交联草酸脱羧酶聚集体的制备及其性质

为了获得缓解泌尿系统草酸盐结石病症的酶制剂,用无载体固定化方法——交联酶聚集体(CLEAs)制备交联草酸脱羧酶聚集体。使用基因工程菌E.coli BL21(DE3)/pET32a/YvrK诱导表达制备草酸脱羧酶粗酶液,用30%的乙醇进行沉淀分离提纯,纯化倍数为2.7倍,酶活回收率91.2%;在戊二醛添加量为0.06%、pH5、牛血清白蛋白添加量0.5g/L、4℃的条件下处理该酶2h,得到交联草酸脱羧酶聚集体(oxdc-CLEAs),酶活回收率达95.4%;酶学性质研究表明,相对游离草酸脱羧酶,交联草酸脱羧酶聚集体的耐酸性、耐热性、耐胰蛋白酶降解能力均有提高。     

麦胚富集γ-氨基丁酸的培养条件优化

采用水浴保温的方法对麦胚中γ-氨基丁酸（GABA）进行富集,研究了培养温度、时间、液料比和培养液pH对麦胚中GABA含量和谷氨酸脱羧酶（GAD）活性的影响,采用响应面法对麦胚富集GABA的培养条件进行了优化。结果表明,在一定范围内培养温度、时间、液料比和培养液pH可有效提高麦胚中GAD活性,促进GABA积累。Box-Behnken实验结果显示,麦胚富集GABA的最优培养条件为培养温度46℃,时间1.5h,液料比6∶1（mL/g）,培养液pH4.6。在此培养条件下,麦胚中GABA最大富集量为36.78mg/g,是麦胚原料的5.51倍。方差分析表明,所建的回归模型能够很好的预测麦胚中GABA富集量的变化。      

基于稻谷加温加湿技术的γ-氨基丁酸富化

目的：提高糙米品质。方法：以γ-氨基丁酸（GABA）含量和谷氨酸脱羧酶活性作为指标,研究富化条件对GABA含量的影响;基于最优参数,通过分析富化过程中籽粒各部位GABA含量,揭示其迁移规律;最后分析该法对糙米品质的影响,并与浸泡法对比。结果：原料水分含量14.5%,富化温度65 ℃,最终水分含量20%,富化时间5 h时,GABA含量最高。富化过程中,GABA主要在糠层生成,在生成的同时向内部迁移,各碾减率的糙米GABA含量均在5 h达到峰值,但迁移率在4 h后趋于稳定,胚乳部分的GABA迁移率最高可达87.1%。与浸泡法相比,加温加湿法富化的糙米偏黄,爆腰率较低,米饭更柔软。结论：加温加湿法优于浸泡法,可用于制备高GABA含量的糙米和精米。     

THE BIOCHEMISTRY OF MATURATION

Negative and positive aspects of maturation are respectively related to aroma and taste modifications. Vicinal diketones, hydrogen sulphide, acetyldehyde being primarily responsible for ‘green’ beer flavours an important feature of maturation is the adjustment of their concentration during the lagering period. The role of secondary fermentation in the removal of these undesirable by-products and the importance of sulphury compounds in determining the typical character of lager beer are reviewed. Particular emphasis is placed upon new enzymatic and genetic approaches to overcome vicinal diketone problems in accelerated fermentation systems using free and immobilized cells. The presence in beer of amino acids, peptides, nucleotides and organic as well as inorganic phosphates is, in part, due to the secretion of these materials by the yeast during lagering. Most of these compounds are contained in internal pools and their actual participation in flavour maturation depends upon intracellular breakdown and accumulation, changes in cell permeability and subsequent exchange possibilities between the yeast cell and the surrounding beer. Participation and practical implications of medium chain length fatty acids in the development of autolytic and yeasty flavours are discussed.     

表达重组谷氨酸脱羧酶工程菌发酵条件的优化

目的对表达谷氨酸脱羧酶(Glutamate decarboxylase,EC4.1.1.15,简称GAD或GDC)的重组大肠杆菌的发酵条件进行优化。方法通过单因素试验优化表达GAD的重组大肠杆菌B3C1的诱导剂IPTG添加时间、IPTG浓度、诱导时间、诱导温度,再经响应面分析法优化工程菌的发酵条件(氯化钠、胰化蛋白胨、酵母粉、摇床转速、初始pH值、培养时间、接种量、装液量)。结果工程菌的最佳诱导条件为:IPTG添加时间为工程菌接种后4 h,IPTG浓度为0.8 mmol/L,诱导时间为4 h,诱导温度为35℃;工程菌的最佳发酵条件为:酵母粉1.01%,氯化钠0.6%,胰化蛋白胨1.5%,培养时间11.25 h,摇床转速250 r/min,初始pH值6.5,接种量3.0%,装液量11.70 ml。工程菌在该条件下发酵培养,GAD酶活达1 227.33 U,比优化前提高了12.29%。结论已成功对表达GAD的重组大肠杆菌的发酵条件进行了优化,为其工业化生产奠定了基础。     

Dynamics of γ-aminobutyric acid in wheat flour bread making

The dynamics of the health-improving non-protein amino acid γ-aminobutyric acid (GABA) during bread making were studied. Wheat flour contains trace levels of GABA (<15 ppm) and ca. 160–175 ppm of its precursor, glutamic acid (GA). During dough mixing, the levels of both GA and GABA largely increased. While wheat flour endogenous glutamic acid decarboxylase (GAD) performs some minor conversion of GA into GABA, yeast is the main contributor to GABA formation. Comparison of amino acid levels of dough samples, without or with yeast, indicated that yeast favours both GA and GABA formation already during mixing. Fermentation decreased both GA and GABA contents, due to amino acid consumption by the yeast, which used more GA than GABA. Proofing and baking resulted in large GABA losses, the latter probably in Maillard browning reactions during baking. Thermal loss of GA was less pronounced than that of GABA. Breads contained only trace levels of GABA and ca. 90–130 ppm of its precursor. Exogenous supplementation of recombinantly produced GAD of Yersinia intermedia decreased GA levels in mixed and fermented dough and increased GABA levels. The highest GAD dosage used resulted in fermented doughs with ca. 300 ppm of GABA, i.e. three times higher than the level present in the reference sample (no GAD added). After baking, a significant GABA level was left in the bread samples (ca. 115 ppm) and GABA-enriched breads were obtained. Addition of sodium glutamate (100–380 ppm) to a bread recipe containing no added GAD clearly indicated that its precursor was not the limiting factor for GABA conversion during bread making since the resulting breads contained no GABA, or only trace levels (ca. 20 ppm).     

酿酒酵母丙酮酸脱羧酶活性测定的研究

丙酮酸脱羧酶是酿酒酵母代谢中非常关键的酶,其催化的反应是利用丙酮酸生成乙醛.研究构建了一种分光光度法测定酿酒酵母中丙酮酸脱羧酶活性的方法,在25℃和pH6.0的条件下测定反应液5min内在波长340nm处每分钟吸光度值的变化.以在25℃和pH6.0的条件下每分钟转化1.0μmoi丙酮酸生成乙醛来定义酶的活性.     

草酸脱羧酶促进草酸钙结晶溶解的体外研究

在体外实验条件下,考察了加酶量、反应pH值、反应温度及其产物甲酸根等因素对草酸脱羧酶催化草酸降解从而促进草酸钙结晶重新溶解过程的影响。结果表明,在10 mL反应体系中,添加4 U草酸脱羧酶,反应pH 3.0,反应温度为37 ℃,反应进行1 周（168 h）,可使近5%的草酸钙结晶重新溶解;实验结果表明,草酸降解产物甲酸根对草酸脱羧酶促进草酸钙结晶溶解产生抑制作用。     

草酸脱羧酶的性质及应用研究进展

草酸脱羧酶（oxalate decarboxylase,Oxdc）属Cupin蛋白超家族,是一种包含Mn2+的均一聚合酶,能够在没有辅因子的条件下催化草酸转化为甲酸和CO2,是植物、微生物中促使草酸代谢降解的主要酶之一。该酶已在农业、食品、工业生产、医疗和生物监测等领域得到了广泛的应用。本文综述近年来在Oxdc来源、结构、作用以及实际应用方面的研究,着重讨论其在泌尿系统草酸盐结石病症方面的作用,为有效预防治疗泌尿系统结石症的研究提供理论参考。