The enzyme activity of α－Acetolactate Decaroboxylases (ALDC)from different microbes was studied, the results demonstrated that it was quite different among them. There were diversities of their enzyme reaction velocities. It was clear that the enzyme activity was affected by the pH of the enzyme reaction system, for example, the optimum pH of ALDC from Lactococcus lactis was 6.6, while for Aerobacter Aerogenes it was 5.8. Addition leucine,valine and isoleucine into enzyme reaction system obviously affected the enzyme activity of ALDC from different microbes.

Ergosterol is an economically important metabolite produced by yeast. To improve the production of ergosterol by Saccharomyces cerevisiae YEH56 (pHXA42) from molasses, a cheap and regenerative material, different strategies were applied. First, Plackett-Burman design and central composite design were applied to screen the significant factors in fermentation medium using ergosterol yield (g/L) as the response value. Ergosterol yield reached 371.56 mg/L by using the optimal fermentation medium in shake-flask culture (total sugar in molasses 40 g/L, KH2PO4 1 g/L, K2HPO4 1.86 g/L, CuSO4 x 5H2O 17.5 mg/L, FeSO4 x 7H2O 13.9 mg/L, MgSO4 x 5H2O 12.3 mg/L, corn steep liquor 10 mL/L), which was increased by 29.5% compared with the initial culture. Second, ergosterol yield was increased by 62.1% using a pH-control strategy in a 5-L bioreactor. Third, ergosterol production was improved further by using molasses feeding strategy. After 38 h fermentation, ergosterol yield reached 1 953.85 mg/L, which was 3.2 times of that in batch fermentation. Meanwhile, ergosterol production rate was increased by 42.7% compared with that in the batch culture.
Culture Media ; Ergosterol ; biosynthesis ; Fermentation ; Industrial Microbiology ; Molasses ; Saccharomyces cerevisiae ; genetics ; growth & development ; metabolism

Flocculent gene FLO1 and its truncated form FLO1c with complete deletion of repeat unit C were expressed in a non-flocculent industrial strain Saccharomyces cerevisiae CE6 to generate recombinant flocculent strains 6-AF1 and 6-AF1c respectively. Both strains of 6-AF1 and 6-AF1c displayed strong flocculation and better cell growth than the control strain CE6-V carrying the empty vector under acetic acid stress. Moreover, the flocculent strains converted glucose to ethanol at much higher rates than the control strain CE6-V under acetic acid stress. In the presence of 0.6% (V/V) acetic acid, the average ethanol production rates of 6-AF1 and 6-AF1c were 1.56 and 1.62 times of that of strain CE6-V, while the ethanol production rates of 6-AF1 and 6-AF1c were 1.21 and 1.78 times of that of strain CE6-V under 1.0% acetic acid stress. Results in this study indicate that acetic acid tolerance and fermentation performance of industrial S. cerevisiae under acetic acid stress can be improved largely by flocculation endowed by expression of flocculent genes, especially FLO1c.
Acetic Acid ; chemistry ; Ethanol ; Fermentation ; Flocculation ; Glucose ; Industrial Microbiology ; Mannose-Binding Lectins ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics

Objective To investigate the protective effects of different doses of fasudil on hepatic ischemia/reperfussion (I/R) injury in rats with liver cirrhosis.Methods Cirrhosis was induced in rats by subcutaneous injection of 60％ carbon tetrachloride (CC14) corn oil solution (0.4 ml/100 g) twice a week for twelve weeks.Five percent of alcohol was given intermittently in drinking water.Then 40 cirrhotic rats were randomized into 4 groups.In sham group,sham operation was performed.In I/R group A and B,whole rat livers were subjected to warm ischemia by clamping the hepatic artery and portal vein for 30 min.In group A,the selected rats were pretreated with low-dose fasudil 1 mg/kg (intraperitoneal injection) 30 min before the induction of ischemia,and in group B,with high-dose 10 mg/kg.The serum levels of alanine aminotransferase (ALT) and endothelin-1 (ET-1),and the liver tissue superoxide dismutase (SOD)activity,malondialdehyde (MDA) content,the expression of HIF-1a (hypoxia-inducible fador-1a) were measured after reperfusion for 6 hours.Hepatic pathologic changes were observed under microscope.Results Compared with I/R group,the serum ALT,AST,ET-1 levels,MDA content and the expression of HIF-1 a were markedly decreased in group B,while the SOD activity significantly increased (P ＜ 0.05).And the pathologic changes were less severe in group B.Conclusion The high-dose fasudil markedly lessened the expression of HIF-1a,up-regulated the concentration of SOD,and lowered the levels of MDA and ET-1,protecting against heoatic ischemia/reperfusion injury in rats with liver cirrhosis.

Saccharomycopsis fibuligera possesses high alpha-amylase and glucoamylase activities that enable it to utilize raw starch as a carbon source. A expression cassette containing the promoter sequence of 3-phosphogylycerate kinase gene (PGK1p), the alpha factor signal sequence from Saccharomyces cerevisiae and the alpha-amylase coding sequence of S. fibuligera was constructed. The alpha-amylase expression cassette was inserted in the ILV2 locus of industrial brewer's yeast strain YSF-5 encoding alpha-acetolactate synthase (AHAS) by homologous recombination. The transformed yeast strain was selected on the media with starch as the sole carbon source and verified by PCR. The transformant exhibited secretive alpha-amylase activity, low AHAS activity and reduced diacetyl production. Effects of temperature, pH, and metal ions on the activity of the alpha-amylase expressed by the transformant were examined. The fermentation performance of host strain YSF-5 and the transformant was also examined.
Beer ; microbiology ; Diacetyl ; metabolism ; Glycogen Synthase Kinase 3 ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomycopsis ; enzymology ; genetics ; alpha-Amylases ; biosynthesis ; genetics ; metabolism

The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pMOXZa-A vector to generate plasmids pMOXZ-HPV16N and pMOXZ-HPV16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPVI6L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37 degrees C for 72 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by H. polymorpha.
Capsid Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Codon ; genetics ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; genetics ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Yeast flocculation is described as a reversible, asexual and calcium dependent process, in which cells adhere to form flocs by interaction of specific cell surface proteins named flocculins on yeast cells with mannose residues present on the cell wall of adjacent yeast cells. Yeast flocculation provides a very economical and convenient pathway for separation of yeast cells from the fermentation broth or removal of heavy metal ions from effluent. A large number of tandem repeats have been found in genes encoding flocculins, which not only have great regulatory effect on the structure and function of flocculins, generating the diversity of flocculation characteristics, but lead to genetic instability in flocculation as well for driving slippage and recombination reactions within and between FLO genes. Here, the research progress in effect of variation of tandem repeats in FLO genes on flocculation characteristics and genetic stability were reviewed to direct and promote the controllable application of flocculation in industrial fermentation process and environmental remediation.
Fermentation ; Flocculation ; Mannose ; Membrane Proteins ; genetics ; Saccharomyces cerevisiae ; genetics ; growth & development ; Saccharomyces cerevisiae Proteins ; genetics ; Tandem Repeat Sequences

Ethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, in the process of industrial production of ethanol, both cell growth and fermentation of ethanologenic S. cerevisiae are dramatically affected by environmental stresses, such as thermal stress. In this study, we improved both the thermotolerance and fermentation performance of industrial ethanologenic S. cerevisiae by combined usage of chemical mutagenesis and genomic DNA mutagenesis-based genetic recombination method. The recombinant S. cerevisiae strain T44-2 could grow at 44 degrees C, 3 degrees C higher than that of the original strain CE6. The survival rate of T44-2 was 1.84 and 1.87-fold of that of CE6 when heat shock at 48 degrees C and 52 degrees C for 1 h respectively. At temperature higher than 37 degrees C, recombinant strain T44-2 always gave higher cell growth and ethanol production than those of strain CE6. Meanwhile, from 30 degrees C to 40 degrees C, recombinant strain T44-2 produces 91.2-83.8 g/L of ethanol from 200 g/L of glucose, which indicated that the recombinant strain T44-2 had both thermotolerance and broad thermal adaptability. The work offers a novel method, called genomic DNA mutagenesis-based genetic recombination, to improve the physiological functions of S. cerevisiae.
Adaptation, Physiological ; DNA, Fungal ; genetics ; Ethanol ; metabolism ; Fermentation ; Hot Temperature ; Industrial Microbiology ; methods ; Mutagenesis ; Recombination, Genetic ; Saccharomyces cerevisiae ; genetics ; metabolism