重组酿酒酵母5-氨基酮戊酸合酶在大肠杆菌的表达

以酿酒酵母基因组为模板,采用PCR扩增出编码成熟5-氨基酮戊酸合酶(第一个氨基酸残基为Asn63)的基因hem1,将其克隆到pMAL-2x中构建重组表达载体,转化大肠杆菌表达菌株BL21(DE3),在0.5 mmol·L-1的IPTG诱导下,SDS-PAGE显示分子量约为49 kD的特异条带.重组菌在紫外灯下显示红色荧光,表明酶在大肠杆菌体内活性表达.体外反应表明,在浓度超过0.1 mmol·L-1的IPTG诱导下,酶活力下降,培养基的初始pH值约为6.5,5-氨基酮戊酸产量达最大,且主要分泌到胞外.     

耐酸性清酒酵母A发酵工艺研究

[目的]为生产优质清酒奠定理论基础。[方法]以优质粳米为原料,在单因素试验的基础上。采用正交试验法研究耐酸性清酒酵母A的发酵规律。[结果]水料比为1—2时,清酒酒精度较高。水料比为0．5时,发酵醪的糖浓度和渗透压较高。水料比为3时,发酵醪的淀粉浓度降低,清酒酒精度低。酒母量为20％时,利于清酒双边发酵,清酒的可溶物含量约为11％。米曲量为30％～50％时,清酒色度低,苦涩味轻。乳酸添加量为8‰～12‰时,发酵结束后米曲中糖化酶的活力约为290mg/（g·h）。乳酸量超过12‰时,酶活力下降比较快,清酒有异杂味感。15℃下发酵21d的清酒酒精度达17．1％（V／V）,淀粉利用率为88．1％。[结论]利用耐酸性清酒酵母发酵生产清酒,简化了生产工艺,缩短了发酵时间,提高了原料利用率。     

布拉酵母菌对黄曲霉毒素B1脱毒特性研究

本试验研究了不同菌体预处理、不同浓度菌体、不同作用时间、不同pH环境对布拉酵母菌脱除黄曲霉毒素B₁(AFB₁)效果的影响。结果发现,布拉酵母菌脱除AFB₁的主要机理是吸附作用;与2 mol/L HCl 121 ℃处理30 min相比,100 ℃处理30 min更有利于布拉酵母菌对AFB₁的脱毒;强酸性和偏碱性的环境也利于布拉酵母菌对AFB₁的脱毒。     

缺刻缘绿藻二酰甘油酰基转移酶2(DGAT2)的基因特性与功能鉴定

酰基-CoA:二酰甘油酰基转移酶(DGAT)是三酰甘油(TAG)合成过程中的关键酶。在缺刻缘绿藻转录组测序数据库中,通过同源搜索,发现了1个编码DGAT2的cDNA全长序列。该序列长1997 bp,其中,5’-非转录区(UTR)长44 bp,3’-UTR长897 bp,开放阅读框(ORF)长1056 bp,编码一个351个氨基酸的蛋白质,预测的分子量为39.43 kD,等电点为9.46。基于缺刻缘绿藻和其他物种相应的DGAT基因编码蛋白序列所构建的Neighbor-joining(NJ)系统进化树,结果表明该基因与DGAT2聚成一支,显著不同与DGAT1和DGAT3。氨基酸序列比对发现,该基因含有DGAT2所具有的HPHG这4个氨基酸所组成的高度保守特征序列。因此,将该基因命名为MiDGAT2基因。将它的cDNA与其DNA序列进行比较后发现,MiDGAT2基因含有6个内含子,其剪切位点均符合“GT-AG”规则。为进一步了解其功能,利用反转录PCR克隆了该基因的ORF序列,然后将其亚克隆到表达载体pYES2中,成功地构建了重组表达质粒pY-MiDGAT2。通过电穿孔法将该重组质粒转入酿酒酵母TAG合成缺陷株H1246中,经筛选与序列验证得到含有重组质粒pY-MiDGAT2的酵母转化株。酵母转化株在用SC培养基并加入半乳糖诱导表达培养后,其脂类的薄层色谱分析结果表明,所转的MiDGAT2基因能使酵母转化株恢复TAG合成的能力,从而证实了MiDGAT2基因具有DGAT的功能;利用荧光染料Bodipy对酵母细胞的染色结果显示,MiDGAT2基因能使酵母转化株的细胞重现油滴,尽管重建的油滴大小明显比野生型酵母的小。这些研究为缺刻缘绿藻TAG合成代谢途径与调控机理的探讨奠定了基础。     

Evaluations of Hilyses™, fermented Saccharomyces cerevisiae,on rainbow trout (Oncorhynchus mykiss) growth performance,enzymatic activities and gastrointestinal structure

This study examined the effects of Hilyses?, fermented Saccharomyces cerevisiae (S. cerevisiae), on rainbow trout growth performance, haematological parameters, digestive enzyme activities and gastrointestinal structure. Rainbow trout (mean weight 100–110 g) were fed dietary Hilyses? (5 g kg^?1) and control diet without Hilyses? for 50 days. Results of this study demonstrated that yeast supplementation in treatment group resulted in increased feed intake, followed by improved feed conversion ratio (FCR) and growth performance. Significant increases were also observed in trypsin and amylase activities in juvenile fish fed treatment diet. Light microscopy demonstrated that both groups of fish displayed normal morphology of proximal intestine and pyloric caeca. In yeast‐treated group, higher density of the goblet cells per villus in the proximal intestine was shown. No effects on haematological parameters and carcass chemical composition were noted. It is therefore possible to use fermented S. cerevisiae supplementation to significantly improve the gastrointestinal structure and growth performance in rainbow trout.     

酿酒酵母ADH2基因沉默表达载体的构建

利用重叠延伸PCR获得酿酒酵母基因沉默组件SADH2,经酶切与酿酒酵母穿梭质粒pYES2.0连接,构建酿酒酵母ADH2基因沉默表达载体。经酶切及测序鉴定成功构建了ADH2基因沉默表达载体pSADH2。     

Evaluating the potential of wine-making residues and corn cobs as support materials for cell immobilization for ethanol production

Three wine-making residues (grape seeds, skins and stems), and corn cobs were evaluated as support material for immobilization of Saccharomyces cerevisiae and the ethanol production by the immobilized cells was assessed. The main objective of this study was to find an abundant and low cost material suitable for the cells immobilization and able to be used in a next step of wine production by immobilized yeast cells. The four natural materials were used as support in two different forms: untreated, and treated by a sequence of acid and basic reactions. Untreated grape skin and corn cobs provided the highest cell immobilization results (25.1 and 22.2 mg cells/g support, respectively). The maximum ethanol production yield (about 0.50 g/g) was also obtained when the cells were immobilized in these untreated materials. It was also found that the support materials released nutrients to the medium, which favored the yeast development and the ethanol production. The use of immobilized cells systems under agitated conditions gave ethanol yields similar to those obtained by the static fermentations, but the immobilized cell concentration was significantly lower. In brief, static fermentation with cells immobilized on grape skins or corn cobs appear to be an interesting alternative for use on wine-making. The use of grape skins, particularly, which is a by-product of the wine elaboration, could be of larger interest to obtain an integrated wine production process with by-product reuse.     

六个甘蓝型油菜油酸脱氢酶（FAD2）假基因的克隆和分析

以甘蓝型油菜湘油15为材料,采用PCR方法克隆并分析了56个FAD2基因克隆和47个FAD2基因cDNA克隆,从中发现6个新的FAD2基因拷贝。它们与公布的甘蓝型油菜FAD2基因(AY577313)具有87%以上的同源性,没有内含子,在开放阅读框中存在1~12个终止密码子,其中有2个拷贝具有转录功能。将这6个FAD2基因拷贝在酿酒酵母中进行体内表达实验,通过气相色谱检测脂肪酸组成证明其不具备油酸脱氢酶功能,与对照相比,也没有改变酵母体内脂肪酸组成。由此推测这6个FAD2基因拷贝为假基因。     

超氧化物歧化酶在酿酒酵母碱胁迫抗性中的功能研究

[目的]研究酿酒酵母超氧化物歧化酶与其碱胁迫抗性的相关性。[方法]采用可见分光光度法,测定碱胁迫条件下酵母菌超氧化物歧化酶活性（SOD）指标;采用平板滴定生长试验,检测酿酒酵母铜/锌超氧化物歧化酶缺失菌株（sod1Δ）和锰超氧化物歧化酶缺失菌株（sod2Δ）的碱胁迫抗性;采用台盼蓝染色,检测碱胁迫条件下酵母细胞的存活率。[结果]在pH值为7.9条件下处理2h可明显诱导酵母细胞内SOD活性升高;与野生型酵母菌株相比,sod1Δ和sod2Δ基因缺失菌均表现碱胁迫敏感表型,并且碱胁迫条件下细胞存活率明显下降。[结论]超氧化物歧化酶在调控酿酒酵母碱胁迫抗性中发挥重要作用。     

复合微生态制剂对肉仔鸡血清生化指标和十二指肠组织形态的影响

本试验旨在研究由鼠李糖乳杆菌和啤酒酵母共生培养物制备的复合微生态制剂对肉仔鸡血清生化指标和十二指肠组织形态的影响。试验选用1日龄健康艾维茵肉仔鸡240只,随机分为6个组,每组4个重复,每个重复10只鸡。对照组饲喂基础日粮,抗生素组饲喂含600 mg/kg金霉素的基础日粮,4个试验组饲喂基础日粮,并在饮水中分别添加0.1%、0.5%、1.0%、1.5%复合微生态制剂。试验结果表明,复合微生态制剂能有效提高肉仔鸡血清钙、磷、总蛋白、球蛋白和白蛋白的含量(P＜0.05),能降低胆固醇的含量和白球比(P＜0.05),并使十二指肠肠绒毛密度增加,长度显著增长(P＜0.05)。复合微生态制剂对肉仔鸡血清中某些生化指标和十二指肠组织形态有一定的影响。