Zhengqiang Jiang

Characterization of a thermostable extracellular beta-glucosidase with activities of exoglucanase and transglycosylation from Paecilomyces thermophila.

The purification and characterization of a novel extracellular beta-glucosidase from Paecilomyces thermophila J18 was studied. The beta-glucosidase was purified to 105-fold apparent homogeneity with a recovery yield of 21.7% by DEAE 52 and Sephacryl S-200 chromatographies. Its molecular masses were 116 and 197 kDa when detected by SDS-PAGE and gel filtration, respectively. It was a homodimeric glycoprotein with a carbohydrate content of 82.3%. The purified enzyme exhibited an optimal activity at 75 degrees C and pH 6.2. It was stable up to 65 degrees C and in the pH range of 5.0-8.5. The enzyme exhibited a broad substrate specificity and significantly hydrolyzed p-nitrophenyl-beta- d-glucopyranoside ( pNPG), cellobiose, gentiobiose, sophorose, amygdalin, salicin, daidzin, and genistin. Moreover, it displayed substantial activity on beta-glucans such as laminarin and lichenan, indicating that the enzyme has some exoglucanase activity. The rate of glucose released by the purified enzyme from cellooligosaccharides with a degree of polymerization (DP) ranging between 2 and 5 decreased with increasing chain length. Glucose and glucono-delta-lactone inhibited the beta-glucosidase competitively with Ki values of 73 and 0.49 mM, respectively. The beta-glucosidase hydrolyzed pNPG, cellobiose, gentiobiose, sophorose, salicin, and amygdalin, exhibiting apparent Km values of 0.26, 0.65, 0.77, 1.06, 1.39, and 1.45 mM, respectively. Besides, the enzyme showed transglycosylation activity, producing oligosaccharides with higher DP than the substrates when cellooligosaccharides were hydrolyzed. These properties make this beta-glucosidase useful for various biotechnological applications.

Biochemical Characterization of a Novel Thermostable β-1,3-1,4-Glucanase (Lichenase) from Paecilomyces thermophila

The purification and characterization of a novel extracellular beta-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila J18 were studied. The strain produced the maximum level of extracellular beta-glucanase (135.6 U mL(-1)) when grown in a medium containing corncob (5%, w/v) at 50 degrees C for 4 days. The crude enzyme solution was purified by 122.5-fold with an apparent homogeneity and a recovery yield of 8.9%. The purified enzyme showed as a single protein band on SDS-PAGE with a molecular mass of 38.6 kDa. The molecular masses were 34.6 kDa and 31692.9 Da when detected by gel filtration and mass spectrometry, respectively, suggesting that it is a monomeric protein. The enzyme was a glycoprotein with a carbohydrate content of 19.0% (w/w). Its N-terminal sequence of 10 amino acid residues was determined as H2N-A(?)GYVSNIVVN. The purified enzyme was optimally active at pH 7.0 and 70 degrees C. It was stable within pH range 4.0-10.0 and up to 65 degrees C, respectively. Substrate specificity studies revealed that the enzyme is a true beta-1,3-1,4-D-glucanase. The K m values determined for barley beta-D-glucan and lichenan were 2.46 and 1.82 mg mL(-1), respectively. The enzyme hydrolyzed barley beta-D-glucan and lichenan to yield bisaccharide, trisaccharide, and tetrasaccharide as the main products. Circular dichroism studies indicated that the protein contains 28% alpha-helix, 24% beta-sheet, and 48% random coil. Circular dichroism spectroscopy is also used to investigate the thermostability of the purified enzyme. This is the first report on the purification and characterization of a beta-1,3-1,4-glucanase from Paecilomyces sp. These properties make the enzyme highly suitable for industrial applications.

Production of xylooligosaccharides from the steam explosion liquor of corncobs coupled with enzymatic hydrolysis using a thermostable xylanase.

The production of xylooligosaccharides (XOs) from corncobs was studied using a two-stage process based on a steam explosion pretreatment followed by enzymatic hydrolysis. Corncobs with different chip sizes were subjected to steam explosion under different experimental conditions of temperature and time, namely 188-204 degrees C for 2.5-7.5 min. The results indicate that corncobs were optimally steam exploded at 196 degrees C for 5 min, resulting in hemicellulose recovery of 22.8%. Especially, corncobs with large chip sizes (approximately 100 mm) during steam explosion pretreatment were suitable to produce XOs. Furthermore, a thermostable xylanase from Paecilomyces themophila J18 was used to hydrolyze steam explosion liquor of corncobs (SELC) for the production of XOs. A maximum XOs yield of 28.6 g XOs/100 g xylan in corncobs was achieved and XOs syrup contained more than 90% of xylobiose and xylotriose when the hydrolysis was carried out under the optimized conditions (pH 7.0, 70 degrees C, 7.5 U mL(-1) and 2.5 h). These results suggest that the process might be effective in production of XOs for industrial applications.

Characterization of a protease-resistant α-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides.

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.

High-level expression of extracellular secretion of a β-xylosidase gene from Paecilomyces thermophila in Escherichia coli

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.

Cloning and expression of a Paecilomyces thermophila xylanase gene in E. coli and characterization of the recombinant xylanase

A cDNA library of Paecilomyces thermophila was constructed, and the gene encoding xylanase (designated Pt xynA) was isolated from the library. Pt xynA consisted of 681 bp, and the translated protein encoded 226 amino acids. This is the first functional gene cloned from P. thermophila. The gene was successfully expressed in Escherichia coli BL21 and the recombinant xylanase (XynA) was purified to homogeneity by Ni-NTA and Sephadex G50. XynA showed an optimum activity at 75 degrees C and pH 7.0. Its residual activity was more than 60% after being treated at 85 degrees C for 30 min. K(m) values of XynA for birchwood xylan, beechwood xylan and oat-spelt xylan were 4.4, 3.6 and 9.7 mg ml(-1), respectively. The enzyme has an endohydrolytic mode of action and can hydrolyse xylotriose to xylobiose through transglycosylation. These results indicate the XynA is a thermostable enzyme and has great potential in various industries.

Isolation, identification and synthesis of four novel antioxidant peptides from rice residue protein hydrolyzed by multiple proteases

Multiple proteases were optimized to hydrolyze the rice residue protein (RRP) to produce novel antioxidant peptides. An antioxidant peptide fraction (RRPB3) with IC50 of 0.25 mg/ml was purified from the RRP hydrolysate using membrane ultrafiltration followed by size exclusion chromatography and reversed-phase FPLC. RRPB3 was found to include four peptides (RRPB3 I-IV) and their amino acid sequences were RPNYTDA (835.9 Da), TSQLLSDQ (891.0 Da), TRTGDPFF (940.0 Da) and NFHPQ (641.7 Da), respectively. Furthermore, four peptides were chemically synthesized and their antioxidant activities were assessed by DPPH radical scavenging, ABTS radical scavenging assay and FRAP-Fe(3+) reducing assay, respectively. Both RRPB3 I and III showed synergistic antioxidant activity compared to each of them used alone. All four synthetic peptides showed excellent stability against simulated gastrointestinal proteases. Therefore, the peptides isolated from RRP may be used as potential antioxidants in the food and drug industries.

Purification and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei with high specific activity and its gene sequence.

Production, purification, and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei CAU432 were investigated. High-level extracellular β-1,3-1,4-glucanase production of 6230 U/mL was obtained when oat flour (3%, w/v) was used as a carbon source at 50 °C. The crude enzyme was purified to homogeneity with a specific activity of 28818 U/mg. The molecular weight of purified enzyme was estimated to be 35.4 kDa and 33.7 kDa by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.5 and 60 °C, respectively. The K(m) values of purified β-1,3-1,4-glucanase for barley β-glucan and lichenan were 2.0 mM and 1.4 mM, respectively. Furthermore, the gene (RmLic16A) encoding the β-1,3-1,4-glucanase was cloned and its deduced amino acid sequence showed the highest identity (50%) to characterized β-1,3-1,4-glucanase from Paecilomyces thermophila. The high-level production and biochemical properties of the enzyme enable its potential industrial applications.

Cloning, expression, purification and application of a novel chitinase from a thermophilic marine bacterium Paenibacillus barengoltzii.

A novel chitinase gene (PbChi70) from a marine bacterium Paenicibacillus barengoltzii was cloned and functionally expressed in Escherichia coli. The recombinant enzyme (PbChi70) was purified to homogeneity with a recovery yield of 51.9%. The molecular mass of purified enzyme was estimated to be 70.0 kDa by SDS-PAGE. PbChi70 displayed maximal activity at pH 5.5 and 55 °C, respectively. It exhibited strict substrate specificity for colloidal chitin, glycol chitin, powdery chitin, and N-acetyl chitooligosaccharides with degrees of polymerization above three. The enzyme exhibited an endo-type cleavage pattern and hydrolyzed colloidal chitin to yield mainly (GlcNAc)2. Furthermore, colloidal chitin was hydrolyzed by PbChi70 to produce 21.6 mg mL(-1) (GlcNAc)2 with the highest conversion yield of 89.5% (w/w). (GlcNAc)2 was further separated by an active charcoal column with a purity of 99% and a final yield of 61%. The unique enzymatic properties of the chitinase may make it a good candidate for (GlcNAc)2 production.

Biochemical Characterization of a Novel l-Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment

ABSTRACT A novel fungal gene encoding the Rhizomucor miehei l-asparaginase (RmAsnase) was cloned and expressed in Escherichia coli. Its deduced amino acid sequence shared only 57% identity with the amino acid sequences of other reported l-asparaginases. The purified l-asparaginase homodimer had a molecular mass of 133.7 kDa, a high specific activity of 1,985 U/mg, and very low glutaminase activity. RmAsnase was optimally active at pH 7.0 and 45°C and was stable at this temperature for 30 min. The final level of acrylamide in biscuits and bread was decreased by about 81.6% and 94.2%, respectively, upon treatment with 10 U RmAsnase per mg flour. Moreover, this l-asparaginase was found to potentiate a lectins induction of leukemic K562 cell apoptosis, allowing lowering of the drug dosage and shortening of the incubation time. Overall, our findings suggest that RmAsnase possesses a remarkable potential for the food industry and in chemotherapeutics for leukemia.