Huiyong Jia

China Agricultural University, Beijing, Beijing Shi, China

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Publications (7)19.56 Total impact

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    ABSTRACT: Aims:  To characterize a β-xylosidase from the thermophilic fungus Thermomyces lanuginosus and to investigate its potential in saccharification of hemicellulosic xylans. Methods and Results:  A gene (designated TlXyl43) encoding β-xylosidase was cloned from T. lanuginosus CAU44 and expressed in Escherichia coli. The gene consists of a 1017-bp open reading frame without introns. It encodes a mature protein of 338 residues with no predicted signal peptide, belonging to glycoside hydrolase (GH) family 43. Over 60% of the recombinant β-xylosidase (TlXyl43) was secreted into the culture medium. TlXyl43 was purified 2·6-fold to homogeneity with an estimated mass of 51·6 kDa by SDS-PAGE. The purified enzyme exhibited optimal activity at pH 6·5 and 55°C and was stable at 50°C. It was competitively inhibited by xylose with a K(i) value of 63 mmol l(-1) . Conclusions:  In this study, a GH family 43 β-xylosidase gene (TlXyl43) from T. lanuginosus CAU44 was cloned and functionally expressed in E. coli, and over 60% of recombinant protein was secreted into the culture. Significance and Impact of the Study:  This is the first report of the cloning and functional expression of a β-xylosidase gene from Thermomyces species. TlXyl43 holds great potential for variety of industries.
    Letters in Applied Microbiology 08/2012; · 1.63 Impact Factor
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    ABSTRACT: A xylanase gene from Paecilomyces thermophila was functionally expressed in Pichia pastoris. The recombinant xylanase (xynA) was predominantly extracellular; in a 5 l fermentor culture, the total extracellular protein was 8.1 g l(-1) with an activity of 52,940 U ml(-1). The enzyme was purified to homogeneity with a recovery of 48 %. The recombinant xynA was optimally active at 75 °C, as measured over 10 min, and at pH 7. The enzyme was stable up to 80 °C for 30 min. It hydrolyzed birchwood xylan, beechwood xylan and xylooligosaccharides to produce xylobiose and xylotriose as the main products.
    Biotechnology Letters 07/2012; 34(11):2043-8. · 1.85 Impact Factor
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    ABSTRACT: To fulfill the need for acid-tolerant and thermostable β-1,3-1,4-glucanases, an error-prone PCR and DNA-shuffling approach was employed to enhance the activity of thermostable β-1,3-1,4-glucanases from Paecilomyces thermophila (PtLic16A) at acidic pH. Mutant PtLic16AM2 was selected and characterized, and showed optimal activity at pH 5.0, corresponding to an acidic shift of 2.0 pH units relative to the wild-type enzyme. Other properties of PtLic16A such as temperature optimum and substrate specificity that are beneficial for industrial applications did not change. Based on the substituted residues of PtLic16AM2, three site-directed mutations, D56G, D221G and C263S, were designed to study these residues' roles. The amino acid residues at positions 56 and 263 were found to be important in determining optimal pH activity. Activity of the D221G variant showed no significant difference from the wild-type. Thus, it appears that the change in optimal pH for PtLic16AM2 was mainly caused by the combination of substitutions D56G and C263S. This study provides a β-1,3-1,4-glucanase (PtLic16AM2) with high potential for industrial applications.
    Journal of Biotechnology 02/2012; 159(1-2):50-5. · 3.18 Impact Factor
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    ABSTRACT: The α-galactosidase gene, RmGal36, from Rhizomucor miehei was cloned and expressed in Escherichia coli. The gene has an open reading frame of 2256bp encoding 751 amino acid residues. RmGal36 was optimally active at pH 4.5 and 60°C, but is stable between pH 4.5 and 10.0 and at a temperature of up to 55°C for 30min retaining more than 80% of its relative activity. It displayed remarkable resistance to proteases and its activity was not inhibited by galactose concentrations of 100mM. The relative specificity of RmGal36 towards various substrates is in the order of p-nitrophenyl α-galactopyranoside>melibiose>stachyose>raffinose, with a K(m) of 0.36, 16.9, 27.6, and 47.9mM, respectively. The enzyme completely hydrolyzed raffinose and stachyose present in soybeans and kidney beans at 50°C within 60min. These features make RmGal36 useful in the food and feed industries and in processing of beet-sugar.
    Bioresource Technology 02/2012; 110:578-86. · 5.04 Impact Factor
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    ABSTRACT: Directed evolution was used to improve the performance of beta-1,3-1,4-glucanase (designated as PtLicl6A) from Paecilomyces thermophila J18 under acidic condition. A mutant library was constructed by error-prone PCR and DNA shuffling, and positive clones were screened by Congo red staining. More than 1 500 mutants were selected. One mutant (PtLic16AM1) exhibited an optimal activity at pH 5.5, while the optimal pH of the wild-type enzyme was 7.0. The mutant PtLic16AM1 kept the high specific activity and thermotolerence of the wild-type enzyme. Sequence analysis revealed that the mutant enzyme has four sense substitutions which caused four amino acid substitutions - namely T58S, Y110N, G195E and D221G.. Homology modeling showed that among the four amino acid substitutions, Y110N was near the active site of the enzyme, while the other three was distant. T58S and G195E may play key roles in the change of optimal pH. This study provided a new perspective of obtaining applicable 3-1,3-1,4-glucanase for industrial use.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2011; 27(12):1797-804.
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    ABSTRACT: A β-galactosidase gene (designated PaGalA) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris under the control of the AOX1 promoter. The coding region of 3036 bp encoded a protein of 1011 amino acids with a deduced molecular mass of 108.7 kDa. The PaGalA without the signal peptide was cloned into a vector pPIC9K and was expressed successfully in P. pastoris as active extracellular β-galactosidase. The recombinant β-galactosidase (PaGalA) was secreted into the medium at an extremely high levels of 22 mg ml−1 having an activity of 9500 U ml−1 from high density fermentation culture, which is by far the highest yield obtained for a β-galactosidase. The purified enzyme with a high specific activity of 820 U mg−1 had a molecular mass of 120 kDa on SDS-PAGE. PaGalA was optimally active at pH 4.5 and a temperature of 60 °C. The recombinant β-galactosidase was able to hydrolyze lactose efficiently at pH 5.0 and 50 °C. It also possessed transglycosylation activities at high concentrations of lactose. PaGalA exhibited better lactose hydrolysis efficiency in whey than two other widely used commercial lactases. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.Graphical abstractA β-galactosidase gene (designated PaGalA) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.Research highlights▶ High-level expression of a β-galactosidase gene was achieved in Pichia pastoris. ▶ The β-galactosidase was optimally active at pH 4.5 and a temperature of 60 °C. ▶ The enzyme efficiently hydrolyzed lactose lactose present in acid- and sweet-whey.
    Journal of Molecular Catalysis B Enzymatic 01/2011; 69:112-119. · 2.82 Impact Factor
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    ABSTRACT: A novel β-xylosidase gene (designated as PtXyl43) from thermophilic fungus Paecilomycesthermophila was cloned and extracellularly expressed in Escherichia coli. PtXyl43 belonging to glycoside hydrolase (GH) family 43 has an open reading frame of 1017 bp, encoding 338 amino acids without a predicted signal peptide. No introns were found by comparison of the PtXyl43 genomic DNA and cDNA sequences. The recombinant β-xylosidase (PtXyl43) was secreted into the culture medium in E. coli with a yield of 98.0 U mL(-1) in shake-flask cultures. PtXyl43 was purified 1.2-fold to homogeneity with a recovery yield of 61.5% from the cell-free culture supernatant. It appeared as a single protein band on SDS-PAGE with a molecular mass of approx 52.3 kDa. The enzyme exhibited an optimal activity at 55 °C and pH 7.0, respectively. This is the first report on the cloning and expression of a GH family 43 β-xylosidase gene from thermophilic fungi.
    Bioresource Technology 09/2010; 102(2):1822-30. · 5.04 Impact Factor