Application of bioinformatics analysis of signal peptide in the identification of Neurospora crassa phyA gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify the function of the gene encoding Neurospora crassa EAA33149.1 protein which has 46.85% similarity with Aspergillus niger phA gene.</p><p><b>METHODS</b>The bioinformatics analysis was conducted using the prediction algorithms SignalP v3.0, arginine and lysine propeptide cleavage sites in eukaryotic protein sequence prediction algorithms ProP 1.0 server, transmembrane domain prediction algorithms Tmpred and TMHMM v2.0, potential GP I-anchor sites prediction algorithm big-P I Predictor and the subcellular protein location prediction algorithms TargetP v1.01. According to the analysis results, the gene was cloned into Saccharomyces cerevisiae.</p><p><b>RESULTS</b>The signal peptide, the cleavage site and the secretion pathway were determined, and the expressed recombinant protein with 54,000 displayed phytase activity.</p><p><b>CONCLUSION</b>The gene has been identified to encode N. crassa phyA.</p>

Overproduction of glucoamylase by recombinant Aspergillus niger harboring multiple copies of glaA / 生物工程学报

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.

Glyoxylate cycle is required for the overproduction of glutamate but is not essential for Corynebacterium glutamicum growth on glucose / 生物工程学报

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.

Study on fermentation condition of alkaline protease gene engineering strain and the purification and characterization of recombinant enzyme / 生物工程学报

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.

Concise Plasmids for Antibiotic Resistance Cassette Recovery and in Vitro Recombination / 微生物学通报

In gene manipulation, different selectable markers with various linkers are necessary. In order to get selectable markers directly, we constructed from pBlueScript SK(-) a series of particular plasmids, pSKsymKm, pSKsymBle, pSKsymEry, pSKsymHyg and pSKsymGm, each contains Kanamycin, Bleomycin, Erythromycin, Hygromycin or Gentamycin resistance cassette. By restriction enzyme digestion and gel extraction, any of five antibiotic resistance genes with specific ends can be conveniently obtained.

Recombinant Lactobacilli for Ethanol Production / 微生物学通报

Recombinant plasmids pHY-PA, pBBR-PA were constructed in which genes pdc and adhB were placed under the control of tac promoter, respectively, and had successfully expressed in Escherichia coli. Then these recombinant plasmids were electroporated into Lactobacillus strains for ethanol production. Preliminary ethanol fermentation using these Lactobacillus strains and their recombinants was carried out using 42℃ as fermentation temperature. The results indicated that introducing pdc and adhB, ethanologenic pathway was successfully constructed in L.plantanum CICIM B0080. 0.4% (V/V) ethanol was detected at the end of fermentation with 6.7% glucose, and that is 67-fold as the wild-type B0080. Two-fold of ethanol production was detected in L.amylovorus B0112 (pHY-PA) and L.acidophilus B0068 (pBBR-PA). Introducing both pdc and adhB, and meanwhile knock-outing the lactate dehydrogrnase gene may better convert carbon flux to ethanologenic direction.

PRELIMINARY STUDY ON ISOLATION, IDENTIFICATION AND ETHANOL FERMENTATION OF THERMOTOLERANT YEAST / 微生物学通报

Two thermotolerant, ethanol-producing yeast cultures: THFY-4 and THFY-16 were isolated from 381 nature samples. THFY-4 can grow on 30% glucose plate at 51 ℃,while THFY-16 can grow on the same medium at 45℃.After preliminary ide ntification, THFY-4 was identified as Kluyveromyces sp. and THFY-16 belon gs to Saccharomyces genus. The ethanol fermentation experiment shows that T HFY-4 can only produce 4.88% (v/v) ethanol from 20% glucose after 60 hours, wh ile THFY-16 can produce 11.44% ethanol under the same condition. When using s accharified Canna edulis Ker wort as fermentation medium, 9.43%(v/v) ethanol we re produced from 16.1% glucose, which is 91.0% of the theoretical yield.

SCREENING OF MONASCUS PRODUCING ONLY YE LLOW PIGMENTS / 微生物学通报

mutants which didnt produce red pigmen ts on malt extract agar plate were obtained.The 8 stable mutants were cultured on solid medium.Two samples wer e yellow,the others were white.The extracted samples were scanned in visible len gth.2 yellow samples showed only one absorptive peak at 370nm,the 6 white sample s showed no absorptive peak.The mutants producing only yellow pigments on solid medium were tested in liquid culture.The results indicated their ability to pro duce only yellow pigments were stable.