Nguyen Duc Huy

Characterization of a recombinant bifunctional xylosidase/arabinofuranosidase from Phanerochaete chrysosporium

A bifunctional xylosidase/arabinofuranosidase gene (PcXyl) was cloned from the cDNA library of Phanerochaete chrysosporium and further expressed in Pichia pastoris. Enzymatic assay indicated that P. pastoris produced rPcXyl at a level of 26,141 U l⁻¹. The xylosidase and arabinofuranosidase activities of rPcXyl were maximized, respectively, at pHs of 5.0 and 5.5 and temperatures of 45°C and 50°C. SDS-PAGE revealed a single band of purified rPcXyl of 83 kDa. Cu²⁺ and Zn²⁺ completely inhibited the enzyme activity of rPcXyl. The enzyme activity of rPcXyl was increased 151%, 126% and 123%, respectively, in the presence of glucose, xylose and arabinose at concentrations of 5 mM. rPcXyl hydrolyzed xylobiose to xylose and xylotriose to xylose and xylobiose, indicating rPcXyl acts as an exo-type enzyme. Additionally, rPcXyl enhanced xylose release from xylan substrates in synergy with rPcXynC.

Heterologous expression of endo-1,4-beta-xylanaseC from Phanerochaete chrysosporium in Pichia pastoris

The cDNA of endo-1,4-β-xylanaseC, isolated from Phanerochaete chrysosporium, was expressed in Pichia pastoris, under the control of the alcohol oxidase I promoter. Using either the intrinsic leader peptide of xylanaseC or the α-factor signal peptide of Saccharomyces cerevisiae, xylanaseC is efficiently secreted into the medium, at a maximum concentration of 2500 U·l(-1).

Putative endoglucanase PcGH5 from Phanerochaete chrysosporium is a β-xylosidase that cleaves xylans in synergistic action with endo-xylanase.

A predicted endoglucanase gene (PcGH5) was cloned from Phanerochaete chysosporium, and expressed in Pichia pastoris. Although PcGH5 showed similarity with the conserved domains of a cellulase superfamily GH5, a β-glucosidase/6-phospho-β-glucosidase/β-galactosidase superfamily, and an endoglucanase, recombinant PcGH5 exhibited a β-xylosidase activity, rather than endoglucanase activity. Therefore, the predicted gene was named as PcXyl5. Further characterization of recombinant PcXyl5 showed not only catalysis of the hydrolysis of xylo-oligomers to xylose, but also displayed transglycosylation activity using alcohol as a receptor. Optimum pH of rPcXyl5 was found to be 5.5, whereas optimum temperature was 50°C. rPcXyl5 increased reducing sugar release of birchwood xylan, beechwood xylan, and arabinoxylan by 6.4%, 13%, 15.8%, respectively, in synergistic action with endo-xylanase. Interestingly, the late addition of rPcXyl5 into reaction with endo-xylanase resulted in a larger increase of reducing sugar release from pretreated barley straw that addition at the start or by treatment with endo-xylanases alone. The increases observed were 6.3% and 13.8%, respectively, showing a great potential application for hemicellulose saccharification.

Trichoderma asperellum Chi42 Genes Encode Chitinase

Four Trichoderma strains (CH2, SH16, PQ34, and TN42) were isolated from soil samples collected from Quang Tri and Thua Thien Hue provinces in Vietnam. The strains exhibited high chitinolytic secretion. Strain PQ34 formed the largest zone of chitinase-mediated clearance (> 4 cm in diameter) in agar containing 1% (w/v) colloidal chitin. Analysis of the internal transcribed spacer regions of these strains indicated that they were Trichoderma asperellum. The molecular weights of the chitinases were approximately 42 kDa. Chitinase genes (chi42) of T. asperellum strains TN42, CH2, SH16, and PQ34 were 98~99% homologous to the ech42 gene of T. harzianum CB-Pin-01 (accession No. DQ166036). The deduced amino acid sequences of both T. asperellum strains SH16 and TN42 shared 100% similarity.

Screening and Production of Manganese Peroxidase from Fusarium sp. on Residue Materials

Abstract In this study, we report the manganese peroxidase production ability from a Fusarium sp. strain using an inexpensive medium of agriculture residues of either rice straw or wood chips as carbon source. The highest manganese peroxidase activity on rice straw medium and on wood chips was 1.76 U/mL by day 9 and 1.91 U/mL by day 12, respectively.

Cloning and expression of two genes coding endo-β-1,4-glucanases from Trichoderma asperellum PQ34 in Pichia pastoris

Two genes coding endo-β-1,4-glucanases were cloned from Trichoderma asperellum PQ34 which was isolated from Thua Thien Hue province, Vietnam. The expression of these genes in Pichia pastoris produced two enzymes with molecular masses of approximately 46 kDa (about 42 kDa of enzymes and 4 kDa of signal peptide). The effects of induction time and temperature, inducer concentration, and culture medium on the endo-β-1,4-glucanase activity were investigated. The results showed that the highest total activities of two endo-β-1,4-glucanases were approximately 4.7 × 10−8 kat (from Glu1-TA gene) and 7.3 × 10−8 kat (from Glu2-TA gene) occurred after 4 days of induction using 25 mL L−1 methanol at 30◦C when the yeast cells were cultured in a YPL medium.

Heterologous Expression of Endo-1,4-beta-xylanaseA from Phanerochaete chrysosporium in Pichia pastoris

The cDNA of endo-1,4-β-xylanaseA, isolated from Phaenerocheate chrysosporium was expressed in Pichia pastoris. Using either the intrinsic leader peptide of XynA or the α-factor signal peptide of Saccharomyces cerevisiae, xylanaseA is efficiently secreted into the medium at maximum concentrations of 1,946 U/L and 2,496 U/L, respectively.