环境介质对β-淀粉酶活性的影响

考察部分环境介质对β-淀粉酶活性影响。结果表明：Ca^2＋、Ba^2＋、海藻酸钠以及聚乙烯基吡咯烷酮（PVPK30）在一定浓度范围内对β-淀粉酶有激活作用，激活作用尤以Ca^2＋最显著，当Ca^2＋浓度为30mmol·L^-1时，可使β-淀粉酶的活性提高近3倍；十二烷基硫酸钠、曲拉通-100、SO3^2-等对β-淀粉酶的活性有抑制作用；SO4^2-、S2O3^2-、明胶等对β-淀粉酶的活性影响不大；Mg^2＋、吐温-80在不同浓度条件下有不同的作用。     

乙醛脱氢酶基因过表达酿酒酵母在黄酒中降高级醇作用

为降低黄酒中高级醇产量,以酿酒酵母（Saccharomyces cerevisiae）单倍体菌株AY12α为出发菌,利用同源重组技术构建乙醛脱氢酶基因过表达菌株,并在不同黄酒发酵工艺下探究其对酿酒酵母产高级醇的影响。结果表明,成功构建了5株乙醛脱氢酶基因（ALD2、ALD3、ALD4、ALD5和ALD6）过表达菌株,在大米加麦曲的传统黄酒发酵工艺下,所有改造菌株均可以促进乙酸、降低高级醇的生成,其中改造菌株α-ALD6效果最显著（P＜0.01）,乙酸产量是出发菌AY12α的2.92倍,总高级醇产量下降72.04%。在不同发酵工艺中,改造菌株α-ALD5与α-ALD6均可显著促进乙酸、降低高级醇生成（P＜0.01）;在纯种根霉曲做糖化剂的发酵工艺中,改造菌株在培菌糖化24 h后接入降高级醇效果更好。与纯种根霉曲相比,改造菌株以麦曲作糖化剂时降高级醇效果更好,且改造菌株α-ALD6在大米加麦曲的传统黄酒发酵工艺下降高级醇效果最好。     

响应面法优化酿酒酵母乙醛脱氢酶和乙醇脱氢酶酶活检测方法

从酿酒酵母前处理方式方面对ALD和ADH酶活测定条件进行了优化,采用单因素试验对前处理方式进行了分析研究,结果表明破壁时间、超声波功率、超声时间、反应温度和缓冲液pH值对酶活测定值产生一定的影响.通过Box-Behnken中心组合设计和响应面分析法,确定了测定这2种酶的最理想酵母前处理条件,即超声功率420W,超声时间为11min,破碎时间为12min.     

The ALD6 gene of Saccharomyces cerevisiae encodes a cytosolic,Mg2+-activated acetaldehyde dehydrogenase

The deduced translation product of an open reading frame on the left arm of chromosome XVI of Saccharomyces cerevisiae, with the systematic name of YPL061w, is 500 amino acids in length and shares significant homology with aldehyde dehydrogenases. Amino acids 2 to 16 of the protein encoded by YPL061w were found to be identical to the N-terminal 15 amino acids of the purified cytosolic, Mg²⁺-activated acetaldehyde dehydrogenase (ACDH) of S. cerevisiae. This enzyme is thought to be involved in the production of acetate from which cytosolic acetyl-CoA is then synthesized. Deletion of YPL061w was detrimental to the growth of haploid strains of yeast; an analysis of one deletion mutant revealed a maximum specific growth rate (in complex medium containing glucose) of one-third of that displayed by the wild-type strain. Mutants deleted in YPL061w were also unable to use ethanol as a carbon source. As expected, the cytosolic, Mg²⁺-activated ACDH activity had been lost from the mutants, although the mitochondrial, K⁺-activated ACDH was readily detected. YPL061w has been registered with the name of ALD6 in the Saccharomyces Genome Database and the nucleotide sequence submitted to GenBank as part of accession number U39205. © 1997 John Wiley & Sons, Ltd.     

酿酒酵母产乙醇脱氢酶发酵条件的研究

选择了一株酿酒酵母菌(Saccharomyces cerevisiae)As2.399,通过单因素和正交实验得到酿酒酵母产乙醇脱氢酶(Alcohol Dehydrogenase,ADH)的最优发酵条件。结果表明:产ADH的最优培养条件是温度为25℃、pH为5、通气量为250mL、接种量为7%,液体发酵培养中pH对ADH活性的影响最大。      

乙醛脱氢酶的克隆表达及其酶学性质

将Gene Bank中人源乙醛脱氢酶2的基因序列根据酿酒酵母(Saccharomyces cerevisiae)的密码子偏好性进行密码子优化后合成目的基因ALDH2,采用融合PCR技术,构建人源ALDH2基因的表达组件TRP1L-URA3-TPIp-ALDH2-TPIt-TRP1R。通过电转化的方法将基因表达组件通过同源重组整合到酿酒酵母W303-1A的基因组上,获得基因工程菌W303-ALDH2。工程菌发酵后的粗酶液经His TrapTMexcel亲和层析柱纯化获得重组ALDH2,其活性为20.70 U/mg;重组酶的相对分子质量是56 k Da;最适反应pH和温度分别为5.0和35℃;除Na+外,K+、Ca2+、Mg2+、Mn2+均能不同程度地提高ALDH2的酶活。以乙醛为底物测得酶的Km值为0.908 mmol/L,最大反应速度Vmax为114.94 U/mg;以辅酶NAD+为底物测得酶的Km值为0.282 mmol/L,最大反应速度Vmax为58.82 U/mg。     

表没食子儿茶素没食子酸酯与L-茶氨酸对乙醇脱氢酶和乙醛脱氢酶活性的体外协同作用

目的 探究表没食子儿茶素没食子酸酯(epigallocatechingallate,EGCG)与L-茶氨酸对乙醇脱氢酶(alcohol dehydrogenase, ADH)和乙醛脱氢酶(acetaldehyde dehydrogenase, ALDH)活性的体外协同作用。方法 设置不同浓度及组分,以0μg/mL实验组为对照,先后进行单因素实验和最优组合验证实验,通过对ADH、ALDH活性的检测,确认EGCG、L-茶氨酸不同剂量的解酒效果并探究其最佳配比。结果 单因素实验中,样品质量浓度为200、300μg/mL实验组相较于0μg/mL浓度组,随着EGCG和L-茶氨酸的添加, ADH和ALDH活性显著高于空白组(P<0.05);最优组合验证实验中,11组不同配比组合物对ADH和ALDH活性均具有促进作用,且协同组比单因素组激活效果要更好。其中,当配比组合物为7:3时, ADH、ALDH的活性最高。结论 EGCG、L-茶氨酸解酒组合物最佳配比为7:3时ADH与ALDH活性最高,研究结果为EGCG、L-茶氨酸组合物用于抗醉酒药物研发提供科学依据。     

基于响应面法优化酿酒酵母培养体系

该研究以小麦胚芽为原料,2,6-二甲氧基对苯醌（2,6-DMBQ）产量为评价指标,对发酵菌种进行筛选并考察原料预处理方式的影响。在此基础上,采用单因素试验及正交试验对发酵条件进行优化。结果表明,最适发酵菌种为低糖面包酵母;最佳原料预处理方式为超声处理;低糖面包酵母发酵小麦胚芽产2,6-二甲氧基对苯醌的最优发酵条件为菌种添加量1∶8（酵母与麦胚质量比）,料液比1∶15（麦胚与水质量比）,发酵时间42 h,发酵温度28 ℃。在此最优发酵条件下,2,6-二甲氧基对苯醌的产量最高为（765.23±9.11） μg/g。     

酿酒酵母蛋白提取工艺优化

采用响应面法优化酵母蛋白提取工艺,实验结果表明,酵母蛋白最佳提取方法为0.1%Na Cl、0.97%Na OH、2.0%十二烷基硫酸钠（SDS）和0.77%曲拉通X-100。在此条件下,酵母蛋白提取的理论值为168.68 mg/g（干重）,实际提取量为147.57 mg/g（干重）,占干酵母质量的14.76%。四溶剂复合法可有效提取酵母蛋白,为提取酵母蛋白提供了方法基础。      

A short-chain dehydrogenase gene from Pichia stipitis having D-arabinitol dehydrogenase activity

An NAD⁺-dependent D-arabinitol dehydrogenase (polyol dehydrogenase) gene was isolated from Pichia stipitis CBS 6054 and cloned in Saccharomyces cerevisiae. The gene was isolated by screening of a λ-cDNA library with a zymogram technique. D -Arabinitol, xylitol, D -glucitol and galactitol are substrates for the recominant protein. With D -arabinitol as substrate the reaction product is D -ribulose. The molecular weight of the native tetramer enzyme is 110 000 Da and the monomer is 30 000 Da. The amino acid sequence is homologous to the short-chain dehydrogenase family. It is 85·5% identical to a D -arabinitol dehydrogenase from Candida albicans. The gene in P. stipitis was induced by D -arabinitol and P. stipitis was able to grow on D -arabinitol. The physiological role of D -rabinitol metabolism is discussed.     

Characterization of the recombinant succinic semi‐aldehyde dehydrogenase from Saccharomyces cerevisiae

The yeast succinic semi‐aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS–PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µm NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi‐aldehyde (K_m = 15.48 ± 0.14 µm ) and could use both NAD⁺ and NADP⁺ as co‐factors, with K_m values of 579.06 ± 30.1 µm and 1.017 ± 0.46 mm, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD⁺ (K_i = 129.5 µm ) but a non‐competitive inhibitor with respect to succinate semi‐aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with K_i = 1.13–10.2 mm, and showed competitive inhibition with respect to NAD⁺ and mixed‐competitive, non‐competitive and non‐competitive inhibition, respectively, with respect to succinate semi‐aldehyde. The kinetic data suggest a 'ping‐pong' mechanism. Copyright © 2014 John Wiley & Sons, Ltd.