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1.
A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA
fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A
had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50–55 °C. The recombinant endoglucanase
Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding
domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262–319 and 448–473,
which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.
Received: 14 January 1999 / Received revision: 29 March 1999 / Accepted: 6 April 1999 相似文献
2.
A recombinant Escherichia coli clone expressing an endoglucanase was identified from a genomic library of the halophilic bacterium Halomonas sp. S66-4, and the enzyme was designated Cel8H. The cel8H gene consisted of 1,053 bp and encoded 350 amino acids sharing the highest identity of 48% to other known endoglucanases.
The protein was expressed in E. coli BL21 (DE3) and purified to homogeneity. The purified recombinant enzyme had an optimal activity of 4.9 U/mg at pH 5 and 45°C
toward the substrate carboxymethylcellulose. It exhibited extraordinary properties which differed from endoglucanases reported
previously at the point of high salt tolerance above 5 M, simultaneously with high pH stability at pH 4–12 and high temperature
stability at 40–60°C. Various substrate tests indicated that the enzyme hydrolyzes β-1,4-glucosidic bonds specifically. 相似文献
3.
Although cellulases have been isolated from various microorganisms, no functional cellulase gene has been reported in the
Vibrio genus until now. In this report, a novel endo-β-1,4-glucanase gene, cel5A, 1,362 bp in length, was cloned from a newly isolated bacterium, Vibrio sp. G21. The deduced protein of cel5A contains a catalytic domain of glycosyl hydrolase family 5 (GH5), followed by a cellulose binding domain (CBM2). The GH5
domain shows the highest sequence similarity (69%) to the bifunctional beta 1,4-endoglucanase/cellobiohydrolase from Teredinibacter turnerae T7902. The mature Cel5A enzyme was overexpressed in Escherichia coli and purified to homogeneity. The optimal pH and temperature of the recombinant enzyme were determined to be 6.5–7.5 and 50°C,
respectively. Cel5A was stable over a wide range of pH and retained more than 90% of total activity even after treatment in
pH 5.5–10.5 for 1 h, indicating high alkali resistance. Moreover, the enzyme was activated after pretreatment with mild alkali,
a novel characteristic that has not been previously reported in other cellulases. Cel5A also showed a high level of salt tolerance.
Its activity rose to 1.6-fold in 0.5 M NaCl and remained elevated even in 4 M NaCl. Further experimentation demonstrated that
the thermostability of Cel5A was improved in 0.4 M NaCl. In addition, Cel5A showed specific activity towards β-1,4-linkage
of amorphous region of lignocellulose, and the main final hydrolysis product of carboxymethylcellulose sodium and cellooligosaccharides
was cellobiose. As an alkali-activated and salt-tolerant enzyme, Cel5A is an ideal candidate for further research and industrial
applications. 相似文献
4.
The genes encoding the catalytic domains (CD) of the three endoglucanases (EG I; Cel7B, EG II; Cel5A, and EG III; Cel12A)
from Trichoderma reesei QM9414 were expressed in Escherichia coli strains Rosetta-gami B (DE3) pLacI or Origami B (DE3) pLacI and were found to produce functional intracellular proteins.
Protein production by the three endoglucanase transformants was evaluated as a function of growth temperature. Maximal productivity
of EG I-CD at 15°C, EG II-CD at 20°C and EG III at 37°C resulted in yields of 6.9, 72, and 50 mg/l, respectively. The endoglucanases
were purified using a simple purification method based on removing E. coli proteins by isoelectric point precipitation. Specific activity toward carboxymethyl cellulose was found to be 65, 49, and
15 U/mg for EG I-CD, EG II-CD, and EG III, respectively. EG II-CD was able to cleave 1,3–1,4-β- d-glucan and soluble cellulose derivatives. EG III was found to be active against cellulose, 1,3–1,4-β- d-glucan and xyloglucan, while EG I-CD was active against cellulose, 1,3–1,4-β- d-glucan, xyloglucan, xylan, and mannan. 相似文献
5.
The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 ( trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal
peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase
was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight
of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C
and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained
66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl
β- d-cellobiose, but no activity was detected against p-nitrophenyl β- d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography,
while enzyme did not hydrolyze cellobiose and cellotriose. 相似文献
6.
A metagenomic library consisting of 3,024 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B with high-molecular-weight DNA extracted from red soil in South China. A novel cellulase gene with an open reading
frame of 1,332 bp, cel5G, encoding an endo-β-1,4-glucanase was cloned using an activity-based screen. The deduced enzyme, Cel5G, belongs to the
glycosyl hydrolase family 5 and shares <39% identity with endoglucanases in the GenBank database. cel5G was expressed in E. coli BL21, and the recombinant enzyme Cel5G was purified to homogeneity for enzymatic analysis. Cel5G hydrolyzed a wide range
of β-1,4-, β-1,3/β-1,4-, or β-1,3/β-1,6-linked polysaccharides, amorphous cellulose, filter paper, and microcrystalline cellulose.
Its highest activity was in 50 mM citrate buffer, pH 4.8, at 50°C. Cel5G is stable over a wide range of pH values (from 2.0
to 10.6) and is thermally stable under 60°C. It is highly tolerant and active in high salt concentrations and is stable in
the presence of pepsin and pancreatin. The K
m and V
max values of Cel5G for carboxymethyl cellulose are 19.92 mg/ml and 1,941 μmol min −1 mg −1, respectively. These characteristics indicate that Cel5G has potential for industrial use. 相似文献
7.
A gene encoding an intracellular glucoamylase was identified in the genome of the extreme thermoacidophilic Archaeon Thermoplasma acidophilum. The gene taGA, consisting of 1,911 bp, was cloned and successfully expressed in Escherichia coli. The recombinant protein was purified 22-fold to homogeneity using heat treatment, anion-exchange chromatography, and gel
filtration. Detailed analysis shows that the glucoamylase, with a molecular weight of 66 kDa per subunit, is a homodimer in
its active state. Amylolytic activity was measured over a wide range of temperature (40–90°C) and pH (pH 3.5–7) and was maximal
at 75°C and at acidic condition (pH 5). The recombinant archaeal glucoamylase uses a variety of polysaccharides as substrate,
including glycogen and amylose. Maximal activity was measured towards amylopectin with a specific activity of 4.2 U/mg and
increased almost threefold in the presence of manganese. Calcium ions have a pronounced effect on enzyme stability; in the
presence of 5 mM CaCl 2, the half-life increased from 15 min to 2 h at 80°C. 相似文献
8.
An endo-β-1,4-xylanase gene, designated xyn10G5, was cloned from Phialophora sp. G5 and expressed in Pichia pastoris. The 1,197-bp full-length gene encodes a polypeptide of 399 amino acids consisting of a putative signal peptide at residues
1–20, a family 10 glycoside hydrolase domain, a short Gly/Thr-rich linker and a family 1 carbohydrate-binding module (CBM).
The deduced amino acid sequence of XYN10G5 shares the highest identity (53.4%) with a putative xylanase precursor from Aspergillus terreus NIH2624. The purified recombinant XYN10G5 exhibited the optimal activity at pH 4.0 and 70 °C, remained stable at pH 3.0–9.0
(>70% of the maximal activity), and was highly thermostable at 70 °C (retaining ~90% of the initial activity for 1 h). Substrate
specificity studies have shown that XYN10G5 had the highest activity on soluble wheat arabinoxylan (350.6 U mg −1), and moderate activity to various heteroxylans, and low activity on different types of cellulosic substrates. Under simulated
gastric conditions, XYN10G5 was stable and released more reducing sugars from soluble wheat arabinoxylan; when combined with
a glucanase (CelA4), the viscosity of barley–soybean feed was significantly reduced. These favorable enzymatic properties
make XYN10G5 a good candidate for application in the animal feed industry. 相似文献
9.
Soil metagenomes represent an unlimited resource for the discovery of novel biocatalysts from soil microorganisms. Three large-inserts
metagenomic DNA libraries were constructed from different grassland soil samples and screened for genes conferring cellulase
or xylanase activity. Function-driven screening identified a novel cellulase-encoding gene ( cel01) and two xylanase-encoding genes ( xyn01 and xyn02). From sequence and protein domain analyses, Cel01 (831 amino acids) belongs to glycoside hydrolase family 9 whereas Xyn01
(170 amino acids) and Xyn02 (255 amino acids) are members of glycoside hydrolase family 11. Cel01 harbors a family 9 carbohydrate-binding
module, previously found only in xylanases. Both Xyn01 and Xyn02 were most active at 60°C with high activities from 4 to 10
and optimal at pH 7 (Xyn01) and pH 6 (Xyn02). The cellulase gene, cel01, was expressed in E. coli BL21 and the recombinant enzyme (91.9 kDa) was purified. Cel01 exhibited high activity with soluble cellulose substrates
containing β-1,4-linkages. Activity with microcrystalline cellulose was not detected. These data, together with the analysis
of the degradation profiles of carboxymethyl cellulose and barley glucan indicated that Cel01 is an endo 1,4-β-glucanase.
Cel01 showed optimal activity at 50°C and pH 7 being highly active from pH range 5 to 9 and possesses remarkable halotolerance. 相似文献
10.
A β-1,4-endoglucanase (Cel5A) was cloned from the genomic DNA of saccharolytic thermophilic eubacterium Thermoanaerobacter tengcongensis MB4 and functionally expressed in Escherichia coli. Substrate specificity analysis revealed that Cel5A cleaves specifically the β-1,4-glycosidic linkage in cellulose with high
activity (294 U mg −1; carboxymethyl cellulose sodium (CMC)). On CMC, kinetics of Cel5A was determined ( K
m 1.39 ± 0.12 g l −1; k
cat/ K
m 1.41 ± 0.13 g −1 s −1). Cel5A displays an activity optimum between 75 and 80 °C. Residues Glu187 and Glu289 were identified as key catalytic amino
acids by sequence alignment. Interestingly, derived from a non-halophilic bacterium, Cel5A exhibits high residual activities
in molar concentration of NaCl (3 M, 49.3%) and KCl (4 M, 48.6%). In 1 M NaCl, 82% of Cel5A activity is retained after 24
h incubation. Molecular Dynamics studies performed at 0 and 3 M NaCl, correlate the Cel5A stability to the formation of R-COO −···Na + ··· −OOC-R salt bridges within the Cel5A tertiary structure, while activity possibly relates to the number of Na + ions trapped into the negatively charged active site, involving a competition mechanism between substrate and Na +. Additionally, Cel5A is remarkably resistant in ionic liquids 1-butyl-3-methyllimidazolium chloride (1 M, 54.4%) and 1-allyl-3-methylimidazolium
chloride (1 M, 65.1%) which are promising solvents for cellulose degradation and making Cel5A an attractive candidate for
industrial applications. 相似文献
11.
Paenibacillus polymyxa GS01 secretes Cel44C-Man26A as a multifunctional enzyme with cellulase, xylanase, lichenase, and mannanase activities. Cel44C-Man26A
consists of 1,352 amino acids in which present a catalytic domain (CD) of the glycosyl hydrolase family 44 (GH44), fibronectin
domain type 3 (Fn3), catalytic domain of glycosyl hydrolase family 26 (GH26), and a cellulose-binding module type 3 (CBM3).
A truncated Cel44C-Man26A protein, consisting of 549 amino acid residues, reacted as a multifunctional mature enzyme despite
the absence of the 10 amino acids containing GH44, Fn3, GH26, and CBM3. However, the multifunctional activity was not found
in the mature Cel44C-Man26A protein truncated to less than 548 amino acids. The truncated Cel44C-Man26A proteins showed the
optimum pH for the lichenase activity was pH 7.0, pH 6.0 for the xylanase and mannanase, and pH 5.0 for the cellulase. The
truncated Cel44C-Man26A proteins exhibited enzymatic activity 40–120% higher than the full-length Cel44C. 相似文献
12.
The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi ( Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5′-untranslated region (UTR), 3′-UTR,
four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading
frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa.
The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species.
The recombinant protein was expressed in E. coli and detected by SDS–PAGE and western blot. GC–MS analysis showed that the wild-type GfSQS could catalyze the reaction from
farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that
the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate. 相似文献
13.
The gene encoding a family-57-like α-amylase in the hyperthermophilic archaeon Methanococcus jannaschii, has been cloned into Escherichia coli. Extremely thermoactive α-amylase was confirmed in partially purified enzyme solution of the recombinant culture. This enzyme
activity had a temperature optimum of 120°C and a pH optimum 5.0–8.0. The amylase activity is extremely stable against denaturants.
Hydrolysis of large sugar polymers with α-1–6 and α-1–4 linkages yields products including glucose polymers of 1–7 units.
Highest activity is exhibited on amylose. The catalyst exhibited a half-life of 50 h at 100°C, among the highest reported
thermostabilities of natural amylases. 相似文献
14.
An artificial bifunctional enzyme, cellulase-β-glucosidase, was prepared by gene fusion from the hyperthermophilic bacterium
Thermotoga maritima MSB8. The fusion protein exhibited both cellulase (Cel5C) and β-glucosidase (BglB) activity when the bglB gene was fused to downstream of cel5C, but not when cel5C was fused to downstream of bglB. The specific activity of the bifunctional enzyme was 70% lower than that of cellulase or β-glucosidase. The fusion enzyme
was purified, and the MW was estimated as 114 kDa. The fusion enzyme displayed optimum cellulase activity at pH 8.0 and 70°C
over 30 min, and optimal β-glucosidase activity at pH 7.0 and 80°C over 30 min. 相似文献
15.
Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959. 相似文献
16.
Homology of ars (arsenic-resistance system) genes was examined among the indigenous bacteria isolated from the soils and sediments of two
abandened Au mines, which are highly contaminated with arsenic. The DNA and amino acid sequence homology of the ars determinants were investigated using an ars genotype. The isolated showed As(III)-oxidation ability contained arsAB genes encoding the efflux pump as well as arsR and arsD regulator genes. The arsR and arsD leader gene are required for an arsenic resistance system when the high-homology genes ( arsR; pl258 52.09% and arsD; Shewanell sp. 42.33%) are controlled by the ars inducer-independent regulatory amino acid sequence. These leader gene were observed under weak acidic conditions in the Myoung-bong
(pH; 5.0 to 6.0) and Duck-um (pH; 4.0 to 7.0) mines In addition, the strains with the ability of As (V)-reduction involved
the arsC gene homologues, as in the strain CW-16 ( Pseudomonas putida). The arsenic-resistance genes in the isolated indigenous bacteria showed varying degrees of amino acid similarity to the
homologous genes found in the database (GenBank) such as P. putida KT2440: 39–53% for arsR, 22–42% for arsD, 16–84% for arsA, 26–45% for arsB, 17–44% for arsAB, 37–41% for arsC, and 14–47% for arsH. These findings suggested that the function of the various ars gene in indigenous bacteria existing in weakly oxidative conditions may be the key factor for redox mechanisms and biogeochemical
systems in arsenic contaminated soils. 相似文献
17.
A gene, aga-MJ11, encoding an α-galactosidase (EC 3.2.1.22) was cloned from Pedobacter nyackensis MJ11 CGMCC 2503, expressed in Escherichia coli, and biochemically characterized. The gene consisted of 2,163 nucleotides encoding a 720 amino acid–protein with a theoretical
molecular weight of 82.6 kDa. The deduced amino acid sequence of Aga-MJ11 shared the highest identity of 51% to an α-galactosidase
from Parabacteroides distasonis (YP_001301506), which belongs to glycoside hydrolase (GH) family 36. Purified recombinant Aga-MJ11-H showed optimal activity
at pH 5.5 and 40°C, was stable at pH 4.0–10.0, retained ~80% of the maximum activity at 30°C (the optimum temperature for
freshwater fish), exhibited tolerance to some proteases, and had a wide substrate specificity (pNPG, melidiose, stachyose
and raffinose). All these features make Aga-MJ11 potentially useful for applications in aquaculture. The enzyme studied in
the present work may represent a novel GH-36 α-galactosidase from the genus Pedobacter.
X. Liu and K. Meng contributed equally to this work. 相似文献
18.
A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI,
was linearized with SalI and transformed into Pichia pastoris GS115 ( his4, Mut +) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His +, Mut +). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration
of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′- AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular
weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that
(10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at
a broad pH range of 7.0–10.5 and at a temperature of 30°C or below. 相似文献
19.
A gene encoding a cellobiohydrolase (CBH) was isolated from Thermoascus aurantiacus IFO 9748 and designated as cbh1. The deduced amino acid sequence encoded by cbh1 showed high homology with the sequence of glycoside hydrolase family 7. To confirm the sequence of the gene encoding the CBH, the cloned gene was expressed in the yeast Saccharomyces cerevisiae, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The recombinant enzyme was confirmed as a CBH by analysis of the reaction product and designated as CBHI. Recombinant CBHI retained more than 80% of its initial activity after 1 h of incubation at 65 °C and was stable in the pH range 3.0–9.0. The optimal temperature for enzyme activity was about 65 °C and the optimal pH was about 6.0. The recombinant enzyme was found to be highly glycosylated and this glycosylation was shown to contribute to the thermostability of the enzyme. CBHI expression was shown to be induced at higher temperature in T. aurantiacus. 相似文献
20.
A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material. 相似文献
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