Biomedical subjects

Zheng-Xiang Wang

Publications and source records attributed to Zheng-Xiang Wang.

11 recordsLinked to original sources

PubMed2006

[Overproduction of glucoamylase by recombinant Aspergillus niger harboring multiple copies of glaA].

The glucoamylase gene (glaA) of Aspergillus niger CICIM F0410 was cloned, sequenced and expressed. The integrated plasmid pBC-Hygro-glaA carrying the glaA was constructed and transformed into A. niger F0410. Transformants with multiple copies of glaA integrated in the chromosome were selected by 150 microg/mL hygromycin B and identified by real-time PCR. Two to three multiples of glaA in the chromosome were found to be optimal for higher expression of glucoamylase. Shake-flask fermentation under optimal conditions showed that glucoamylase secreted by the transformant GB0506 was 17.5% higher than parental strain F0410 at the end of fermentation. In conclusion, increasing copy number of glaA by chromosomal integration significantly improves the yield of glucoamylase in the industrial strain of A. niger.

Aspergillus niger↗

PubMed2006

Biochemical properties of a thermostable phytase from Neurospora crassa.

A gene (Ncphy) encoding a putative phytase in Neurospora crassa was cloned and expressed in Pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. The recombinant phytase (rNcPhy) hydrolyzed phytic acid with a specific activity of 125 U mg-1, Km of 228 micromol L-1, Vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees C and a pH optimum of 5.5 and a second pH optimum of 3.5. The enzyme displayed pH stability around pH 3.5-9.5 and showed satisfactory thermostability at 80 degrees C. The phytase from N. crassa has potential for improving animal feed processing at higher temperatures.

6-Phytase↗

PubMed2006

[Comparison of different transformation methods for Monascus sp].

In order to facilitate the producer of polyketide pathway, four different transformation methods were tested and compared in an attempt to develop the genetic transformation system of Monascus sp. Using vector pBC-Hygro, the fungus was transformed to be hygromycin B-resistant, by conventional transformation as well as electroporation based on protoplast, electroporation based on germinated conidia, and restriction enzyme-mediated integration (REMI). Electroporation based on germinated conidia was found to be inappropriate for transforming Monascus sp. due to a low transformation frequency. The conventional transformation and electroporation technique based on protoplasts were thought not to be fit for transforming Monascus sp., due to a low stability of transformants though they yielded up to 135 transformants and 125 transformants per microgrammol/Lol/Le DNA, respectively. Transformant number was increased by 20-fold by REMI (2,500 transformants per microgrammol/Lol/Le DNA) and 70%-75% of them were stable. REMI technique would be very beneficial to the establishment of the genetic transformation system of Monascus sp.

DNA, Recombinant↗

PubMed2005

[Preparation and Regeneration of Protoplasts from Monascus purpureus and Genetic Transformation System.].

Generation of fungal protoplast is an essential tool for genetic transformation system. To establish protoplast-mediated genetic transformation system of Monascus purpureus, conditions for the protoplast isolation and regeneration of the mycelia of various enzymes and osmotic stabilizers were examined. To investigate suitable cell age for the protoplast preparation of mycelia of M. purpureus, the mycelia were cultured in different ways at 30oC. Mycelia obtained through cellophane - mediated culture for 30~40h were adequate to protoplast preparation. When lysing enzyme, cellulase and snailase were added to the mycelia in combination or alone, combination of lysing enzyme, cellulase and snailase accordingly at the concentration of 0.3%, 0.1% and 1% was most benefit for protoplast yield. When we applied various osmotic stabilizers at different concentrations to protoplast preparation, 1 mol/L MgSO4 was most effective for the protoplast release. The suitable incubation time with enzyme for the maximum release of protoplasts was 2.5-hr. When we investigate various osmotic stabilizers for the regeneration of the protoplasts of mycelia of strain M34 and N18, the complete medium containing 0.6 mol/L sucrose induced highest hyphal growth with regeneration frequency of 8.5% and 36.4%, respectively. PEG and CaCl2- mediated protoplast co-transformation of strain M34 with pBC-Hygro and pNL1, hygromycin B as selective marker, was fulfilled and 100 stable transformants per microgram DNA were obtained.

Hygromycin B↗

PubMed2005

[Glyoxylate cycle is required for the overproduction of glutamate but is not essential for Corynebacterium glutamicum growth on glucose].

The glyoxylate cycle was hypothesed to be indispensable for glutamate overproduction in coryneform bacteria, for it was thought to fulfill anaplerotic functions and to supply energy during the growth phase. During glutamate overproduction phase, however, it has been noted that the high level of the cycle is detrimental to the glutamate production. In order to clarify the relationship between the glutamate production and the glyoxylate cycle, a chromosomal aceA-disrupted mutant of wild-type C. glutamicum ATCC 13032 was constructed. The isocitrate lyase (ICL) activity of the parental strain was 0.011 u/mg of protein and reached 1.980 u/mg of protein after acetate induction; the mutant strain WTdeltaA, however, had no detectable ICL activity and was no longer able to grow on minimal medium with acetate as the sole carbon source. Compared with the wild-type C. glutamicum WT, the mutant strain WTdeltaA, exhibited the same growth rate with glucose as the sole carbon source, indicating glyoxylate cycle is not required for its growth on glucose. On the contrary, the glutamate production in WTdeltaA was severely impaired and more residual glucose was found in the fermentation broth at the end of fermentation with the mutant strain than with the wild-type strain. Further investigations into the relationship between the glutamate production and the glyoxylate cycle are under the way, which may help to elucidate the mechanism of glutamate overproduction.

Corynebacterium glutamicum↗

PubMed2004

Cloning and over-expression of an alkaline protease from Bacillus licheniformis.

The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.

Amino Acid Sequence↗

PubMed2004

Purification and characterisation of an alkaline protease used in tannery industry from Bacillus licheniformis.

An extracellular alkaline protease produced by Bacillus licheniformis AP-1 was purified 76-fold, yielding a single 28 kDa band on SDS-PAGE. It was optimally active at pH 11 and at 60 degrees C (assayed over 10 min). The protease was completely inhibited by phenylmethylsulfonyl fluoride and diodopropyl fluorophosphate, with little increase upon Ca2+ and Mg2+ addition.

Bacillus↗

PubMed2003

The glyoxylate bypass of Ralstonia eutropha.

The glyoxylate bypass genes aceA1 (isocitrate lyase 1, ICL1), aceA2 (isocitrate lyase 2, ICL2) and aceB1 (malate synthase, MS1) of Ralstonia eutropha HF39 were cloned, sequenced and functionally expressed in Escherichia coli. Interposon-mutants of all three genes (DeltaaceA1, DeltaaceA2 and DeltaaceB1) were constructed, and the phenotypes of the respective mutants were investigated. Whereas R. eutropha HF39DeltaaceA1 retained only 19% of ICL activity and failed to grow on acetate, R. eutropha HF39DeltaaceA2 retained 84% of acetate-inducible ICL activity, and growth on acetate was not retarded. These data suggested that ICL1 is in contrast to ICL2 induced by acetate and specific for the glyoxylate cycle. R. eutropha HF39DeltaaceB1 retained on acetate as well as on gluconate about 41-42% of MS activity and exhibited retarded growth on acetate, indicating the presence of a second hitherto not identified MS in R. eutropha HF39. Whereas in R. eutropha HF39DeltaaceA1 and R. eutropha HF39DeltaaceA2 the yields of poly(3-hydroxybutyric acid), using gluconate as carbon source, were significantly reduced, R. eutropha HF39DeltaaceB1 accumulated the same amount of this polyester from gluconate as well as from acetate as R. eutropha HF39.

Acetates↗

PubMed2003

Two phenotypically compensating isocitrate dehydrogenases in Ralstonia eutropha.

The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (IDH) and the glyoxylate bypass enzyme isocitrate lyase are involved in catabolism of isocitrate and play a key role in controlling the metabolic flux between the TCA cycle and the glyoxylate shunt. Two IDH genes icd1 and icd2 of Ralstonia eutropha HF39, encoding isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), were identified and characterized. Icd1 was functionally expressed in Escherichia coli, whereas icd2 was expressed in E. coli but no activity was obtained. Interposon-mutants of icd1 (HF39Deltaicd1) and icd2 (HF39Deltaicd2) of R. eutropha HF39 were constructed and their phenotypes were investigated. HF39Deltaicd1 retained 43% of IDH activity, which was not induced by acetate, and HF39Deltaicd2 expressed 74% of acetate-induced IDH activity. Both HF39Deltaicd1and HF39Deltaicd2 kept the same growth rate on acetate as the wild-type. These data suggested that IDH1 is induced by acetate. The interposon-mutants HF39Deltaicd1 and HF39Deltaicd2 accumulated the same amount of poly(3-hydroxybutyric acid) as the wild-type.

Amino Acid Sequence↗

PubMed2002

Cloning, sequencing and characterization of a gene encoding dihydroxyacetone kinase from Zygosaccharomyces rouxii NRRL2547.

The dihydroxyacetone pathway, an alternative pathway for the dissimilation of glycerol via reduction by glycerol dehydrogenase and subsequent phosphorylation by dihydroxyacetone (DHA) kinase, is activated in the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii during osmotic stress. In experiments aimed at investigating the physiological function of the DHA pathway in Z. rouxii, a typical osmotolerant yeast, we cloned and characterized a DAK gene encoding dihydroxyacetone kinase from Z. rouxii NRRL 2547. Sequence analysis revealed a 1761 bp open reading frame, encoding a peptide composed of 587 deduced amino acids with the predicted molecular weight of 61 664 Da. As the amino acid sequence was most closely homologous (68% identity) to the S. cerevisiae Dak1p, we named the gene and protein ZrDAK1 and ZrDak1p, respectively. A putative ATP binding site was also found but no consensus element associated with osmoregulation was found in the upstream region of the ZrDAK1 gene. The ZrDAK1 gene complemented a S. cerevisiae W303-1A dak1delta dak2 delta strain by improving the growth of the mutant on 50 mmol/l dihydroxyacetone and by increasing the tolerance to dihydroxyacetone in a medium containing 5% sodium chloride, suggesting that it is a functional homologue of the S. cerevisiae DAK1. However, expression of the ZrDAK1 gene in the S. cerevisiae dak1delta dak2 delta strain had no significant effect on glycerol levels during osmotic stress. The ZrDAK1 sequence has been deposited in the public data bases under Accession No. AJ294719; regions upstream and downstream of ZrDAK1are deposited as Accession Nos AJ294739 and AJ294720, respectively.

Adenosine Triphosphate↗

PubMed2002

[Study on fermentation condition of alkaline protease gene engineering strain and the purification and characterization of recombinant enzyme].

In a 5L fermentor the production conditions of alkaline protease gene engineering strain BA071 were investigated. The maximum activity of alkaline protease reached 24,480 u/mL in 40 hours of fermentation by combination of enhancing aeration and changing the agitation rate. The fast purification method of recombinant protease was conducted with FPLC (Fast Protein Liquid Chromatography). The crude enzyme, treated with ammonium sulfate fractionation and decolored with DEAE-A-50 and polyethylene glycol concentration, was purified with CM-Sephadex-C-50 and Sephadex-G-75. The purified enzyme appears homologous on SDS-PAGE. The purity of enzyme was increased 76.2 times. SDS-PAGE analysis showed that the molecular weights of expressed recombinant products were about 28 kD. The optimal reaction pH and temperature of recombinant enzyme were at pH11 and 60 degrees C, respectively. The recombinant enzyme exhibited high temperature tolerance and was stable at a wide range of pH. Ca2+, MG2+ can enhance the stability of the recombinant enzyme. While the protease activity of the enzyme was strongly inhibited by Hg2+, Ag+, PMFS [symbol: see text] DFP, and was not affected by SDS and Urea.

Bacillus↗