利用固定化酵母细胞转化反式肉桂酸生产L—苯丙氨酸

研究了深红酵母（Ｒｈｏｄｏｔｏｒｕｌａｒｕｂｒａ）的培养基成分,培养固定化及转化条件,实验表明最佳基成分（％）葡萄糖０．５,胰蛋白胨０．５,酵母膏０．５,磷酸二氢钾０．０５,Ｌ－Ｐｈｅ０．０５,ｐＨ７．０,３０℃２０Ｌ发酵罐中培养１５～１７ｈ,最佳固定化条件为：用２．５％卡拉胶包埋１８％的湿菌体,最佳转化条件为：１．０％反式肉桂酸,４ｍｏｌ／Ｌ铵离子,ｐＨ０．５,３０℃,用卡拉胶固定化深红酵母（Ｒ     

利用固定化酵母细胞转化反式肉桂酸生产L-苯丙氨酸

研究了探红酵母(Rhodotorula rubra)的培养基成分、培养固定化及转化条件。实验表明最佳培养基成分(％)：葡萄糖0 5,胰蛋白胨0．5,酵母膏0．5,磷酸二氢钾0．05,L-Phe0．05,pH．0,30℃,20L发酵罐中培养15～17h.最佳固定化条件为：用2．5％卡拉胶包埋18％的湿菌体。最佳转化条件为：1．0％反式肉桂酸,4mol／L铵离子,pH10．5,30℃。用卡拉胶固定化的深红酵母(Rhodotorula rubra)可以将77．7％的反式肉桂酸转化为L-苯丙氪酸。     

谷氨酰胺合成酶产生菌的固定化研究

谷氨酰胺合成酶产生菌的固定化在酶法合成谷氨酰胺中的应用具有重要意义。实验首先从味精废水中筛选出谷氨酰胺合成酶高产菌株LNU018,然后分别用海藻酸钠、聚乙烯醇(PVA)为载体对谷氨酰胺合成酶高产菌棒杆菌进行固定化。探讨了固定化条件对固定化小球结构、机械强度、弹性、稳定性和培养后菌体的谷氨酰胺合成酶的活性情况的影响,分析确定最佳的固定化条件。研究结果表明,5%的海藻酸钠、11%的聚乙烯醇形成的固定化菌球大小合适,有弹性,但5%的海藻酸钠能更好的保持酶活性,比11%聚乙烯醇高16%,其为最佳的固定化条件。     

用固定化弗劳地柠檬酸杆菌XP05从溶液中回收铂

比较了5种固定弗劳地柠檬酸杆菌XP05菌体的方法,其中明胶海藻酸钠包埋法为固定菌体的最佳方法。扫描电子显微镜观察表明,XP05菌体较均匀地分布于包埋基质中。固定化XP05菌体吸附Pt^4＋受吸附时间、固定化菌体浓度、溶液的pH值和Pt^4＋起始浓度的影响。吸附作用是一个快速的过程;吸附Pt^4＋的最适pH值为1.5;在50～250 mg P^4＋/L范围内,吸附量与Pt^4＋起始浓度成线性关系,吸附过程符合Langmuir和Freundlich吸附等温模型。在Pt^4＋起始浓度250 mg/L、固定化菌体2.0 g/L、pH 1.5和30℃条件下,振荡吸附60 min, 吸附量为35.3 mg/g。0.5 mol/L HCl能使吸附在固定化菌体上的Pt解吸98.7%。从废铂催化剂处理液回收铂的结果表明,在Pt^4＋起始浓度111.8 mg/L、固定化菌体4.0 g/L、pH 1.5和30℃条件下,振荡吸附60 min, 吸附量为20.9 mg/g。在填充床反应器中,在Pt^4＋起始浓度50 mg/L、流速1.2 ml/min、固定化菌体1.86 g的条件下,饱和吸附量达24.7 mg/g; 固定化XP05菌体经4次吸附解吸循环后吸附率仍达78％。     

具有葡萄糖异构酶活性的菌体的固定化

1.介绍了用明胶包埋菌体后,再用戊二醛交联固定制备固定化链霉菌的方法。用这种方法制得的固定化链霉菌活力回收高,包埋量大,机械性能好。2.除了对固定化的条件进行摸索外,还就蛋白酶对固定化反应的影响进行了研究,它对菌体的固定化影响较大。3.菌体固定化后,表观米氏常数有所增加,pH 稳定性有所提高,其他葡萄糖异构酶性质几乎没有改变。     

具有葡萄糖异构酶活性的菌体的固定化

1.介绍了用明胶包埋菌体后,再用戊二醛交联固定制备固定化链霉菌的方法。用这种方法制得的固定化链霉菌活力回收高,包埋量大,机械性能好。2.除了对固定化的条件进行摸索外,还就蛋白酶对固定化反应的影响进行了研究,它对菌体的固定化影响较大。3.菌体固定化后,表观米氏常数有所增加,pH稳定性有所提高,其他葡萄糖异构酶性质几乎没有改变。     

嗜热脂肪芽孢杆菌氨基酰化酶在大肠杆菌中的克隆、表达及细胞固定化研究

将嗜热脂肪芽孢杆菌的氨基酰化酶(amaA)基因克隆到E.coli中进行表达,同时利用渗透交联法固定化E.coli细胞,并对固定化细胞氨基酰化酶进行了温度和pH等理化性质的初步探讨.结果显示amaA基因在E.coli中获得了高效表达,酶活性达1043U/g湿菌体.最佳固定化条件为3%卡拉胶,30%细胞.当以1.25%多乙烯多胺渗透交联固定化细胞10min和0.1%戊二醛硬化处理20min,酶学性质研究表明,酶反应的最适温度为65℃,最适pH为7.0.细胞固定化后仍保留有83%活性,pH稳定范围更广,热稳定性更高,55℃酶活性不损失,4℃保存23d仍保留有固定化时73.6%的酶活性,连续10批次转化酶活性仅损失约20%,预示该固定化E.coli具有良好的操作和保存稳定性.     

性能优良的测定谷氨酸的传感器

现在酶或菌体固定化的生物传感器的研制很盛行,但存在酶不稳定等问题,因此在应用上进展不大。日本味之素公司为降低生产成本,不断开发出优良的检测系统,其中固定化菌体与质谱仪组合而成的谷氨酸传感器已在工厂中应用了2年。     

包埋法固定微生物细胞技术的新进展

自六十年代固定化酶技术问世以来,固定化细胞技术随之迅速发展。到了七十年代,作为酶源的微生物菌体本身的固定化又引起人们极大兴趣,其研究和应用已涉及食品、化工、医药、环境保护等领域。目前,工业化生产上采用的固定化方法主要有两大类:吸附法和包埋法。微生物菌体的固定化一般采用包埋法。其优点是:(1)微生物菌体包埋在聚合物中不易漏出;(2)操作条件温和、对外界环境的缓冲作用大;(3)可防止微生物菌体的机械损伤,易于再生,产物分离提取容易。 载体的性能影响固定化细胞的机械强度、细胞活性、工作稳定性,因此,载体的选择及其制备方法一直     

固定化根霉发酵生产脂肪酶

以聚氨酯为少根根霉固定化载体,对固定化后的细胞连续重复批次发酵进行了研究。优化了重复批次发酵培养基组成。在取代发酵液40mL,取代培养基组成为全脂豆粉3%,花生油0.5％条件下,固定化菌体摇瓶实验可连续使用140h,重复9批次。酶的时空产率提高6倍。5L发酵罐小试固定化菌体可连续发酵6批次。固定化细胞连续发酵,大大缩短了发酵的时间,酶的时空产率获得大幅提高。     

Microbiological transformation of steroids

Из 64 исследовавшихся нами штаммов (28 видов) различных микроорганизмов способность к гидроксилированию до положения 11β-стероидной молекулы была найдена только для 19 штаммов (10 видов). Однако ни один из микробов не гидроксилировал молекулу стероида толяко до положения 11б: во всех случаях было отмечено возникновение дальнейших гидроксилированных метаболитов,—преимущественно с гидроксигруппой в положении 11α. Оба эпимера образуются в соотношении, которое зависит от структуры стероидного субстрата, подвергаущегося энзиматическим превращениям. Для прогестерона отношение образующегося 11α-эпимера к 11β-эпимеру составляет в лучшем случае 7∶1, тогда как для стероидов S (Reichstein) отношение обоих эпимеров для штамма Absidia orchidis равно 4∶5. Состав культивационной среды оказывает влияние на накопление микроорганизмами системы необходимых ферментов, а тем самым и на скорость трансформации стероидов, но не на качество и соотношение возникающих метаболитов.     

Microbiological transformation of steroids

Описана трансформация стероида S (Reichstein) штаммом Absidia orchidis 310 в смеси кортизона, 11-α-эпикортизола и кортизола. Приводится метод аналитической оценки превращения стероида. Было исследовано влияние величины pH среды (оптимум 6,5), температуры (27–30°C), вида и количества растворителя (наиболее удобен метиловый спирт в количестве 2–4 мл. (100 мл среды), числа оборотов при перемешивании в течение ферментации (оптимально 600 об./мин.), далее, влияние аэрации и концентрации исходного стероида на скорость превращения (оптимально до 60мг/100мл среды). При оптимальных условиях выход стероида S, по аналитическим данным, составляет 48,9% кортизола и 43,9% 11α-эпикортизола; кортизон образуется только в микроскопических количествах.     

Chitin deacetylase product inhibition

Chitin deacetylase is the only known enzyme catalyzing the hydrolysis of the acetamino linkage in the N-acetylglucosamine units of chitin and chitosan. This reaction can play an important role in enzymatic production of chitosan from chitin, or in enzymatic modification of chitosan, which has applications in medicine, pharmacy or plant protection. It was previously shown that acetic acid, a product of the deacetylation process, may act as an inhibitor of chitin deacetylase. Here we show the mechanism of inhibition of chitin deacetylase isolated from Absidia orchidis vel coerulea by acetic acid released during the deacetylation process. The process follows competitive inhibition with respect to acetic acid with an inhibition constant of K(i) = 0.286 mmol/L. These results will help to find the optimal system to carry out the enzymatic deacetylation process for industrial applications.     

The influence of supplemental components in nutrient medium on chitosan formation by the fungus Absidia orchidis

Chitosan, a derivative of chitin, is a natural component of some fungus cell walls. It is formed by the complex action of chitin synthase and chitin deacetylase. The in vitro activity of these two enzymes is known to be influenced by several factors. We investigated the influence of ferrous ions, manganese ions, cobalt ions, trypsin, and chitin, as individual supplements to the nutrient medium, on the in vivo activity of chitin synthase and chitin deacetylase to form chitosan in the fungus Absidia orchidis. Manganese and ferrous ions gave the most significant results. These ions increase chitosan yields through an increase in biomass production rather than an increase of chitosan content in cell walls. Manganese and ferrous ions lowered the activity of chitin deacetylase; however, their influence on the activity of chitin synthase was more complex. The effects of trypsin and chitin on biomass and cell wall chitosan content were negligible, while cobalt ions completely inhibited the growth of fungi.     

Kinetic behaviour of α-galactosidase produced by Absidia griseola: a comparison between free and immobilized forms of the enzyme

In this research the characteristics of free (partially purified) and immobilized (mould pellets of Absidia griseola) -galactosidase have been investigated. Inhibition studies of the enzyme showed that p-nitrophenol and sucrose do not have any inhibitory effect on the enzyme, but that galactose is a competitive inhibitor. In the immobilized form, inhibition was lower than in the free enzyme and the level of inhibition decreased as the temperature increased. The activity and stability of free and immobilized enzyme were investigated with respect to temperature, and the results showed that the optimal temperature range of the free enzyme was 45–50 °C, while the immobilized enzyme had an optimum at 55–60 °C. The optimum pH for the free enzyme was 6.0 and the value was decreased to 5.0 by immobilizing. The experimental effectiveness factors were found to be represented as a single function of the modified Thiele modulus, including parameters such as pellet size, enzyme concentration in the pellets and substrate concentration. Both experimental and theoretical data concerning effectiveness factors are nearly the same.     

梨头霉11α—羟基化制备16β—甲基—11α,17α21—三烃基孕甾—1, …

选育到一株对１６β－甲基－１７α,２１－二羟基孕甾－１,４＝－二烯－３,２０－二酮（Ⅱａ）１１α－羟基化活性强的梨头霉Ａ２８菌株,并发现底物２１－乙酰化（Ⅱｂ）可明显提高１１α－羟工 能力。在适宜的转化条件下,１１ｂ投料浓度０．５％,产物１６β－基１１α,１７α,２１－三羟基孕甾－１,４－二烯－３,２０－二酮（Ⅲ）收率为７３％,结构经波谱分析确认。     

溶胶凝胶法固定化黑曲霉脂肪酶的性质研究

近年来溶胶-凝胶法固定脂肪酶已成为研究热点。选用TMOS、MTMS、ETMS和PTMS 4种硅烷试剂对黑曲霉脂肪酶进行了固定化研究。固定化的最佳配方为ETMS/TMOS=5：1、水与硅烷试剂分子比为8;固定化脂肪酶的固定率为80.2%、相对活性为136.3%;以乳化橄榄油作为底物,在50℃和pH4.0的条件下,固定化脂肪酶与游离脂肪酶K_m分别为1.899×10^-4M和2.789×10^-4M;最适反应pH均为pH4.0,固定化脂肪酶在pH4.0~pH5.5之间其活性能保持95%以上;固定化脂肪酶最适反应温度为60℃,较游离脂肪酶提高了10℃;固定化脂肪酶的酸碱稳定性和热稳定性较非固定化酶有显著的提高。固定化脂肪酶的使用寿命和保存稳定性良好,使用12次后仍能够保留71.7%活性,在室温避光条件下保存180天后仍可保留79.2%活性。     

高分子膜载体固定化木瓜蛋白酶

在浸润条件下,以0.5%(v/v)戊二醛交联的高分子膜尼龙载体固定化木瓜蛋白酶。对固定化条件进行了优化,比较了固定化酶与游离酶的酶学参数。结果表明,4℃、pH6.0条件下,将膜载体浸润于2mg/mL酶液中5h,固定化酶活为303.4U/g。固定化酶最适反应pH为6.0～7.0,最适反应温度为65℃。其pH稳定性、热稳定性均比游离酶高。     

Sanitization of immobilized biocatalysts for the production of 6‐aminopenicillanic acid

Five different chemical reagents and γ‐rays were tested for the sanitization of immobilized biocatalysts with high penicillin G acylase (PGA) activity. The most effective chemical reagents were N‐cetyl‐N,N,N‐trimethylammonium bromide (CTAB) and 2‐isopropyl‐5‐methylphenol (thymol). The optimum concentration of CTAB for the treatment of the immobilized enzyme was 0.25% [w/v] and 1 h, for immobilized cells 0. [w/v] and 3 h. The optimum concentration of thymol for the immobilized enzyme was found to be 0.1% [w/v] and 1 h, for immobilized cells 0.27% [w/v] and 2 h. The optimum dose of γ‐rays for the sanitization of the immobilized enzyme was established as 3.2 kGy, for immobilized cells as 4.5 kGy.     

大豆异黄酮糖苷酶的纯化及性质研究

利用Absidiasp.R菌株,通过液体发酵的方法,得到了一种高活性的大豆异黄酮糖基水解酶。该酶经硫酸铵分级沉淀、DEAE-Cellocuse(DE-52)离子交换层析纯化,被纯化了11倍,收率为10.9%;经SDS-聚丙烯酰胺凝胶电泳测得该酶的分子量为53kD;该酶的最适反应温度为50℃;最适pH为5.0;温度低于60℃,pH在5.0~7.0范围内该酶较稳定,Co2 、Ca2 对该酶有激活作用;Ag 、Cu2 对该酶有抑制作用。当以染料木甙为底物时该酶的米氏常数(Km)为1.3×10-2mol/L。等电聚焦电泳测得其等电点为3.2。