Respiration-deficient mutants of Zymomonas mobilis show improved growth and ethanol fermentation under aerobic and high temperature conditions

Respiration-deficient mutant (RDM) strains of Zymomonas mobilis were isolated from antibiotic-resistant mutants. These RDM strains showed various degrees of respiratory deficiency. All RDM strains exhibited much higher ethanol fermentation capacity than the wild-type strain under aerobic conditions. The strains also gained thermotolerance and exhibited greater ethanol production at high temperature (39°C), under both non-aerobic and aerobic conditions, compared with the wild-type strain. Microarray and subsequent quantitative PCR analyses suggest that enhanced gene expression involved in the metabolism of glucose to ethanol resulted in the high ethanol production of RDM strains under aerobic growth conditions. Reduction of intracellular oxidative stress may also result in improved ethanol fermentation by RDM strains at high temperatures.     

红曲色素及其在食品工业中的应用

红曲色素是一种天然食用色素。从古到今 ,它在食品工业中有着广泛的应用。本文从其来源和主要成分、性质和特点、应用、发展趋势四个方面对红曲色素作了阐述 ,并着重于它在食品工业中的应用。     

红曲霉菌的应用现状及发展前景

阐述了红曲霉菌的生理活性物质在医药、食品、酿酒领域的应用现状及存在问题,并展望了红曲霉菌培养物产业的发展前景。     

Cloning and sequencing of the URA3 gene of Kluyveromyces marxianus CBS 6556

The URA3 gene, coding for orotidine-5′-phosphate decarboxylase, from Kluyveromyces marxianus CBS 6556, was isolated from a genomic DNA library. The K. marxianus URA3 gene encodes a protein of 267 amino acids with a calculated molecular weight of 29·3 kDa. Comparison of the K. marxianus protein with the corresponding enzymes of Saccharomyces cerevisiae and Kluyveromyces lactis showed amino acid sequence identifies of 81% and 88%, respectively. Using contour-clamped homogeneous electric field gel electrophoresis, the genomic copy was found to be located on chromosome VI. We have used the cloned gene for the construction of a K. marxianus leu2 mutant. This mutant contains no heterologous sequences, which is essential to make it acceptable for application in the food industry.     

红曲中桔霉素的薄层层析分析

红曲产品中可能含有桔霉素，己使我国的红曲出口受到很大影响。根据国内外对桔霉素的最新研究报告，采用薄层层析法，对红曲中桔霉素的提取方法和展开剂分别进行研究。结果表明，以酸性水溶液作为提取剂，以甲苯：乙酸乙酌：甲酸(6：3：1)为展开剂，桔霉素的分离检测效果较好。在此基础上对22个红曲样进行了分析，结果显示，大部分红曲都含桔霉素，但含量高低不一，从中获得桔霉素含量低而色价高的红曲霉菌株有3个。     

Isolation and characterization of acid-sensitive mutants of Pediococcus acidilactici

Acid-sensitive mutants of Pediococcus acidilactici BCC 9545, a starter culture of the Thai fermented pork sausage nham, were isolated as spontaneous neomycin resistant mutants. The mutants generally produced less acid and acidified the culture media less than the parent strain in a 72 h culturing period. Interestingly, the ATPase activities of the mutants did not differ considerably from that of the parent strain in acidic conditions. It was also found that the internal pH values of the mutant strains were somewhat lower in neutral environment, while at pH 5.0 their internal pHs were significantly lower compared to the parent's. Inhibiting the H⁺-ATPase activities in energized cells by N,N′-dicyclohexyl carbodiimide also revealed that protons were leaking from the mutants at neutral pH, which increased under acidic conditions. In contrast, the parent strain exhibited a smaller proton leak and only under acidic conditions. The membrane fatty acid analysis of the mutants indicated that under acidic conditions the mutants had a significantly smaller major unsaturated/saturated fatty acids ratio ((C_18:1 + C_18:3n6)/(C_16:0 + C_18:0)) compared to the parent strain's membrane. Taken together, these observations suggest there is a reasonable possibility that the membrane fatty acid profile differences in the mutants resulted in their acid-sensitivity.     

Hierarchical strategy for protein folding and design: synthesis and expression of T4 lysozyme gene and two putative folding mutants

A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesizedand cloned by ligation of 26 deoxyribooligo-nucleotide fragmentsin two steps with a linearized plasmid followed by transformation.On selection by colony hybridization and DNA sequence analysis,clone pTLY.10 was identified to contain a complete T4 lysozymesynthetic DNA. On expression under lac-promoter, unfused T4lysozyme was obtained in {small tilde}4–6% yield. Thedesign and synthesis of two putative folding mutants, flexible(Gly-Gly-Gly) and rigid (Asn-Asp-Gly) at position 73-74-75,were based on hierarchical principles. Both mutants lost enzymaticactivity of the wildtype. These results are readily understandableif the hierarchical organization of the structure is taken intoaccount. A possible explanation is that the catalytic sitesare blocked in both mutants.     

Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin

The loop exchange mutant chymosm 155–164 rhizopuspepsinwas expressed in Trichoderma reesei and exported into the mediumto yield a correctly folded and active product. The biochemicalcharacterization and crystal structure determination at 2.5Å resolution confirm that the mutant enzyme adopts a nativefold. However, the conformation of the mutated loop is unlikethat in native rhizopuspepsin and involves the chelation ofa water molecule in the loop. Kinetic analysis using two syntheticpeptide substrates (six and 15 residues long) and the naturalsubstrate, milk, revealed a reduction in the activity of themutant enzyme with respect to the native when acting on boththe long peptide substrate and milk. This may be a consequenceof the different charge distribution of the mutated loop, itsincreased size and/or its different conformation.     

Restoration of peroxisome formation in two conditional peroxisome-deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources

Expression of the peroxisome-deficient (Per^?) phenotype by per mutants of Hansenula polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6^ts and per14-11^ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D -alanine (for both mutants) or methylamine (for per14-11^ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition. However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno) cytochemical analyses indicated that during growth of cells on methanol in the presence of either D -alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.