鲫鱼乙酰胆碱酯酶酶活测定条件的优化研究

在单因素试验的基础上,选定保温时间、保温温度、pH值三个因素的三个水平进行中心组合试验,建立了淡水鲫鱼脑、肌肉和肝脏乙酰胆碱酯酶（ACHE）酶活的二次回归方程。此模型拟合良好,通过响应面分析得到了酶活测定的优化组合条件。结果表明：保温时间、保温温度、pH值对酶活都有极其显著影响,鱼脑酶活测定的最佳条件为保温时间35min,保温温度32℃,pH7．6。肝脏酶活测定最伟条件为保温时间28min,保温温度30℃,pH7．6。肌肉酶活测定的最佳条件为保温时间31min,保温温度31℃,pH7．6。     

香蕉多糖提取技术研究

采用酶法提取香蕉多糖,通过设计正交试验优化提取工艺,考察超声波工作功率、超声波提取时间、酶解p H值及酶解时间四个因素对香蕉多糖提取得率的影响。结果表明,在采用复合酶酶解及提取温度为50℃的条件下,超声波提取影响较大,酶法影响次之最佳的提取工艺条件为超声功率200W,超声提取时间60min,酶解p H值为6.00,酶解时间为120min。     

超声辅助酶解葛根粉及其响应面优化工艺的研究

以超声为辅助手段,利用中温α-淀粉酶对葛根粉进行酶解,在单因素实验的基础上,根据Box-Benhnken中心组合实验设计原理采用三因素三水平的响应面实验,以葡萄糖当量(DE值)为考察指标,确定最优酶解工艺参数.确定了辅助超声条件为:超声功率90W、超声时间10min.在该条件下,优化出的最佳酶解工艺为:固形物浓度29.6％,酶解时间32min,酶添加量1.19％.扫描电镜观察结果显示,酶解后的葛根粉颗粒呈不规则片状,变得疏松、易于溶解.     

超声辅助酶法提取河蚬多糖

选择超声辅助酶法提取河蚬多糖,确定了不同超声时间、超声功率、料水比、酶解时间等因素对河蚬多糖提取率的影响,并进行了正交试验分析。结果表明,最佳提取工艺条件为:超声时间40 min、超声功率80W、料液比1∶30、酶解时间90 min,在此条件下,河蚬多糖的提取率为4.86%。     

超声辅助酶法提取燕麦蛋白的研究

采用超声波辅助酶法从燕麦粉中提取燕麦蛋白,研究了料水比、粉碎度、超声时间、超声功率、超声温度以及酶解pH值、酶解时间、酶解温度、加酶量对燕麦蛋白提取率的影响.通过单因素实验和正交实验得到最佳提取条件,即料水比1: 8、粉碎度40目、超声时间25 min、超声功率40 W、超声温度50℃、加酶量1.1%(中性蛋白酶)、酶解pH8、酶解时间1.5 h、酶解温度45℃,此条件下燕麦蛋白的提取率可达80.3%.     

超声辅助水酶法提取月见草籽油的研究

月见草籽油在医药、保健及食品工业领域具有重要价值。本文对超声辅助水酶法提取月见草籽油进行了研究,采用超声及粉碎处理方式对月见草籽进行预处理,确定最佳预处理条件:超声功率300W、超声时间30min、超声温度60℃;采用Alcalase 2.4L碱性蛋白酶进行酶解,利用响应面优化试验,确定最优的月见草籽油脂提取工艺:料液比为5.4(w/w)、酶添加量为1.38(v/w)、酶解温度为62.5℃、酶解时间2.8h,响应面有最优值为84.32%;测定油脂的基本组成及相关品质指标,结果表明,不同提取方法对月见草籽油的皂化值、折射率、色泽的影响不显著,但酸值及磷脂含量均低于溶剂法月见草籽油。优化的工艺简便可行、提取率高,为超声辅助水酶法提取月见草籽油提取工艺的产业化提供理论依据。     

超声波协同酶法提取芹菜中总黄酮的工艺优化

为提高芹菜中总黄酮的得率,采用超声波-纤维素酶协同方法醇提芹菜总黄酮,研究了酶解pH值、加酶量、超声功率、料液比、超声时间对芹菜总黄酮得率的影响。在单因素试验的基础上,通过正交试验优化确定了芹菜中黄酮提取的最佳工艺条件,即酶解pH 值为4.0、加酶量210 U/g、超声功率200 W、料液比为1∶25（g∶mL）、超声时间40 min,在此条件下总黄酮得率为1.018 4%。     

超声波辅助酶解脱脂小麦胚芽制备抗氧化肽的研究

研究了超声波辅助酶解脱脂小麦胚芽对酶解产物抗氧化性的影响.在单因素实验基础上,通过响应面法考察了超声功率、超声时间和超声温度对酶解产物的1,1-二苯基-2-苦基肼(DPPH)清除率的影响.结果表明,最佳的超声酶解条件为:超声功率214 W,超声时间3 min,超声温度25 ℃.此时酶解产物的DPPH清除率理论值为62.4%,实际的清除率达到61.2%.     

酶辅助超声波提取何首乌多糖及其抗氧化性研究

采用酶辅助超声提取法,从何首乌中提取多糖,用苯酚-硫酸比色法测定多糖含量.探讨了何首乌多糖的工艺条件,为酶辅助超声提取在工业生产上的应用积累参数.结果表明,提取何首乌多糖的最佳工艺条件:提取时间为15 min、浸取液倍数为30倍、pH值为6.0、提取温度为43℃、酶添加量为2.5%、提取功率150 W,多糖提取率为25.26%,何首乌多糖对HO·的还原率为36.23%.此方法是一种简单快捷和高效的提取方法.     

超声波辅助酶法分离提取葡萄酒泥酵母SOD工艺条件的优化

以葡萄酒泥酵母为试材,超氧化物歧化酶（SOD）比活力为指标,在超声功率、超声时间、蜗牛酶质量浓 度、酶解pH值和酶解温度等单因素试验基础上,采用正交试验优化超声波辅助蜗牛酶法提取SOD的工艺条件。结果 表明：提取工艺因素对SOD比活力影响大小顺序为超声时间＞蜗牛酶质量浓度＞超声功率＞酶解pH值＞酶解温度, 1 g湿酵母悬浮于5 mL 0.2 mol/L的磷酸盐缓冲液,其提取SOD最佳工艺条件为超声功率400W、超声时间12 min、 2.0 mg/100 mL蜗牛酶液1 mL、pH 6.6、温度37 ℃,在此条件下,分离提取得到的SOD比活力达192.27 U/mg。结果表 明超声波辅助酶法在葡萄酒泥酵母SOD的提取中具有较高的应用价值。     

醋酸菌产乙醇脱氢酶发酵条件的研究

该研究对醋酸菌（Acetobacter pomorum）产乙醇脱氢酶（ADH）的发酵培养基的碳源和氮源进行了优化。采用单因素试验和响应面试验考察发酵温度、发酵时间、起始pH及接种量对乙醇脱氢酶比酶活的影响。在单因素试验的基础上,采用响应面试验进行发酵条件优化。结果表明,最佳发酵培养基配方为葡萄糖0.8%,酵母浸膏粉1.2%;最佳发酵工艺为发酵温度35 ℃、发酵时间72 h、起始pH 6.0、接种量6%。在此工艺条件下,ADH的比酶活为7 284 U/mg,与预测值（7 320 U/mg）的相对误差为0.49%。     

啤酒酵母乙醛代谢关键酶相关性研究

乙醛是啤酒中的主要风味物质,其代谢主要来自酵母细胞。酵母中乙醇脱氢酶及乙醛脱氢酶是乙醛代谢的关键酶,对乙醛变化起着重要作用。跟踪啤酒酵母发酵过程中相对酶活力及乙醛变化,发现两种乙醇脱氢酶和乙醛脱氢酶的相对酶活力与发酵过程乙醛含量变化具有一定相关性。同时对低产乙醛啤酒酿酒酵母kb2-4与出发菌株啤酒酵母kb进行发酵试验,跟踪检测相对酶活力及乙醛含量,其乙醇脱氢酶Ⅰ和乙醇脱氢酶Ⅱ及乙醛脱氢酶相对酶活力均高于出发菌株,平均增幅分别为15.5%,11.6%和5%。3种酶活性的变化协同作用可以使乙醛含量降幅最大为33.8%。     

Substrate specificity of alcohol dehydrogenase from the yeast Hansenyls polymorpha CBS 4732 and Candida utilis CBS 621

The substrate specificity of alcohol dehydrogenase (ADH) from Hansenula polymorpha and Candida utilis has been compared with that of the classical ADH from baker's yeast. Cell-free extracts of H. polymorpha and C. utilis exhibited a much higher ratio of butanol to ethanol oxidation than baker's yeast ADH. This was also observed with the purified enzymes. The ratio of activities with ethanol and butanol was pH-dependent. With the baker's yeast enzyme the activity strongly decreased with increasing chain length, whereas the enzymes form H. polymorpha and C. utilis showed a high reactivity with long-chain alcohols. In addition, the affinity constant for ethanol was more than tenfold lower than that of the baker's yeast enzyme. The purified preparation yielded several protein bands on polyacrylamide slab gels, each of which showed activity with both ethanol and butanol.     

黑豆多肽的制备及其对乙醇脱氢酶活性的 影响研究

目的制备黑豆多肽,研究其对乙醇脱氢酶(alcohol dehydrogenase,ADH)活性的影响。方法采用碱性蛋白酶与胰蛋白酶分步水解法制备黑豆多肽。用截留分子量分别为5000、3500、2000、1000 Da的透析袋将黑豆多肽分离成4个相对分子量的肽段,对不同相对分子量的黑豆多肽进行抗氧化能力以及体外醒酒活性实验。结果黑豆多肽在碱性蛋白酶与胰蛋白酶分步水解后的水解度达35.64%。不同分子量的黑豆多肽均具有对乙醇脱氢酶的激活作用和对羟自由基(·OH)的清除能力,且都随着黑豆多肽相对分子质量的减小而增强。相对分子质量在1000 Da以下的黑豆多肽对乙醇脱氢酶的激活率和羟自由基(·OH)的清除能力最强,分别达22.43%和24.39%。结论黑豆多肽具有较强的乙醇脱氢酶激活率,并且与黑豆蛋白质和其他来源的多肽相比,相对分子质量在500~2000 Da的黑豆多肽中亮氨酸和丙氨酸的含量较多,因此推测黑豆多肽具有较好的醒酒效果。     

Quinoprotein alcohol dehydrogenase is involved in catabolic acetate production, while NAD-dependent alcohol dehydrogenase in ethanol assimilation in Acetobacter pasteurianus SKU1108

The relationship between quinoprotein alcohol dehydrogenase (ADH) and NAD-dependent ADH was studied by constructing quinoprotein ADH-deficient mutants. Quinoprotein ADH-deficient mutants were successfully constructed from Acetobacter pasteurianus SKU1108 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and also by adhA gene disruption with a kanamycin cassette. The NTG mutant exhibited a complete loss of its acetate-producing ability and acetic acid resistance, while the disruptant also exhibited a loss of its acetic acid resistance but retained a weak ADH activity. The immunoblot analysis of quinoprotein ADH indicated that there are no appreciable ADH subunits in the membranes of both mutant strains. The NTG mutant grew better than the wild-type strain in ethanol-containing medium, despite the absence of quinoprotein ADH. In the mutant, the activities of two NAD-dependent ADHs, present in a small amount in the wild-type strain, markedly increased in the cytoplasm when cultured in a medium containing ethanol, concomitant to the increase in the activities of the key enzymes in TCA and glyoxylate cycles. The disruptant showed a poorer growth than the wild-type strain, producing a lower amount of acetic acid in ethanol culture, and it induced one of the two NAD-dependent ADHs and some of the acetate-assimilating enzymes induced in the NTG mutant. This study clearly showed that quinoprotein ADH is extensively involved in acetic acid production, while NAD-dependent ADH only in ethanol assimilation through the TCA and glyoxylate cycles in acetic acid bacteria. The differences between the NTG mutant and the disruptant are also discussed.     

The alcohol dehydrogenase system in the yeast, Kluyveromyces lactis

We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis. Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADH genes in K. lactis. Two of these genes have been isolated from a genomic bank by hybridization to ADH2. The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S. cerevisiae and Schizosaccharomyces pombe respectively. One K. lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S. cerevisiae, was named K1ADH1. The other gene, homologous to K1ADH1 in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondrial targeting presequence. We named this gene K1ADH3. The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresis-resolved K. lactis genome. ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source.     

啤酒酵母HD34-1乙醇脱氢酶活性的研究

菌株HD34-1是将B.sub的乙醇脱氢酶(ADH)基因转入啤酒酵母HD34而构建的无醇啤酒酵母工程菌株,该菌株的ADH基因受Gal启动子的控制,因此为了考察该菌株ADH基因的表达情况,本研究设计了半乳糖和乙醇的浓度梯度进行发酵试验并测定了相应条件下的的ADH活性,结果表明2%的半乳糖浓度,诱导16h最适宜ADH基因的表达。     

Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions,using alcohol dehydrogenase as sensor reaction

With the current quantitative metabolomics techniques, only whole‐cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde‐3‐phosphate dehydrogenase and 3‐phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH‐catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose‐limited chemostats under steady‐state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose‐limited steady‐state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction. Copyright © 2015 John Wiley & Sons, Ltd.