胡萝卜表面产纤维素酶细菌初步鉴定分析

从胡萝卜表面筛选到一株产纤维素酶活性较高的菌株HP1。使用PCR方法获得此菌株长度为1435bp的16SrDNA序列。利用生物信息学方法在美国国立生物技术信息中心NCBI基因库比对分析了该序列,发现其与Bacilluscereus相似性最高,为99%。结合菌落观察和生理生化分析,初步确定菌株HP1为Bacillussp.。序列和生化分析显示其是一株新型的芽孢杆菌,其在NCBI上的登录号是:FJ908092。     

嗜盐性乳酸菌的生理与分子生物学鉴定

对从酱醅中分离筛选出的耐高渗嗜盐性乳酸细菌, 进行了生理生化鉴定, 结果如下：该菌革兰氏染色呈阳性,过氧化氢酶反应呈阴性,可以在pH 5.0下生长, 在18%或更高的NaCl浓度中能够正常生长代谢, 从葡萄糖主要产L（＋）乳酸,属于四联球菌属的嗜盐性乳酸球菌. 进一步对该菌进行分子生物学鉴定,测得菌株的16S rRNA序列与GenBank登记号为FSB201的嗜盐四联球菌（Tetragenococcus halophilus）具有99%的同源性.再将该16S rRNA序列与乳酸菌分类中17个模式菌株的16S rRNA序列共同创建进化树,发现筛选菌与GenBank登记号为AB106344、AB106343的嗜盐四联球菌亲缘关系最近,证明筛选菌应属四联球菌属中嗜盐四联球菌中的一种。     

肉鸡中魏斯菌属细菌的分离与鉴定

应用16S DNA基因测序法对肉鸡中分离出的可疑魏斯菌属细菌菌株进行鉴定。从沈阳某养鸡场抽取病死鸡在无菌条件下采集样品10份,微需氧条件下进行细菌分离纯化,比对魏斯菌属的基本形态和革兰氏染色特点,得到与魏斯菌属类似菌株14株。采用16S rDNA基因测序方法对分离出的细菌进行基因序列分析。在NCBI网站用BLAST软件在GenBank上进行同源性比较,比对结果表明编号为X1001、X1002、X1006、X1008、X1009的株菌同源性达90%。生化实验结果表明,X1006号菌株为魏斯菌属中的融合魏斯菌属菌株。     

一株渗透压补偿溶质Ectoine生成菌的分离及其分子生物学鉴定

从大连普兰店盐场盐池底土壤中筛选获取一株产渗透压补偿溶质Ectoine的耐盐菌株SL39,其16SrDNA序列分析表明,菌株SL39与Halobacillustrueperi具有100%同源性。该菌株最适生长温度为30℃,最适初始pH为7.0,能够在0￣25%(w/v)NaCl浓度范围内生长,最适生长NaCl浓度为5%(w/v)。SL39菌株在2mol/LNaCl的培养基中诱导能合成Ectoine,在30℃诱导培养48h其合成量为304.8mg/L。     

高渗胁迫下啤酒酵母的生理特性研究

研究了0．3mol／L、0．6mol／L、1．0mol/L的氯化钠对啤酒酵母活细胞数、细胞形态、糖代谢、pH值、菌体蛋白的影响。细胞动力学结果表明：随着NaCl浓度的增加,细胞的渗透胁迫加剧,细胞收缩。与对照组比较0．6mol／LNaCl、1．0mol／L NaCl延长细胞对数生长期,减缓葡萄糖消耗速率,且分别在2h-4h、6h-8h内出现糖代谢停滞,但并不影响细胞的增殖。在生理可接受的渗透范围内,与对照组比较0．3mol／LNaCl基本上不影响啤酒酵母的生物量、糖代谢速率和pH值。说明该菌株有较高的抗高渗能力。随着NaCl浓度的升高,渗透胁迫加剧,耐高渗有关的蛋白开始表达。     

产胆盐水解酶BSH乳酸菌的鉴定及发酵条件优化

目的:鉴定降胆固醇和产胆盐水解酶的乳酸菌菌株,并通过优化发酵条件提高胆盐水解酶酶活.方法:对初步筛选的乳酸菌菌株Lp-1进行体外降胆固醇试验,采用PCR方法鉴定菌株中的胆盐水解酶基因,用16SrRNA方法鉴定该菌株,并对该菌株发酵培养基和发酵条件进行优化.结果:乳酸菌菌株Lp-1具有体外降胆固醇功能,基因组中含有胆盐水解酶基因,其脱氧核糖核苷酸序列与已发表的胆盐水解酶基因同源性为98.9%;该菌株与已发表的植物乳杆菌16S rRNA同源性为99%;通过发酵条件优化,以葡萄糖为碳源,蛋白胨、酵母粉为氮源,碳氮比1∶1,初始pH 6.5,接种量7%,在37 ℃下培养14 h,胆盐水解酶酶活最高达到11.2 U/mL.结论:鉴定到1株植物乳杆菌Lp-1(Lactobacillius plantarum Lp-1),该菌含有胆盐水解酶基因,并产胆盐水解酶;发酵条件优化后该菌产胆盐水解酶活力比优化前提高6倍.     

一株产纳豆激酶菌株的分离筛选及鉴定

对能产生纳豆激酶的菌群进行了筛选,分离得到了1株具有较高纤溶活性的菌株N391,其纳豆激酶相当于1722.4U/mL尿激酶的酶活。利用细菌16S rDNA通用引物对其16S rRNA进行PCR扩增,得到1511bp的片段,该PCR产物序列通过Blast软件在NCBI网站中进行同源性比较,通过DNA MAN和MAGE3.1软件绘制系统发育树,结果表明,菌株N391的16S rRNA序列(DQ906100)与枯草芽胞杆菌(Bacillus subtilis)的16S rRNA序列的同源性在99%以上,在系统发育树中,菌株N391与Bacillus subtilis在同一分支,且遗传距离最短。结合常规的形态、生理生化鉴定,N391的形态及大部分生理生化特征与Bacillus subtilis极为相似,表明菌株N391属于Bacillus subtilis的1个菌株,由此初步确定该菌在微生物系统发育学上的地位。     

高渗胁迫下啤酒酵母的生理特性研究

研究了0．3mol／L、0．6mol／L、1．0mol/L的氯化钠对啤酒酵母活细胞数、细胞形态、糖代谢、pH值、菌体蛋白的影响。细胞动力学结果表明：随着NaCl浓度的增加,细胞的渗透胁迫加剧,细胞收缩。与对照组比较0．6mol／LNaCl、1．0mol／L NaCl延长细胞对数生长期,减缓葡萄糖消耗速率,且分别在2h-4h、6h-8h内出现糖代谢停滞,但并不影响细胞的增殖。在生理可接受的渗透范围内,与对照组比较0．3mol／LNaCl基本上不影响啤酒酵母的生物量、糖代谢速率和pH值。说明该菌株有较高的抗高渗能力。随着NaCl浓度的升高,渗透胁迫加剧,耐高渗有关的蛋白开始表达。     

马铃薯α-淀粉酶基因的克隆及生物信息学分析

利用RT-PCR 方法从马铃薯(Solanum tuberosum)茎段总RNA 中扩增、克隆amyA1 基因(NCBI 登录号GQ406048.1)。采用半定量RT-PCR 方法检测amyA1 基因在马铃薯茎、叶等不同组织中的表达强度,表明在茎组织中的表达丰度略高。利用生物信息学软件分析amyA1 密码子的偏好性;同时对AmyA1 氨基酸的理化性质、细胞内定位、保守结构及高级结构进行预测。基于NCBI 数据库中有物种代表性的29 种α - 淀粉酶基因序列构建了基因进化树。amyA1 基因全长1224bp,可编码一条理论分子质量为46.40kD、407 个氨基酸残基组成的、可能为亲水性的胞外酶。与NCBI已登记的马铃薯α - 淀粉酶基因(登录号M79328.1)核苷酸及氨基酸序列同源性达98%。第20～348 位点范围内的氨基酸残基含有与淀粉酶13 家族及亚家族相似的催化活性域(PF00128、SM00624),第349～407位点范围内的氨基酸残基含有α - 淀粉酶C- 末端β折叠区域(PF07821)。蛋白质结构预测表明氨基酸残基序列有维持淀粉酶活性的(β /α) 8 桶状结构以及其他几个功能域结构。所构建的基因进化树表明,两个马铃薯α - 淀粉酶基因与木薯、苹果的序列同源性较高,与菜豆的次之,与水稻、大麦、玉米等单子叶植物的序列同源性较低。     

甘油代谢对酿酒酵母酒精发酵的影响

以工业酒精酵母菌株(Saccharomyces cerevisiae Y)为对象,通过生孢子法获得其a型和α型单倍体.根据同源重组原理构建整合载体pUC19-GPD1::Q Km 线性化后电击转化进入a型单倍体.G418抗性筛选缺失GPD1基因的转化子S.cerevisiae Y(a,△gpd1).重组菌S.cerevisiae Y(a.△gpd1)与α型单倍体杂交获得二倍体,采用PCR扩增法鉴定得到GPD1全突变菌株S.cerevisiae Y(△gpd1,△gpd1).分别以20 g/L和150 g/L的初糖在摇瓶中进行发酵实验考察.结果表明,S.cerevisiae Y(a,△gpd1)的葡萄糖转甘油率分别从12.43%和6.04%降低到9.35%和5.54%,葡萄糖转酒精率分别从36.4.9%和39.96%提高到36.91%和40.29%;而S.cerevisiae Y(△god1,△gpd1)的葡萄糖转甘油率分别从14.16%和7.49%降低到10.46%和5.79%,葡萄糖转酒精率分别从36.23%和39.89%提高到38.52%和41.50%.     

Expression of the GPD1 and GPP2 orthologues and glycerol retention during growth of Debaryomyces hansenii at high NaCl concentrations

The highly NaCl-tolerant yeast Debaryomyces hansenii produces and obtains high levels of intracellular glycerol as a compatible solute when grown at high NaCl concentrations. The effect of high NaCl concentrations (4%, 8% and 12% w/v) on the glycerol production and the levels of intra- and extracellular glycerol was determined for two D. hansenii strains with different NaCl tolerance and compared to one strain of the moderately NaCl-tolerant yeast Saccharomyces cerevisiae. Initially, high NaCl tolerance seems to be determined by enhanced glycerol production, due to an increased expression of DhGPD1 and DhGPP2 (AL436338) in D. hansenii and GPD1 and GPP2 in S. cerevisiae; however, the ability to obtain high levels of intracellular glycerol seems to be more important. The two D. hansenii strains had higher levels of intracellular glycerol than the S. cerevisiae strain and were able to obtain high levels of intracellular glycerol, even at very high NaCl concentrations, indicating the presence of, for example, a type of closing channel, as previously described for other yeast species.     

luxS基因对盐胁迫下植物乳杆菌生长及细菌素合成的影响

以植物乳杆菌KLDS1.0391野生株及其luxS基因缺失突变株为研究对象,探究luxS基因对该菌盐耐受能力的影响,并测定在0%、2%、3%、4%和6%质量分数NaCl胁迫条件下,luxS基因缺失对该菌生长及细菌素合成的影响,采用实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）技术在转录水平上测定该菌细菌素合成相关基因的表达水平。结果表明：随NaCl质量分数的升高,2 株菌的存活率均逐渐下降,当NaCl质量分数大于3%时,相同条件下野生株对盐的耐受能力显著强于突变株（P＜0.05）;除此之外,NaCl质量分数越高,菌株进入稳定期的时间越向后延迟,luxS基因缺失突变株在各NaCl质量分数下生长及细菌素合成能力均显著弱于野生株（P＜0.05）。实时荧光定量PCR结果显示,当培养至稳定期24 h时,细菌素结构基因plnEF以及双组分调节基因plnD和plNC8HK均因luxS基因的缺失,表达显著下调（P＜0.05）。因此,luxS基因缺失显著降低植物乳杆菌KLDS1.0391盐耐受能力,并且在NaCl胁迫条件下,该菌生长及细菌素合成能力也因luxS基因缺失而显著下降。     

Over-expression of GSH1 gene and disruption of PEP4 gene in self-cloning industrial brewer's yeast

Foam stability is often influenced by proteinase A, and flavor stability is often affected by oxidation during beer storage. In this study, PEP4, the gene coding for proteinase A, was disrupted in industrial brewing yeast. In the meantime, one copy of GSH1 gene increased in the same strain. GSH1 is responsible for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for synthesis of glutathione which is one kind of important antioxidant and beneficial to beer flavor stability. In order to improve the brewer's yeast, plasmid pYPEP, pPC and pPCG1 were firstly constructed, which were recombined plasmids with PEP4 gene, PEP4's disruption and PEP4's disruption+GSH1 gene respectively. These plasmids were verified to be correct by restriction enzymes' assay. By digesting pPCG1 with AatII and PstI, the DNA fragment for homologous recombination was obtained carrying PEP4 sequence in the flank and GSH1 gene internal to the fragment. Since self-cloning technique was applied in the study and the modified genes were from industrial brewing yeast itself, the improved strains, self-cloning strains, were safe to public. The genetic stability of the improved strains was 100%. The results of PCR analysis of genome DNA showed that coding sequence of PEP4 gene had been deleted and GSH1 gene had been inserted into the locus of PEP4 gene in self-cloning strains. The fermentation ability of self-cloning strain, SZ-1, was similar to that of the host. Proteinase A could not be detected in beer brewed with SZ-1, and GSH content in the beer increased 35% compared to that of the host, Z-1.     

腌鱼中耐盐酵母菌的分离鉴定及其生理生化特性

为了获得生产性能优良的菌株,采用PDA固体培养基平板涂布法从腌鱼中分离、纯化酵母菌,研究优势耐盐酵母菌株的生长温度、耐酸能力、耐亚硝酸盐能力等生理生化特性及ITS rDNA分子生物学鉴定。结果表明,15种不同种类腌制海鱼中共分离出436株酵母菌,经纯化后获得9株不同种属的酵母菌,通过不同NaCl含量的YPD液体培养基对9株酵母菌进行筛选,最终筛选出7#季也蒙毕赤酵母和9#奥默柯达酵母两株耐盐性较好的优势耐盐酵母菌菌株,耐盐能力分别达到9%和12%,最适生长温度均在28~32 ℃之间,最适pH在5~6之间,耐亚硝酸盐含量可达100 mg/kg。腌鱼中获得优势菌株具有良好的生长性能,可为腌鱼制品发酵剂的开发奠定基础。     

Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis

Glycerol is formed as a by-product in production of ethanol and baker's yeast during fermentation of Saccharomyces cerevisiae under anaerobic and aerobic growth conditions, respectively. One physiological role of glycerol formation by yeast is to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD(+). The objective of this study was to evaluate whether introduction of a new pathway for reoxidation of NADH, in a yeast strain where glycerol synthesis had been impaired, would result in elimination of glycerol production and lead to increased yields of ethanol and biomass under anaerobic and aerobic growth conditions, respectively. This was done by deletion of GPD1 and GPD2, encoding two isoenzymes of glycerol 3-phosphate dehydrogenase, and expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii, encoded by cth. In anaerobic batch fermentations of strain TN5 (gpd2-Delta1), formation of glycerol was significantly impaired, which resulted in reduction of the maximum specific growth rate from 0.41/h in the wild-type to 0.08/h. Deletion of GPD2 also resulted in a reduced biomass yield, but did not affect formation of the remaining products. The modest effect of the GPD1 deletion under anaerobic conditions on the maximum specific growth rate and product yields clearly showed that Gdh2p is the important factor in glycerol formation during anaerobic growth. Strain TN6 (gpd1-Delta1 gpd2-Delta1) was unable to grow under anaerobic conditions due to the inability of the strain to reoxidize NADH to NAD(+) by synthesis of glycerol. Also, strain TN23 (gpd1-Delta1 gpd2-Delta1 YEp24-PGKp-cth-PGKt) was unable to grow anaerobically, leading to the conclusion that the NAD(+) pool became limiting in biomass synthesis before the nucleotide levels favoured a transhydrogenase reaction that could convert NADH and NADP(+) to NADPH and NAD(+). Deletion of either GPD1 or GPD2 in the wild-type resulted in a dramatic reduction of the glycerol yields in the aerobic batch cultivations of strains TN4 (gpd1-Delta1) and TN5 (gpd2-Delta1) without serious effects on the maximum specific growth rates or the biomass yields. Deletion of both GPD1 and GPD2 in strain TN6 (gpd1-Delta1 gpd2-Delta1) resulted in a dramatic reduction in the maximum specific growth rate and in biomass formation. Expression of the cytoplasmic transhydrogenase in the double mutant, resulting in TN23, gave a further decrease in micromax from 0.17/h in strain TN6 to 0.09/h in strain TN23, since the transhydrogenase reaction was in the direction from NADPH and NADP(+) to NADH and NADP(+). Thus, it was not possible to introduce an alternative pathway for reoxidation of NADH in the cytoplasm by expression of the transhydrogenase from A. vinelandii in a S. cerevisiae strain with a double deletion in GPD1 and GPD2.     

天然优良葡萄酒酵母发酵特性研究

以实验室保藏的16株葡萄酒酵母为供试菌株,通过模拟酒精发酵初筛获得发酵力强、产酒度高或产香好的酵母5株。以2株商品酵母为对照,采用杜氏管发酵法研究其对NaCl、SO2、酒精、酸碱、温度和高糖的耐受性。结果表明,所有菌株在SO2200～600 mg/L、葡萄糖400～600 g/L浓度范围内都能发酵产气,且在pH1.5～13.0范围内可生长,其中有5株最高耐NaCl为100 g/L以及22%vol的乙醇、2株耐温最高达70℃。葡萄酒酿造结果显示,菌株Y-6发酵酒度高于2个商品酵母,总酸和挥发酸低,且其发酵葡萄酒具有浓郁的果香且香气持久、口感协调,有望应用于特色葡萄酒生产中。