谷氨酰胺和N-乙酰-L-谷氨酰胺的发酵及其发酵转换机制

<正> 一、前言 1957年木下等发表谷氨酸棒杆菌(Corynebacterium glutamicum)进行谷氨酸的工业生产以来,日本的氨基酸发酵生产的研究有很大的进展。很多氨基酸已能用发酵法生产。谷氨酰胺和N-乙酰-I-谷氨酰胺(N-AGM)作为胃溃疡、十二指肠溃疡病等的抗溃疡病药物正在大量应用。作者等应用谷氨酸产生菌谷氨酸棒杆菌的野生株,通过控制环境因素使谷氨酸发酵转换成谷氨酰胺和N-AGM发酵,建立了这些氨基酸的工业生产方法。同时也研究了从谷氨酸发酵转换生产脯氨酸的方法。通过改变培养条件,用谷氨酸棒杆菌使发     

谷氨酸发酵菌——Brevibacterium ketoglutaricum nov.sp.北京2990-6的代谢 还原型烟酰胺腺嘌呤核苷酸的氧化

(1)对2990-6号菌的NADH_2,NADPH_2的氧化酶系和转氢酶做了初步的观察并与2990-6号菌在发酵产生大量谷氨酸时的酶系活力的变化做了比较。(2)本工作表明NADH_2氧化酶系以及某些与NAD联结的脱氢酶,NADPH_2氧化酶系以及某些与NADP联结的脱氢酶在细胞内的分布上有着明显的区分。(3)应用比较简便的方法测定了2990-6号菌在各不同生理状态下的NAD,NADH_2,NADP,NADPH_2的含量。(4)对2990-6号菌的谷氨酸发酵机制作了简要的讨论。     

乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响

【目的】研究乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响。【方法】在谷氨酸棒杆菌CGF2中异源表达als SD操纵子构建乙偶姻生产菌株CGT1,考察敲除乙酸生成途径cat和pqo对乙偶姻的影响。然后引入短乳杆菌的NADH氧化酶,在优化的溶氧条件下研究其对乙偶姻产量的影响。【结果】CGT1在摇瓶发酵中可积累6.27 g/L乙偶姻,敲除cat使乙偶姻产量显著提高30.94%,达到8.21 g/L;双敲除cat和pqo没有进一步提高产量。通过优化发酵的溶氧水平,乙偶姻产量达到10.06 g/L。在高溶氧水平下引入NADH氧化酶导致菌株的生长和糖代谢速率提高,但乙偶姻产量略有降低。在分批补料发酵中,重组菌株乙偶姻产量达到40.51 g/L,产率为0.51 g/(L?h)。【结论】在谷氨酸棒杆菌中阻断乙酸合成途径cat能够有效提高乙偶姻产量,NADH氧化酶在高溶氧水平下表达不利于乙偶姻的合成,需要进一步调节表达水平以确定其效果。     

混菌发酵生产γ-聚谷氨酸的工艺条件优化

采用谷氨酸棒杆菌S9114和枯草芽胞杆菌NTG-4在10 L自控发酵罐上进行混菌发酵,探索混菌发酵生产γ-聚谷氨酸的可行性并进行工艺优化。结果表明:温度、接种量、pH及溶氧对聚谷氨酸发酵有较大影响,发酵前期维持32℃,6 h提温至37℃变温控制,谷氨酸棒杆菌和枯草芽胞杆菌接种量分别为5%和0.5%,pH 7.0,溶氧20%最有利于γ-聚谷氨酸发酵,在此条件下发酵32 hγ-聚谷氨酸最高产量为38.3 g/L。     

谷氨酸棒杆菌一步法发酵糖质原料生产γ-聚谷氨酸

γ-聚谷氨酸在食品、化妆品、生物医药等领域具有广泛的应用,目前主要的生产菌株是谷氨酸依赖型菌株,在生产过程中需要添加谷氨酸作为前体,因而生产γ-聚谷氨酸的成本较高。文中主要研究从糖质原料一步法发酵合成γ-聚谷氨酸的生产工艺。首先,从产γ-聚谷氨酸的菌株枯草芽孢杆菌中克隆γ-聚谷氨酸合成酶的基因簇pgs BCA,在谷氨酸棒杆菌模式菌株ATCC13032中进行诱导型和组成型表达,结果显示,仅诱导型表达菌株可以积累γ-聚谷氨酸,产量为1.43 g/L。进一步对诱导条件进行优化,确定诱导时间为2 h,IPTG浓度为0.8 mmol/L,γ-聚谷氨酸产量为1.98g/L。在此基础上,在一株高产谷氨酸的谷氨酸棒杆菌F343中外源表达pgs BCA,对重组菌进行发酵,结果表明,在摇瓶发酵中γ-聚谷氨酸产量达到10.23g/L,在5L发酵罐中产量达到20.08g/L;继而对γ-聚谷氨酸进行分子量测定,结果显示,产自F343重组菌的γ-聚谷氨酸的重均分子量比产自枯草芽孢杆菌的提高34.77%。文中构建了一步法发酵糖质原料生产γ-聚谷氨酸的新途径,同时为开发其潜在应用奠定了基础。     

纪念我国发酵法生产味精四十周年--回忆617短杆菌谷氨酸发酵的研究

综观我国当代发酵法生产氨基酸、核苷酸、有机酸、酶制剂、抗生素等研制过程,基本上是跟踪国外先进技术并加以仿制,本文中L-谷氨酸发酵的研制可以说是一个典型范例。从某种意义上来说,如今我国氨基酸和有机酸发酵之发达,莫不始于L-谷氨酸发酵的成功。然而反思我国的发酵工业,虽然L-谷氨酸、柠檬酸、啤酒的产量堪称世界第一,但充其量中国只能称之为发酵工业大国。因此,今后如果不进行自主科技创新,中国的发酵工业就不可能成为真正意义上科技领先的强国。     

上海大学生物工程系完成计算机辅助教学课件

由上海大学生物工程系王福源教授主持的计算机辅助教学课件《仿真谷氨酸发酵实验技术》已完成 ,并获上海市教学成果 2等奖。该课件包括 :一、发酵罐部件介绍 ;二、谷氨酸发酵实验演示 ;三、实验操作 ;四、成绩评定等几个部分。食品工程专业及生物工程专业的学生在计算机上进行发酵罐模拟操作和进行谷氨酸发酵仿真实验 ,基本上能达到与实罐操作相同的效果 ,可满足实验教学的要求。上海大学生物工程系完成计算机辅助教学课件     

谷氨酸发酵过程葡萄糖自动流加系统

补料（流加葡萄糖）操作是谷氨酸发酵过程最重要的操作之一,工业上有各种各样的补料操作方法。控制葡萄糖浓度于一个较为平稳且适中的水平有利于提高谷氨酸发酵的性能指标。通过在线计量谷氨酸发酵中的氨水耗量并据此在线推定发酵液中的葡萄糖浓度,构建了一个谷氨酸发酵自动在线补料系统。使用该控制系统,谷氨酸发酵过程的葡萄糖浓度可以控制在任意水平,平稳、无波动的谷氨酸发酵可以得到实现。     

糖蜜原料发酵生产谷氨酸 Ⅰ.糖蜜流加法

废糖蜜是发酵工业的廉价碳源,一向被用于酒精、丙酮丁醇、酵母和柠檬酸等的发酵生产。考虑到我国味精工业发展的需要,从节约粮食、降低成本出发,我们曾对黄色短杆菌617以糖蜜为原料发酵生产谷氨酸进行了一系列的研究。通过大量试验,最后确定采用糖蜜流加法对以糖蜜为原料发酵生产谷氨酸是适宜     

上海大学生物工程系完成计算机辅助教学课件

由上海大学（嘉定）生物工程系王福源教授主持的计算机辅助教学课件《仿真谷氨酸发酵实验技术》已完成。该课件包括：一、发酵罐部件介绍;二、谷氨酸发酵实验演示;三、实验操作;四、成绩评定等几个部分。食品工程专业及生物工程专业的学生在计算机上进行发酵罐模拟操作和进行谷氨酸发酵仿真实验,基本上能达到与实罐操作相同的效果,可满足实验教学的要求上海大学生物工程系完成计算机辅助教学课件     

Poly(vinylferrocene)–poly(ethylene glycol) glutamate oxidase electrode for determination of L-glutamate in commercial soy sauces

An amperometric L-glutamate oxidase (GLOD) electrode based on a multilayer of polymer films was developed for the high selective determination of L-glutamate. The multilayer film consisted of three layers as the following configuration i.e., inner membrane of electropolymerized 1,3-diaminobenzene, middle layer of L-GLOD entrapped in photopolymerized poly(vinylferrocene)–poly(ethylene glycol) hydrogel polymer, and outer dialysis membrane. In this manner, the sensor could eliminate interferences and was able to work at a low potential poised at +0.085 V vs. Ag/AgCl. When used in a flow injection system, the sensor responded to L-glutamate in the range 0.5–8.0 mM, with a sensitivity of 9.48 nA mM^–1. The sensor was stable for 5 days of continuous uses (250 assays) and retained 60% activity after 16 days. When used to analyse the L-glutamate contents of a number of different off-the-shelf soy sauces, the sensor gave results in good agreement with the standard colorimetric method.     

L-Glutamate oxidase from Streptomyces cremeus 510 MGU: growth media optimization for the enhancement of the producer fermentative activity]

The investigation was devoted to culture conditions optimization aimed to maximum secretion of extracellular L-glutamate oxidase by Streptomyces cremeus 510 MGU. It was shown that Ca ions at the concentration 5-20 mM and 0.1% ammonium sulphate enhanced activity of extracellular enzyme to 4 folds. L-glutamate acid supplement had no effect on enzyme activity. Influence of some bivalent cations and aeration regimes on L-glutamate oxidase activity was investigated. Growth media optimization along with screening of active variants resulted with isolation of the strain with L-glutamate oxidase activity about 2 U/mL Rate of peroxide degradation in the presence of filtrated culture of S. cremeus was determined by chemiluminescence method.     

Determination of l-glutamate in various commercial soy sauce products using flow injection analysis with a modified electrode

A flow injection analysis (FIA) system with a modified electrode has been developed and optimized for determination of l-glutamate using l-glutamate oxidase (GLOD) (EC 1.4.3.11). GLOD was immobilized on controlled-pore glass using glutaraldehyde. The optimal potential applied on the working electrode was +700mV against a platinum (Pt) reference electrode. The optimal pH and flow rate of the carrier buffer were 7.4 and 1.5ml/min, respectively. A modified electrode was integrated into the FIA system in order to eliminate electroactive interference and it was used to determine l-glutamate in 39 samples of Thai commercial soy sauce products. The results obtained were compared with those obtained from enzymatic assay using glutamate dehydrogenase and those from a chromatographic assay using an amino acid analyser. Good correlations were observed amongst these methods. The results indicated that use of an FIA system with a modified electrode was able to eliminate electroactive interference and was applicable to the determination of l-glutamate in food samples. The modified FIA was faster and simpler than the more common methods of enzymatic and chromatographic analysis.     

Extracellular L-glutamate oxidase from Streptomyces sp. Z-11-6: preparation of it and its properties

Mutagenesis induced with nitrous acid and subsequent selection allowed a genetically stable mutant strain, Streptomyces sp. Z-11-6, to be obtained, whose L-glutamate oxidase activity was 40-fold higher than that of the original natural isolate and was as great as 1.6-1.8 units/ml of culture liquid. A procedure for the isolation and purification of the enzyme was developed; the biochemical properties of the enzyme were studied. Out of 20 amino acids tested (including D-glutamate), the glutamate oxidase from Streptomyces sp. Z-11-6 was active only with L-glutamate. This allows the concentration of L-glutamate to be determined in the presence of other amino acids. Calcium chloride at a concentration of 0.1-0.5% promoted the secretion of the extracellular glutamate oxidase.     

Extracellular L-glutamate oxidase ofStreptomyces sp. Z-11-6: Obtainment and properties

Mutagenesis induced with nitrous acid and subsequent selection allowed a genetically stable mutant strain,Streptomyces sp. Z-11-6, to be obtained, whose L-glutamate oxidase activity was 40-fold higher than that of the original natural isolate and was as great as 1.6–1.8 units/ml of culture liquid. A procedure for the isolation and purification of the enzyme was developed; the biochemical properties of the enzyme were studied. Out of 20 amino acids tested (including D-glutamate), the glutamate oxidase fromStreptomyces sp. Z-11-6 was active only with L-glutamate. This allows the concentration of L-glutamate to be determined in the presence of other amino acids. Calcium chloride at a concentration of 0.1–0.5% promoted the secretion of the extracellular glutamate oxidase.     

Studies on the Metabolism of D-amino acid in Microorganisms

Several strains of bacteria belonging to genus Aerobacter were found to oxidize D-glutamate rapidly, while tbey show feeble oxidative activity toward the L-form even when they were grown in the medium containing DL-glutamate.

The isolation of L-glutamate, a natural amino acid, from its DL-form was achieved by the degradation of D-glutamic acid using one of these strains.

This may be the first observation on a natural amino acid obtained from the racemic one by the metabolic action of the organism.

A new enzyme, D-glutamic acid oxidase, which is responsible for D-glutamate degradation in this organism and differs from Krebs’ D-amino acid oxidase, has been postulated.     

L-谷氨酸生产关键技术创新与产业化应用

L-谷氨酸是目前产量最大的氨基酸品种,也是我国生产规模最大的生物发酵产品。随着合成生物技术以及新型生产装备和技术的发展,国内L-谷氨酸菌种和生产技术近年来取得了明显的提升。本文从L-谷氨酸产业的现状分析和关键技术创新需求的角度出发,概述了L-谷氨酸菌种和生产技术的研究进展,介绍了近年来L-谷氨酸生产关键技术的创新开发和产业应用的进展。     

Effect of carbon sources differing in oxidation state and transport route on succinate production in metabolically engineered Escherichia coli

In mixed-acid fermentation, succinate synthesis requires one mole of phosphoenolpyruvate (PEP), one mole of CO₂, and two moles of NADH for every mole of succinate to be formed. Different carbon sources with different properties were used to address these requirements. Sorbitol generates one more mole of NADH than glucose. Fermentation of sorbitol was shown in this study (and by others) to produce significantly more succinate than fermentation of glucose, due to increased NADH availability. Xylose fermentation conserves the intracellular PEP pool, since its transport does not require the phosphotransferase system normally used for glucose transport. The extra PEP can then be assimilated in the succinate pathway to improve production. In this study, fermentation of xylose did yield higher succinate production than glucose fermentation. Subsequent inactivation of the acetate and lactate pathways was performed to study metabolite redistribution and the effect on succinate production. With the acetate pathway inactivated, significant carbon flux shifted toward lactate rather than succinate. When both acetate and lactate pathways were inactivated, succinate yield ultimately increased with a concomitant increase in ethanol yield.     

Microbial oxidases catalyzing conversion of glycolaldehyde into glyoxal

The present paper reviews oxidases catalyzing conversion of glycolaldehyde into glyoxal. The enzymatic oxidation of glycolaldehyde into glyoxal was first reported in alcohol oxidases (AODs) from methylotrophic yeasts such as Candida and Pichia, and glycerol oxidase (GLOD) from Aspergillus japonicus, although it had been reported that these enzymes are specific to short-chain linear aliphatic alcohols and glycerol, respectively. These enzymes continuously oxidized ethylene glycol into glyoxal via glycolaldehyde. The AODs produced by Aspergillus ochraceus and Penicillium purpurescens also oxidized glycolaldehyde. A new enzyme exhibiting oxidase activity for glycolaldehyde was reported from a newly isolated bacterium, Paenibacillus sp. AIU 311. The Paenibacillus enzyme exhibited high activity for aldehyde alcohols such as glycolaldehyde and glyceraldehyde, but not for methanol, ethanol, ethylene glycol or glycerol. The deduced amino acid sequence of the Paenibacillus AOD was similar to that of superoxide dismutases (SODs), but not to that of methylotrophic yeast AODs. Then, it was demonstrated that SODs had oxidase activity for aldehyde alcohols including glycolaldehyde. The present paper describes characteristics of glycolaldehyde oxidation by those enzymes produced by different microorganisms.     

Production of acetic acid by Clostridium thermoaceticum in batch and continuous fermentations

Batch and continuous fermentations with Clostridium thermoaceticum (ATCC 39073) using automatic pH control were conducted. The value of mu(max) obtained from batch fermentation was about 0.14 h(-1); acetate yield, which was both growth and non-growth associated, was about 2 mole of acetic acid/mole of glucose, compared with a theoretical maximum value of 3. This low yield, compared with literature data, may be explained by glucose loss through a combination of degradation routes. Continuous fermentation could be sustained for 1600 h or more without contamination problems. Continuous fermentation at high dilution rates indicates that mu(max) may be well above 0.17 h(-1) when fresh feed medium is used. Acetate yields in continuous fermentation were about 77% of theoretical or 2.3 mole of acetic acid/mole of glucose.