Ziduo Liu

Huazhong Agricultural University, Wu-han-shih, Hubei, China

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Publications (50)129.65 Total impact

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    ABSTRACT: A 1.020-bp esterase gene, estQ, encoding for a protein of 339 amino acids, was cloned from Aspergillus fumigatus and expressed in E. coli. EstQ exhibited the optimal activity around 40°C and pH 9.0. In order to obtain more thermostable esterases, three mutants (A134T, V160T, A134T-V160T) were constructed by site-directed mutagenesis and also characterized for further research. Compared to A134T and V160T displaying their optimum activity at 40°C, A134T-V160T exhibited a 5°C higher optimal temperature and a longer half-life more than 24 times than that of WT at 50°C. All the mutants displayed favorable effects on thermostability and retained 53-76% activity after pre-incubation for 30min at 45°C, about 20-40% higher than that of the WT. With an increase in Km of the three mutants, a decrease in catalytic efficiency in kcat/Km was observed in mutant V160T and A134T-V160T against p-nitrophenyl butyrate. Homology models of WT and A134T-V160T were built to understand the structure-function relationship. The analysis results showed that the improved thermostability may be due to the favorable interaction and additional hydrogen bonds formed in the mutants by substitution of hydrophobic residues with hydrophilic residues. This study provide useful theoretical reference for enzyme evolution in vitro.
    Enzyme and Microbial Technology 10/2014; 64-65:11-6. · 2.59 Impact Factor
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    ABSTRACT: Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry worldwide. Oxygen deprivation is a stress that A. pleuropneumoniae will encounter during both early infection and the later, persistent stage. To understand modulation of A. pleuropneumoniae gene expression in response to the stress caused by anaerobic conditions, gene expression profiles under anaerobic and aerobic conditions were compared in this study. The microarray results showed that 631 genes (27.7% of the total ORFs) were differentially expressed in anaerobic conditions. Many genes encoding proteins involved in glycolysis, carbon source uptake systems, pyruvate metabolism, fermentation and the electron respiration transport chain were up-regulated. These changes led to an increased amount of pyruvate, lactate, ethanol and acetate in the bacterial cells as confirmed by metabolite detection. Genes encoding proteins involved in cell surface structures, especially biofilm formation, peptidoglycan biosynthesis and lipopolysaccharide biosynthesis were up-regulated as well. Biofilm formation was significantly enhanced under anaerobic conditions. These results indicate that induction of central metabolism is important for basic survival of A. pleuropneumoniae after a shift to an anaerobic environment. Enhanced biofilm formation may contribute to the persistence of this pathogen in the damaged anaerobic host tissue and also in the early colonization stage. These discoveries give new insights into adaptation mechanisms of A. pleuropneumoniae in response to environmental stress.
    The Journal of Microbiology 04/2014; · 1.28 Impact Factor
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    ABSTRACT: A new xylanase gene (xynA) from the marine microorganism Zunongwangia profunda was identified to encode 374 amino acid residues. Its product (XynA) showed the highest identity (42.78 %) with a xylanase from Bacillus sp. SN5 among the characterized xylanases. XynA exhibited the highest activity at pH 6.5 and 30 °C, retaining 23 and 38 % of the optimal activity at 0 and 5 °C, respectively. XynA was not only cold active, but also halophilic, and both its activity and thermostability could be significantly increased by NaCl, showing the highest activity (180 % of the activity) at 3 M NaCl and retaining nearly 100 % activity at 5 M NaCl, compared to the absence of NaCl. In the presence of 3 M NaCl, the k cat/K m value of XynA exhibited a 3.41-fold increase for beechwood xylan compared to no added NaCl, and the residual activity of XynA increased from 23 % (no added NaCl) to 58 % after 1 h incubation at 45 °C. This may be the first report concerning a cold-adapted xylanase from a non-halophilic species that displays the highest activity at a NaCl concentration range from 3 to 5 M. The features of cold activity and salt tolerance suggest the potential application of XynA in the food industry and bioethanol production from marine seaweeds.
    Extremophiles 01/2014; · 2.20 Impact Factor
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    ABSTRACT: Phosphinothricin (PPT) is a kind of non-selective, environmentally friendly herbicide. PPT-tolerance genes are vital in both plant biotechnology as selectable markers and the development of transgenic herbicide-resistant crops. However, there are no other well-identified and commercially available PPT-resistance genes for use in plant genetic engineering besides two PPT N-acetyltransferase genes, which known as pat and bar derived from Streptomyces sp. Here, we isolated a novel PPT N-acetyltransferase gene from PPT-resistant marine bacteria, Rhodococcus sp. strain YM12. The gene, designated as RePAT, encoded a protein (RePAT) of 162 amino acids, which showed 37% identity with that of PAT proteins. Key kinetic constants of RePAT were determined (Km = 0.076 mM, Kcat= 131 min−1) using PPT as a substrate, the enzyme retained considerable activity at pH 8.0 and had an optimum temperature of 35 °C. Interestingly, it possessed over 50% of its maximal activity at temperature conditions between 0 and 10 °C, suggesting that this enzyme is able to protect crop against PPT injury in cold environment. These results illustrated that RePAT could be a new resource for herbicide detoxification by transgenic crops.
    Journal of Molecular Catalysis B Enzymatic 01/2014; · 2.82 Impact Factor
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    ABSTRACT: A novel gene (BmelA) (1323 bp) encoding an α-galactosidase of 440 amino acids was cloned from the deep-sea bacterium Bacillus megaterium and the protein was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of about 45 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 4, with the highest identity (74%) to α-galactosidase Mel4A from Bacillus halodurans among the characterized α-galactosidases. The recombinant BmelA displayed its maximum activity at 35 °C and pH 8.5-9.0 in 50 mM Tris-HCl buffer, and could hydrolyze different substrates with the Km values against p-nitrophenyl-α-D-galactopyranoside (pNP-α-Gal), raffinose and stachyose being 1.02 ± 0.02, 2.24 ± 0.11 and 3.42 ± 0.17 mM, respectively. Besides, 4 mutants (I38 V, I38A, I38F and Q84A) were obtained by site-directed mutagenesis based on molecular modeling and sequence alignment. The kinetic analysis indicated that mutants I38 V and I38A exhibited a 1.7- and 1.4-fold increase over the wild type enzyme in catalytic efficiency (kcat/Km) against pNP-α-Gal, respectively, while mutant I38F showed a 3.5-fold decrease against pNP-α-Gal and mutant Q84A almost completely lost its activity. All the results suggest that I38 and Q84 sites play a vital role in enzyme activity probably due to their steric and polar effects on the predicted “tunnel” structure and NAD+ binding to the enzyme.
    Enzyme and Microbial Technology 01/2014; · 2.59 Impact Factor
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    ABSTRACT: Although biodiesel is a sustainable and renewable diesel fuel, the current feedstock predominantly from edible oils limits the economic feasibility of biodiesel production and thus the development of a cost-effective non-food feedstock is really essential. In this study, approximately 21.6% of crude grease was extracted from housefly (Musca domestica L.) larvae reared on swine manure, and the extracted grease was evaluated for biodiesel production concerning the variables affecting the yield of acid-catalyzed production of methyl esters and the properties of the housefly larvae-based biodiesel. The optimized process of 8:1 methanol/grease (mol/mol) with 2 vol% H2SO4 reacted at 70 °C for 2 h resulted in a 95.7% conversion rate from free fatty acid (FFA) into methyl esters. A 90.3% conversion rate of triglycerides (crude grease) to its esters was obtained from alkaline trans-esterification using sodium hydroxide as catalyst. The major fatty acid components of this larvae grease were palmitic (29.1%), oleic (23.3%), palmitoletic (17.4%) and linoleic (17.2%). The housefly larvae-based biodiesel has reached the ASTM D6751-10 standard in density (881 kg/m3), viscosity (5.64 mm2/s), ester content (96.8%), flash point (145 °C), and cetane number (52). These findings suggest that the grease derived from swine manure-grown housefly larvae can be a feasible non-food feedstock for biodiesel production.
    Renewable Energy. 01/2014; 66:222–227.
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    ABSTRACT: The 5-enolpyruvylshikimate - 3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1,317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.
    Enzyme and Microbial Technology 01/2014; · 2.59 Impact Factor
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    ABSTRACT: A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25 %) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39 % activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93 % activity even at 4 M NaCl. The catalytic efficiency (k cat/K m) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg(-1) s(-1) (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl2. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.
    Extremophiles 12/2013; · 2.20 Impact Factor
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    ABSTRACT: A novel esterase gene, estB, was cloned from the marine microorganism Alcanivorax dieselolei B-5(T) and overexpressed in E. coli DE3 (BL21). The expressed protein EstB with a predicted molecular weight of 45.1 kDa had a distinct catalytic triad (Ser(211)-Trp(353)-Gln(385)) and the classical consensus motif conserved in most lipases and esterases Gly(209)-X-Ser(211)-X-Gly(213). EstB showed very low similarity to any known proteins and displayed the highest similarity to the hypothetical protein (46 %) from Rhodococcus jostii RHA1. EstB showed the optimal activity around pH 8.5 and 20 °C and was identified to be extremely cold-adaptative retaining more than 95 % activity between 0 and 10 °C. The values of kinetic parameters on p-NP caproate (K m, K cat and K cat/K m) were 0.15 mM, 0.54 × 10(3) s(-1) and 3.6 × 10(3) s(-1) mM(-1), respectively. In addition, EstB showed remarkable stability in several studied organic solvents and detergents of high concentrations with the retention of more than 70 % activity after treatment for 30 min. The cold activity and its tolerance towards organic solvents made it a promising biocatalyst for industrial applications under extreme conditions.
    Extremophiles 12/2013; · 2.20 Impact Factor
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    ABSTRACT: Endo-β-1, 4-xylanase was cloned from Geobacillus stearothermophilus 1A05583 by PCR. Enzymes with improved catalytic efficiency were obtained using error-prone PCR and a 96-well plate high-throughout screening system. Two variants 1-B8 and 2-H6 were screened from the mutant library containing 9,000 colonies, which, when compared with the wild-type enzyme increased the catalytic efficiency (kcat/Km) by 25% and 89%, respectively, acting on beechwood xylan. By sequencing 1-B8 and 2-H6, an identical mutation point H179Y was detected and found to overlap in the active site cleft. Following the introduction of the remaining 19 amino acids into position 179 by site-saturation mutagenesis, the catalytic efficiency of H179F was found to be 3.46-fold that of the wild-type. When Whistidine was substituted by tryptophan, arginine, methionine or proline, the enzyme lost activity. Therefore, the position 179 site may play an important role in regulating the catalytic efficiency.
    Journal of Biotechnology 10/2013; · 3.18 Impact Factor
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    ABSTRACT: A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The K m and k cat values of GSxyn for ρNPX were 0.48 mM and 36.64 s(-1), respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The K m and k cat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s(-1), respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The K m and k cat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s(-1), respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall.
    Journal of Industrial Microbiology 10/2013; · 1.80 Impact Factor
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    ABSTRACT: Pseudomonas plecoglossicida, a bacterium strain that exhibits high Serine hydroxymethyltransferase (SHMT) activity, was isolated from the seawater. A full-length glyA encoding SHMT was obtained by a modified thermal asymmetric interlaced-PCR (TRIL-PCR), which consisted of 1,254 bp, encoded a 417 amino acid polypeptide, and shared the highest identity (75 %) with a glyA gene from Acinetobacter radioresistens CMC-1. Recombinant glyA gene was expressed in Escherichia coli BL21 (DE3) and purified by electrophoretic homogeneity. The enzyme showed the optimal activity at pH 8.0 and 40 °C, and remained stable in high alkali conditions. Using SHMT to produce L-serine by catalyzing the reaction of glycine and tetrahydrofolate is one of the most promising routes to synthesize L-serine, achieving 33.4 mM L-serine at the 12th h of the enzymatic reaction with the substrates of glycine (133 mM) and formaldehyde (13.3 mM). The properties make the SHMT a candidate for further enzymatic studies and industrial applications.
    MIRCEN Journal of Applied Microbiology and Biotechnology 08/2013; · 1.08 Impact Factor
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    ABSTRACT: An esterase gene, est10, was identified from the genomic library of a deep-sea psychrotrophic bacterium Psychrobacter pacificensis. The esterase exhibited the optimal activity around 25 °C and pH 7.5, and maintained as high as 55.0 % of its maximum activity at 0 °C, indicating its cold adaptation. Est10 was fairly stable under room temperatures, retaining more than 80 % of its original activity after incubation at 40 °C for 2 h. The highest activity was observed against the short-chain substrate p-nitrophenyl butyrate (C4) among the tested p-nitrophenyl esters (C2-C16). It was slightly activated at a low concentration (1 mM) of Zn(2+), Mg(2+), Ba(2+), Ca(2+), Cu(2+), Fe(3+), urea and EDTA, but was inhibited by DTT and totally inactivated by PMSF. Interestingly, increased salinity considerably stimulated Est10 activity (up to 143.2 % of original activity at 2 M NaCl) and stability (up to 126.4 % after incubation with 5 M NaCl for 6.5 h), proving its salt tolerance. 0.05 and 0.1 % Tween 20, Tween 80, Triton X-100 and CHAPS increased the activity and stability of Est10 while SDS, CTAB had the opposite effect. Est10 was quite active after incubation with several 30 % organic solvents (methanol, DMSO, ethanediol) but exhibited little activity with 30 % isopropanol, ethanol, n-butanol and acetonitrile.
    Extremophiles 07/2013; · 2.20 Impact Factor
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    ABSTRACT: Pantoea agglomerans endo-β-1, 4-mannanase (Man26P) hydrolyzes hemicellulose by cleaving the internal bonds in the mannan-based chains. In order to obtain improved enzymes and understand the structure-function relationship better, Man26P was engineered using a modified DNA shuffling method and a 96-well plate high-throughput screening technology. Compared to the wild-type enzyme, two mutants Gly267Ser and His134Arg/Phe141Leu with 1.14- and 3.30-fold increased catalytic efficiency (kcat/Km) toward locust bean gum (LBG) were obtained from the mutant library containing 19,700 clones. Combined with site-directed mutagenesis, more substitutions were introduced into the 134 site, among which, His134Arg and His134Lys exhibited 2.81- and 2.75-fold increased catalytic efficiency versus the wild-type enzyme. And with an increase in the pKa values of the side chains of the mutated residues at the 134 site, a decrease was observed in their Km values against LBG. The molecular modeling and the docking analysis of the substrate suggest that the enhancement of polar contacts or positive charges at the 134 site contribute to the interaction of the enzyme with the substrate. Furthermore, the nature of the residue at the 267 site could also significantly affect the enzyme activity by regulating substrate binding affinity and product release. The study provides useful information for the directed evolution of mannanases.
    Journal of Biotechnology 07/2013; · 3.18 Impact Factor
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    ABSTRACT: LuxS, a conserved bacterial enzyme involved in the activated methyl cycle, catalyzes S-ribosylhomocysteine (SRH) into homocysteine and AI-2 (the inter-species quorum-sensing signal molecule). This enzyme has been reported to be essential for the survival of Actinobacillus pleuropneumoniae in its natural host. Therefore, it is a potential drug target against A. pleuropneumoniae, an important swine respiratory pathogen causing great economic losses in the pig industry worldwide. In this study, the enzymatic activity determination method was established using the recombinant LuxS of A. pleuropneumoniae. Thirty-five compounds similar to the shape of SRH were screened from the Specs compound library by the software vROCS and were evaluated for LuxS inhibition. Three compounds could inhibit LuxS activity. Two of them were confirmed to be competitive inhibitors and the third one was uncompetitive. All the three compounds displayed inhibitory effects on the growth of A. pleuropneumoniae and two other important swine pathogens, Haemophilis parasuis and Streptococcus suis, with MIC50 values ranging from 11 to 51 μg/ml. No significant cytotoxic effect of the compounds was detected on porcine PK-15 cells at the concentration which showed inhibitory effect on bacterial growth. These results suggest that LuxS is an ideal target to develop antimicrobials for porcine bacterial pathogens. The three LuxS inhibitors identified in this study can be used as lead compounds for drug design.
    Current Microbiology 06/2013; · 1.52 Impact Factor
  • Wei Jiang, Bingzhao Xia, Ziduo Liu
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    ABSTRACT: Currently, l-serine is mainly produced by enzymatic conversion, in which serine hydroxymethyltransferase (SHMT) is the key enzyme, suggesting the importance of searching for a SHMT with high activity. Shewanella algae, a methanol-utilizing marine bacterium showing high SHMT activity, was selected based on screening bacterial strains and comparison of the activities of SHMTs. A glyA was isolated from the S. algae through thermal asymmetric interlaced PCR (TAIL-PCR) and it encoded a 417 amino acid polypeptide. The SaSHMT, encoded by the glyA, showed the optimal activity at 50°C and pH 7.0, and retained over 45% of its maximal activity after incubation at 40°C for 3h. The enzyme showed better stability under alkaline environment (pH 6.5-9.0) than Hyphomicrobium methylovorum GM2's SHMT (pH 6.0-7.5). The SaSHMT can produce 77.76mM of l-serine by enzymatic conversion, with the molecular conversion rate in catalyzing glycine to l-serine being 1.41-fold higher than that of Escherichia coli. Therefore, the SaSHMT has the potential for industrial applications due to its tolerance of alkaline environment and a relatively high enzymatic conversion rate.
    Microbiological Research 04/2013; · 1.99 Impact Factor
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    ABSTRACT: Bacillus thuringiensis is a well known entomopathogenic bacterium used worldwide as an environmentally compatible biopesticide. During sporulation, B. thuringiensis accumulates a large number of parasporal crystals consisting of insecticidal crystal proteins (ICPs) that can account for nearly 20~30% of the cell dry weight. However, the metabolic regulation mechanisms of ICP synthesis remain to be elucidated. In this study, the combined efforts in transcriptomics and proteomics mainly uncovered the following 6 metabolic regulation mechanisms: 1) proteases and the amino acid metabolism (particularly, the branched-chain amino acids) became more active during sporulation; 2) stored poly-β-hydroxybutyrate and acetoin, together with some low-quality substances provided considerable carbon and energy sources for sporulation and parasporal crystal formation; 3) the pentose phosphate shunt demonstrated an interesting regulation mechanism involving gluconate when CT-43 cells were grown in GYS medium; 4) the tricarboxylic acid cycle was significantly modified during sporulation; 5) an obvious increase in the quantitative levels of enzymes and cytochromes involved in energy production via the electron transport system was observed; 6) most F0F1-ATPase subunits were remarkably up-regulated during sporulation. This study, for the first time, systematically reveals the metabolic regulation mechanisms involved in the supply of amino acids, carbon substances, and energy for B. thuringiensis spore and parasporal crystal formation at both the transcriptional and translational levels.
    Molecular &amp Cellular Proteomics 02/2013; · 7.25 Impact Factor
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    ABSTRACT: Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg(51) mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.
    PLoS ONE 01/2013; 8(11):e79175. · 3.73 Impact Factor
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    ABSTRACT: A genomic library of a psychrotrophic Psychrobacter celer 3Pb1 was constructed and screened for lipolytic proteins, and a novel esterase Est12 was cloned and characterized. The esterase gene, est12, contained an open reading frame of 990 bp that encoded a protein of 329 amino acids with an estimated molecular mass of 35,150 Da. Est12 displayed the highest amino acid identity (77%) with a hypothetical esterase from Psychrobacter sp. PAMC 21119 (WP_010200623.1). Phylogenetic analysis suggested that the protein belonged to a new lipase/esterase family. Substrate specifity study showed that Est12 preferred short-chain p-nitrophenyl esters and was most active toward p-nitrophenyl butyrate. Est12 displayed the optimal activity at pH 7.5 and 35 °C, and remained 41% activity at 0 °C while being unstable at temperatures above 40 °C, indicating its cold-adaptation. Besides, Est12 was a salt-tolerant esterase as 4.5 M NaCl significantly declined Km from 0.069 to 0.033 mM and increased kcat from 4.20 to 9.21 s−1, resulting in the increased catalytic efficiency kcat/Km from 60.72 to 276.31 s−1 mM−1. The enzyme activity was also quite stable after 24 h incubation in 0–4.5 M NaCl solutions. In addition, Est12 was very active and stable in the presence of several detergents and organic solvents. This new cold-active and halotolerant esterase would be a potential candidate in industrial applications under extreme conditions (low temperatures, high salinity), and was valuable for studying other unknown esterases/lipases in this new family.
    Journal of Molecular Catalysis B Enzymatic 01/2013; 98:119–126. · 2.82 Impact Factor
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    ABSTRACT: The anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema toxin (EF). The PA moiety carries EF and LF into the cytosol of mammalian cells via a mechanism that depends on the oligomerization of PA and transmembrane pore formation by the PA oligomer. Certain mutants of PA, termed dominant-negative (DN) mutants, can co-oligomerize with wild-type PA and disrupt the translocation ability of the pore. Here, we constructed a PA mutant library by introducing random mutations into domain II of PA and screened three new DN mutants of PA: V377E, T380S, and I432C. All the mutants inhibited the anthrax toxin action against sensitive cells. V377E had the strongest inhibitory effect and was further confirmed to be able to protect mice against a challenge with anthrax lethal toxin. Furthermore, we functionally characterized these mutants. The result showed that these mutations did not impair proteolytic activation or oligomer formation of PA, but impeded the prepore-pore conversion of the oligomer. These DN mutants of PA identified in our study may provide valuable information for elucidating the structure-function relationship of PA and for designing therapeutics for anthrax treatment.
    Applied biochemistry and biotechnology 09/2012; · 1.94 Impact Factor

Publication Stats

232 Citations
129.65 Total Impact Points

Institutions

  • 2008–2014
    • Huazhong Agricultural University
      • • College of Life Science and Technology
      • • College of Veterinary Medicine
      • • National Key Laboratory of Agricultural Microbiology
      Wu-han-shih, Hubei, China