【作者】 惠明；

【导师】 牛天贵；

【作者基本信息】 中国农业大学 ， 食品科学， 2005， 博士

【摘要】 聚γ-谷氨酸（γ-PGA）主要是由一些芽孢杆菌产生的一种水溶性的胞外氨基酸聚合物。近年来被作为高吸水性材料、增稠剂、药物载体、生物絮凝剂及重金属吸附剂等广泛地应用于农业、食品、医药、化妆品及水处理等领域中,是一种具有极大开发价值和广阔应用前景的新型多功能高分子材料。本文从γ-PGA产生菌的筛选及鉴定、发酵条件优化及合成机制、菌种选育、γ-PGA的提纯及理化因素对其分子结构的影响、γ-PGA的应用等方面进行了研究。主要研究结果如下: （1）从76个样品分离得到的231株产芽孢细菌中筛选出一株聚γ-谷氨酸生产菌芽孢杆菌B53,其所产聚合物最大吸收波长为212nm,分子量集中在570～669kD之间,呈多分子量聚集体存在;从细胞形态、生理生化及遗传特征上分析鉴定B53菌株为枯草芽孢杆菌。 （2）B.subtilis B53合成γ-PGA的适宜碳氮源是甘油、柠檬酸、谷氨酸及硫酸铵,K₂HPO₄及Ca²⁺、Fe³⁺对γ-PGA的合成有显著的促进作用。通过正交试验和回归分析得出B53菌株合成γ-PGA的适宜培养基组成（g/L）:L-Glu 20,CTA 9.86,Glycerol 80.36,（NH₄）₂SO₄ 7,MgSO₄·7H₂O_0.5,FeCl₃·6H₂O_0.02,K₂HPO₄ 0.89,CaCl₂ 0.03,MnSO₄·H₂O 0.3。种子液培养24h,接种量4%（V/V）,装液量50mL/300mL三角瓶,初始pH6.5,在37℃,150r/min摇床培养84h,γ-PGA的产量达到26.67g/L。5L发酵罐分批发酵试验表明:在发酵过程中菌体形态与聚合物结构有明显的变化:同柠檬酸和谷氨酸的消耗相比,甘油的代谢比较快,在甘油消耗比较快的阶段伴随γ-PGA的快速合成;γ-PGA的分子量在发酵54h以前基本没有变化,但之后分子量范围逐步变宽;分批发酵产量达18.61g/L。补料发酵试验发现,在发酵中期添加适量甘油及在发酵培养基中添加20～100U/mL的青霉素或10μg/L的生物素都可有利于γ-PGA产量的提高,而添加表面活性剂（如Tween 20、SDS）则不利于γ-PGA的合成。 （3）通过细胞融合技术选育出枯草芽孢杆菌B53与谷氨酸棒杆菌B9的稳定融合株R162,该菌株在以葡萄糖和硫酸铵为碳氮源的培养基上γ-PGA的产量比B53提高一倍以上（有多糖合成）。通过⁶⁰Co γ射线辐照选育出γ-PGA高产突变株F2-28,其发酵产量比B53提高了82.31%。 （4）当发酵液中γ-PGA含量人于20g/L,离心除菌后添加发酵液体积2.5倍的95%乙醇可以得到较好的提取效果;对γ-PGA发酵液采用超滤浓缩可以节约乙醇的用量,离心除菌及超滤浓缩要消耗大量能量。酸化或加热处理可明显降低发酵液的黏度,从而降低离心或超滤操作能耗,但酸化或热处理会使γ-PGA的分子结构发生变化,分子量降低。γ-PGA在pH值中性下比较稳定,pH<5或pH>8,容易发生降解,这是pH值改变引起发酵液黏度降低的主要原因。 （5）采用超声波、紫外线及⁶⁰Co γ射线辐照处理可以对γ-PGA进行分子修饰,对低分子量聚γ-谷氨酸的生产具有参考作用。Ca²⁺、Cu²⁺与Zn²⁺等金属离子可与γ-PGA发生络合作用。 （6）γ-PGA对高岭土、Ca（OH）₂、Mg（OH）₂表现出较强的絮凝活性,采用0.6g/L的γ-PGA溶液对高岭土的絮凝活性可达到90%以上。K⁺、Fe²⁺、Mg²⁺及Ca²⁺具有明显的促絮凝作用,而Al³⁺、Fe³⁺则起削弱作用。CaCl₂浓度超过2g/L及介质溶液维持pH值中性都有利于γ-PGA提高絮凝活性。另外,γ-PGA可制备成水凝胶用作涂膜材料,对水果蔬菜的保鲜有一定作用。更多还原

【Abstract】 Poly(γ-glutamic acid) (γ-PGA) is a water-soluble polymer that consists of D- and L-glutamic acid and is mainly produced by several Bacillus species outside the cells. Potential and unique applications of γ-PGA and its derivatives have been of interest in the past few years in a broad range of industrial fields such as agriculture, food, medicine, cosmetics and water treatment, which can be used as highly water absorbable materials, thickener, drug carrier, biopolymer flocculants and heavy metal absorber. Therefore, the objective of this dissertation is to carry out a fermentation bioprocess with a high γ-PGA yield. The contents mainly include: (1) selection and identification of the strain producing y-PGA; (2) fermentation process optimization and mechanism of y-PGA synthesis; (3) breeding of the high yield strain; (4) the purification and the effects of physical chemistry factors on γ-PGA molecule structure; (5) potential application of y-PGA and the strain. The main results of this paper are as follows:[1] Bacillus sp. B53 was selected from 231 strains that were isolated from 76 specimen and shown to produce poly( γ -glutamic acid). The polymer had an absorption peak at 212nm and its molecular weight was between 570 kD and 669 kD. With the following analysis of colony morphology, physiological and biochemistry experiments, G+C content (mol%) and 16S rRNA gene sequence as well, the strain was identified as Bacillus subtilis.[2] The yield of γ-PGA by B. subtilis B53 was different on various carbon sources and nitrogen sources, and among those, citric acid, glycerol, L-glutamic acid and ammonium sulfate were much better than others; Inorganic salts such as K2HPO4, CaCl2 and FeCl3 facilitated γ-PGA synthesis clearly. Using orthogonal experimental design and regression analysis, the optimum fermentation medium was obtained as follows(g/L): L-Glutamic acid 20, Citric acid 9.86, Glycerol 80.36, (NH4)2SO4 7, MgSO4 7H2O 0.5, FeCl3 6H2O 0.02, K2HPO4 0.89, CaCl2 2H2O 0.03, MnSO4 H2O 0.3; pH 6.5 with NaOH. Under these conditions, γ-PGA 26.67 g/L was achieved in shake flask culture with temperature of 37℃, seed age 24h, inoculation amount 4%(V/V), adding 50mL medium each 300 mL flask and 150 r/min for 84 h. Batch fermentation of γ-PGA in 5 L bioreactor was carried out. The organism cell and the polymer structure changed evidently in the course. Compared with the consumption of citric acid and glutamic acid, the metabolism of glycerol was faster; and γ-PGA synthesis accelerated in the term of glycerol consumption increasing fast; the γ-PGA could be discovered after 12 h and its yield was achieved peak at 84 h, then reduced a few; the molecular weight of γ-PGA was basically no change before 54 h, but its range became wide later; γ-PGA 18.61 g/L was achieved in the batch fermentation. The results of fed-batch fermentation showed adding a few glycerol could increase γ-PGA yields in the middle of the course; and adding penicillin 20~100 U/mL or biotin 10 μ g/L could improve the γ-PGA synthesis in the medium, but adding surfactants (Tween20 or SDS) would be disadvantageous to γ-PGA high-yield.[3] Using protoplasts fusion technique, Bacillus subtilis B53 and Corynebacterium glutamicum B9 were successfully fused. A stable fusion hybrid R162 was obtained, which was clearly different with the parents, its γ-PGA yield was doubled in the medium of glucose and (NH4)2SO4 as carbon and nitrogensources and a few polysaccharide were also formed.Bacillus subtilis B53 was irradiated by 60Co γ-rays; a high-yield mutant was selected and named F2-28, its γ-PGA yield was improved 82.31% by contrast with B53 in the same condition. Cell morphology and forming spore capability of the mutant were researched; the results showed the capsules lays of F2-28 were thicker clearly than before and its forming spore capability changed weakly.[4] γ-PGA was separated by ethanol precipitation. A good separating result was obtained adding 2.5 times 95% ethanol of culture broth after the organisms were removed when γ-PGA concentration of the bro更多还原