GENE TECHNOLOGY FOR INDUSTRIAL YEASTS

Yeast genetics is now available as a practical tool for the development of brewing industry practices. The contribution of Brewing Research Foundation work (1978–84) to recent advances is illustrated by the construction of brewing strains with superattenuating (amylolytic) or anti-contaminant properties. Approaches based on hybridisation (by rare mating) or recombinant DNA technology have been evaluated. Techniques developed for (i) gene transfer to brewing strains, (ii) ensuring stable inheritance of novel characteristics and (iii) exploiting the secretory ability of yeast strains, can be widely applied not only with brewing, distilling, baking or wine yeasts, but also in the use of yeasts to produce novel biotechnical products. ‘Spin-off’ from these studies includes valuable methods for differentiating or enumerating wild yeasts in brewery quality control.     

Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA-triphosphatase activities.

The predicted ORF3 polypeptide (Orf3p) of the linear genetic element pGKL2 from Kluyveromyces lactis was expressed in Bacillus megaterium as a fusion protein with a His(6X)-tag at the C-terminus for isolation by Ni-affinity chromatography. This is the first time that a yeast cytoplasmic gene product has been expressed heterologously as a functional protein in a bacterial system. The purified protein was found to display both RNA 5'-triphosphatase and guanylyltransferase activities. When the lysine residue present at position 177 of the protein within the sequence motif (KXDG), highly conserved in capping enzymes and other nucleotidyl transferases, was substituted by alanine, the guanylyltransferase activity was lost, thereby proving an important role for the transfer of GMP from GTP to the 5'-diphosphate end of the mRNA. Our in vitro data provides the first direct evidence that the polypeptide encoded by ORF3 of the cytoplasmic yeast plasmid pGKL2 functions as a plasmid-specific capping enzyme. Since genes equivalent to ORF3 of pGKL2 have been identified in all autonomous cytoplasmic yeast DNA elements investigated so far, our findings are of general significance for these widely distributed yeast extranuclear genetic elements.     

Antimicrobial activity of recombinant human lactoferrin from <Emphasis Type="Italic">Aspergillus awamori</Emphasis>, human milk lactoferrin and their hydrolysates

The antibacterial activity of human lactoferrin from milk (hLF), recombinant human lactoferrin from Aspergillus awamori (rhLF) and their hydrolysates obtained with pepsin was investigated against Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) were determined for all the bacteria and the proteins assayed. Taking into account the MICs found for both lactoferrins studied, we can say that they behave very similarly, except for L. monocytogenes for which rhLF was more active. We studied the effect that heat treatments exerted on the antibacterial activity of the two types of lactoferrin and the only heat treatment that had a negative effect on that activity was 85 °C for 10 min. The activity of hLF and rhLF in UHT milk and whey against E. coli O157:H7 and L. monocytogenes, was also assayed. Our results showed a reduction in the number of viable cells for both microorganisms when were incubated with rhLF or hLF, but this decrease was lower than in broth media.     

伤寒Vi多糖与不同载体蛋白制备的结合物的特性分析

目的比较以白喉类毒素(diphtheria toxoid,DT)和重组铜绿假单胞菌外毒素A(recombinant Pseudomonas aeruginosa exotoxin A,r EPA)为载体蛋白,分别与伤寒Vi多糖(typhoid Vi polysaccharide,STVi)偶联的结合物的理化特性、抗原性及免疫效应。方法分别以DT、r EPA为载体蛋白,以己二酰肼(adipyl dihydrazide,ADH)为连接剂,使Vi多糖与载体蛋白共价偶联制备多糖蛋白结合物STVi-DT和STVi-r EPA,检测其理化特性指标、抗原性,再将其于0、14、28 d免疫NIH小鼠,经眼眶采血,分离血清,采用间接ELISA法检测小鼠血清抗体水平,分析其免疫原性、免疫记忆效应及免疫持久性。结果 STVi-DT、STVi-r EPA理化特性指标相似。STVi-DT针对STVi、DT抗血清均具有明显抗原性,而针对r EPA抗血清无抗原性,STVi-r EPA针对STVi、r EPA抗血清均具有明显抗原性,而针对DT抗血清无抗原性。Vi多糖和Vi多糖蛋白结合物均具有动物免疫原性,STVi蛋白结合物较STVi具有更强的免疫原性,STVi-DT的动物免疫原性试验结果略优于STVi-r EPA;免疫2剂STVi-DT和STVi-r EPA后,小鼠血清GMT明显升高,且STViDT组明显高于STVi-r EPA组(P0.05),具有较强的免疫记忆效应;免疫STVi-DT和STVi-r EPA后,小鼠血清GMT随免疫剂次增加和时间延长持续升高,直至稳定在一定水平,免疫持久性较好。结论 STVi-DT、STVi-r EPA理化特性相似,均具有显著的免疫原性、免疫记忆效应及免疫持久性,而STVi-DT免疫原性、免疫记忆效应更强。     

Bioconversion of corn stover hydrolysate to ethanol by a recombinant yeast strain

Three corn stover hydrolysates, enzymatic hydrolysates prepared from acid and alkaline pretreatments separately and hemicellulosic hydrolysate prepared from acid pretreatment, were evaluated in composition and fermentability. For enzymatic hydrolysate from alkaline pretreatment, ethanol yield on fermentable sugars and fermentation efficiency reached highest among the three hydrolysates; meanwhile, ethanol yield on dry corn stover reached 0.175 g/g, higher than the sum of those of two hydrolysates from acid pretreatment. Fermentation process of the enzymatic hydrolysate from alkaline pretreatment was further investigated using free and immobilized cells of recombinant Saccharomyces cerevisiae ZU-10. Concentrated hydrolysate containing 66.9 g/L glucose and 32.1 g/L xylose was utilized. In the fermentation with free cells, 41.2 g/L ethanol was obtained within 72 h with an ethanol yield on fermentable sugars of 0.416 g/g. Immobilized cells greatly enhanced the ethanol productivity, while the ethanol yield on fermentable sugars of 0.411 g/g could still be reached. Repeated batch fermentation with immobilized cells was further attempted up to six batches. The ethanol yield on fermentable sugars maintained above 0.403 g/g with all glucose and more than 92.83% xylose utilized in each batch. These results demonstrate the feasibility and efficiency of ethanol production from corn stover hydrolysates.     

重组单抗药物的非还原电泳纯度分析

目的对重组单抗药物的非还原电泳纯度进行分析,排除样品制备过程中形成的一些假象。方法结合重组单抗的分子结构特征,改变常规SDS-PAGE条件,将抗体样品在不同的缓冲液及电泳条件下进行比较。结果抗体在非还原SDS-PAGE纯度检测中出现的次带,可通过在供试品缓冲液中加入烷基化试剂封闭自由巯基而几乎全部消除;通过降低电泳过程的环境温度,可有效降低主带上方高相对分子质量带的出现。结论单抗药物中由于含有多对二硫键引起的一些因电泳样品制备而形成的假象带,可以通过试验方法的修正而去除。     

有效利用木糖的肠道细菌的筛选及转化

以木糖为惟一碳源筛选得到了 32株能利用木糖快速生长的肠道细菌 ,初步鉴定结果表明 ,有效利用木糖的菌株多为肺炎克雷伯氏杆菌 ,其次是大肠杆菌 .选择合适的质粒对其中的 94 7菌株、15 6 9菌株及E .coliJM 10 9进行转化 ,检验转化子中的质粒在无选择压力条件下的传代稳定性 ,结果野生型菌株的转化率均明显低于E .coliJM 10 9,质粒 pET 2 8a在所试验的几株菌中稳定性相对较差 ,pKK2 2 3 3能在 94 7菌株中稳定存在 .     

嗜杀苹果酒酵母的构建

采用核融合缺陷原生质体融合技术 ,将嗜杀质粒供体菌 5 0 45 (α ,his4,kar1— 1[KIL—K1 ]rho+ )中的嗜杀质粒转移到受体菌苹果酒酵母AW中去。用 3 5 %PEG ,3 0℃诱导融合 2 0min ,经嗜杀活性检出、小型酿造实验、遗传稳定性实验、细胞大小及体积的测定、dsRNA质粒的提取及琼脂糖凝胶电泳等实验 ,筛选出 4株遗传性状稳定、具有嗜杀活性且发酵性能良好的融合子F12、F3 0、F5 7、F65。     

副溶血性弧菌毒力因子及耐药机制研究进展

副溶血性弧菌是水产品中主要的致病菌之一,既可造成养殖水产品的疾病与死亡,也可引起人类的胃肠炎等疾病,不仅具有潜在的食品安全隐患,也对消费者健康造成潜在危害。该文主要阐述了副溶血性弧菌的毒力因子耐热直接溶血素(thermostable direct hemolysin, TDH)以及分泌系统T3SS1、T3SS2在菌株体内的作用机理,并对副溶血性弧菌产生耐药性的5种机制——产生生物被膜、质粒介导耐药、产生灭活酶或钝化酶类、外排泵主动排出抗菌药物、药物靶点发生改变进行了综述。