillus terreus
GR strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by
acetone precipitation, gel filtration followed by DEAE-Sephacel chromatographic step. The purified enzyme showed a single
band after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme
after SDS-PAGE was 108 kDa. The enzyme showed optimum pH and temperature of 5.0 and 65 °C, respectively, for artificial substrate
pNPαGal. α-Galactosidase from A. terreus
GR is found to be thermostable, as it was not inactivated after heating at 65 °C for 40 min. The K
m for pNPαGal, oNPαGal, raffinose, and stachyose are 0.1, 0.28, 0.42, and 0.33 mM, respectively. Inhibitors such as 1,10-phenanthroline,
phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, mercaptoethanol, and urea have no effect, whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg2+, Ni2+, Ca2+, Co2+, and Mn2+ had no effect on enzyme activity, but Ag+, Hg2+, and Cu2+ have inhibited complete activity. 相似文献
6.
Characterization of Thermo-stable Endoinulinase from a New Strain <Emphasis Type="Italic">Bacillus Smithii</Emphasis> T7 总被引:1,自引:0,他引:1
A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium
containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH4)H2PO4 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract
of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum
temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively.
Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate
the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application. 相似文献
7.
Mohamed Amine Aounallah Imen Ben Slimene-Debez Kais Djebali Dorra Gharbi Majdi Hammami Sana Azaiez Ferid Limam Olfa Tabbene 《Applied biochemistry and biotechnology》2017,181(2):650-666
A strain producing chitinase, isolated from potato stem tissue, was identified as Bacillus licheniformis by biochemical properties and 16S RNA sequence analysis. Statistical experimental designs were used to optimize nine independent variables for chitinase production by B. licheniformis AT6 strain in submerged fermentation. Using Plackett–Burman design, (NH4)2SO4, MgSO4.7H2O, colloidal chitin, MnCl2 2H2O, and temperature were found to influence chitinase production significantly. According to Box–Behnken response surface methodology, the optimal fermentation conditions allowing maximum chitinase production were (in gram per liter): (NH4)2SO4, 7; K2HPO4, 1; NaCl, 1; MgSO4.7H2O, 0.1; yeast extract, 0.5; colloidal chitin, 7.5; MnCl2.2H2O, 0.2; temperature 35 °C; pH medium 7. The optimization strategy led to a 10-fold increase in chitinase activity (505.26 ± 22.223 mU/mL versus 50.35 ± 19.62 mU/mL for control basal medium). A major protein band with a molecular weight of 61.9 kDa corresponding to chitinase activity was clearly detected under optimized conditions. Chitinase activity produced in optimized medium mainly releases N-acetyl glucosamine (GlcNAc) monomer from colloidal chitin. This enzyme also acts as an exochitinase with β-N-acetylglucosaminidase. These results suggest that B. licheniformis AT6 secreting exochitinase is highly efficient in GlcNAc production which could in turn be envisaged as a therapeutic agent or as a conservator against the alteration of several ailments. 相似文献
8.
An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin
A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the
purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and
native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of
about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa,
thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing,
with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature
for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively.
The inulin hydrolysis activity was completely abolished with 1 mM Hg++, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower
K
m
(0.25 mM) and higher V
max (333.3 IU/mg) values for inulin. 相似文献
9.
Sandhya C Binod P Nampoothiri KM Szakacs G Pandey A 《Applied biochemistry and biotechnology》2005,127(1):1-15
Antifungal activity of chitinase can be effectively utilized in biologic pest control strategies. Because solid-state cultivation
has been termed a cost-effective means for fungal growth and metabolite production, chitinase production by Trichoderma harzianum was studied using wheat bran-based solid medium containing 1% colloidal chitin. Chitinase synthesis was found to be growth
associated because maximum enzyme (5.4 U/g of dry substrate) and biomass production occurred at 72h. Substrate moisture had
a critical impact on chitinase production; five grams of medium having an initial moisture content of 68.4% when incubated
for 72 h increased the enzyme yield to 9.3 U/g of dry substrate. Optimization of colloidal chitin concentration showed that
improvements in chitinase yield and maximum activity were attained with a 2% (w/w) concentration. Supplementation of additional
nitrogen sources also influenced enzyme production, and the best yield was obtained with yeast extract. The effect of crude
chitinase on hyphal morphology of the phytopathogenic fungus Collelotrichum gloeosporioides was swelling as well as lysis of hyphal wall, depending on the age of the mycelium. Studies of pH and thermal stability showed
that crude culture filtrate was active over pH 4.0–6.0 and retained about 48.2% activity after 40 min of incubation at 40°C. 相似文献
10.
Mohamed A. Abdel-Naby Nefisa M. A. El-Shayeb A. A. Sherief 《Applied biochemistry and biotechnology》1992,37(2):141-154
An extracellular chitinase fromAspergillus cerneus was purified by ammonium sulphate precipitation, gel filtration through Sephadex G-100, preparative HPLC chromatography and large slabs of polyacry-lamide gel electrophoresis.The mol wt of the enzyme was estimated to be 25000 by SDS gel electrophoresis, and it contained 9.37% (w/w) carbohydrate residue, as glucose. The pattern of its amino acid composition showed high contents of asparagine, serine, and threonine. The enzyme was active at pH 5.2 and 50°C. The Km value of the enzyme was 4.37 mM (expressed asN-acetylglucosamine). The enzyme was stable at pH 3–9, whereas it was unstable at 70°C or more. Calcium and Mg ions slightly activated the enzyme, whereas Hg2+, I2, andp-chloromercuribenzoate inhibited the enzyme activity. The enzyme hydrolyzed chitin, colloidal chitin, glycol chitin, and chitooligsac-charides, but did not hydrolyze chitosan, starch, xylan, inulin, and cellulose. The lysis ofA. niger and Micorcoccus lysodeikticus cell walls by the action of the enzyme was also investigated. 相似文献
11.
Maalej I Belhaj I Masmoudi NF Belghith H 《Applied biochemistry and biotechnology》2009,158(1):200-212
A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl
cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific
activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25kDa by sodium dodecyl sulfate–polyacrylamide
gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative
activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature
of the purified enzyme was 75°C. The enzyme showed high thermal stability at 50°C (7days) and the half-life of the xylanase
at 100°C was 60min. The enzyme was free from cellulase activity. K
m and V
max values at 50°C of the purified enzyme for birchwood xylan were 22.51mg/ml and 1.235μmol min−1 mg−1, respectively. The enzyme was activated by Ag+, Co2+, and Cu2+; on the other hand, Hg2+, Ba2+, and Mn2+ inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and
highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries. 相似文献
12.
Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE)
(40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with
HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted
up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to
28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed
to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized
on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at
80.0 °C. The immobilized xylanase registered marginal increase and decrease in K
m and V
max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis
activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides
from birchwood xylan, indicating its potential in the nutraceutical industry. 相似文献
13.
A novel β-galactosidase of 120 kDa (BgaBM) from Bacillus megaterium 2-37-4-1 was purified, and its gene (bgaBM) was analyzed and expressed. It displayed wide acceptor specificity for transglycosylation with a series of acceptors, including
pentose, hexose, hydroxyl, and alkyl alcohol using o-nitrophenyl-β-d-galactoside (ONPG) as a donor. BgaBM preferentially hydrolyzed ONPG in all tested substrates and showed maximum activity
at pH 7.5–8.0 and 55 °C. It was stable at pH 6.0–9.0 below 40 °C. The K
m and V
max values for ONPG and lactose were 9.5 mM, 16.6 mM/min and 12.6 mM, 54.4 mM/min, respectively. The nucleotide sequence of the
bgaBM gene consists of an ORF of 3,105 bp corresponding to 118 kDa protein, which indicates that BgaBM is a modular enzyme in the
glycosyl hydrolase family 2, including conserved sugar-binding domain, acid–base catalyst, and immunoglobulin-like beta-sandwich
domain. The possible acid/base and nucleophile sites of BgaBM were estimated to be E481 and E547, respectively. Furthermore,
expression of the bgaBM gene in Escherichia coli and purification of the recombinant enzyme were performed. The recombinant enzyme showed similar biochemical characteristics
to natural enzyme. 相似文献
14.
Zhang Y Ji C Zhang X Yang Z Peng J Qiu R Xie Y Mao Y 《Applied biochemistry and biotechnology》2008,151(1):81-92
A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (<35% identity), while it exhibits the
conservation of the active site and structure element of Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 °C, respectively.
The enzyme retained a high activity at 45–60 °C, while it could be quickly inactivated by a heat treatment at 80 °C for 15 min,
exhibiting a half-life of 8 min at 70 °C. The K
m and V
max for pNPP were determined to be 31.5 μM and 430 μM/min at optimal conditions. A divalent cation is essential, with a combination
of Mg2+ and Co2+ or Zn2+ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant
to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification. 相似文献
15.
Three laccase temperature isoforms were isolated and purified to homogeneity from the xerophyte plant species Cereus pterogonus. This catalytically active protein exhibited an apparent molecular mass of 137 kDa, 90 kDa, and 43 kDa. Under reducing conditions the enzyme yielded a subunit molecular mass of 43 kDa alone, suggesting that the enzyme is a multimer of its subunits. The enzyme exhibited an optimum pH of 10 with 2,6-dimethoxyphenol used as a substrate. The 137 and 90 kDa forms yielded optimum activity at 90°C; whereas the 43 kDa molecular form yielded optimum activity at 60°C. The enzyme kinetic constant Km remained closely similar for all three enzyme forms, whereas Vmax varied by 25 % overall. The catalytic activity remained above its t1/2 value in excess of the 30 min denaturation assay period at 60°C and 90°C. These high-temperature isoforms of the plant laccase enzyme with alkaline pH optima can find great industrial use. 相似文献
16.
Kouakou TH Kouadio YJ Kouamé P Waffo-Téguo P Décendit A Mérillon JM 《Applied biochemistry and biotechnology》2009,158(2):285-301
Polyphenol oxidases (PPOs) were isolated from cell suspensions of two cultivars of cotton (Gossypium hirsutum L.), and their biochemical characteristics were studied. PPO from Coker 312, an embryogenic cultivar, showed a highest affinity
to catechol 20 mM, and PPO from R405-2000, a nonembryogenic cultivar, showed a highest affinity to 4-methylcatechol 20 mM.
The optimal pH for PPO activity was 7.0 and 6.0 for Coker 312 and R405-2000, respectively. The enzyme had an optimal temperature
of 25 °C and was relatively stable at 20–30 °C. Reducing sodium metabisulfite, ascorbic acid, dithiothreitol, SnCl2, and FeCl3 markedly inhibited PPO activity, whereas its activity was highly enhanced by Mg2+, Ca2+, and Mn2+ and was moderately inhibited by Ba2+, Cu2+, and Zn2+. The analysis revealed a single band on the sodium dodecyl sulfate polyacrylamide gel electrophoresis which corresponded
to a molecular weight of 55 kDa for Coker 312 and 42 kDa for R405-2000. 相似文献
17.
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
18.
Biochemical Characterization of Raw-starch-digesting Alpha Amylase Purified from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> 总被引:1,自引:0,他引:1
Gangadharan D Nampoothiri KM Sivaramakrishnan S Pandey A 《Applied biochemistry and biotechnology》2009,158(3):653-662
Alpha amylase (E.C. 3.2.1.1) of Bacillus amyloliquefaciens produced by submerged fermentation was purified to near homogeneity by ion exchange chromatography. Through the process 38.6-fold
increase in purity with a specific activity of 72 U/mg proteins was obtained. The apparent molecular weight of the purified
enzyme was found to be 58 kDa by SDS-PAGE. The enzyme was relatively stable between pH 5.0–8.0 and temperature between 50
and 60°C. The enzyme did not show any obligate requirement of metal ions but Ca2+ and Cu2+ enhanced the enzyme activity marginally and the thermostability was enhanced in the presence of Ca2+ ions. The purified enzyme exhibited maximal substrate specificity for amylose and efficiency in digesting various raw starches.
The K
m and V
max of the enzyme was determined using both amylose and soluble starch as substrate. The analysis of the hydrolyzed products
of soluble starch by thin layer chromatography showed the yield of maltosaccharides after 6 h of hydrolysis. 相似文献
19.
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
20.
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