聚γ-谷氨酸的产生菌筛选、发酵及絮凝特性研究
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摘要
聚γ-谷氨酸是一种天然存在的水溶性的多聚氨基酸，具有生物可降解性，可食用且对人体和环境无毒害。近年来被作为生物絮凝剂、增稠剂、药物缓释材料、生物可降解纤维、高吸水性树脂、重金属螯合剂以及化妆品添加剂等被广泛地应用于食品工业、药物生产、化妆品工业及污水处理中，是一种具有极大开发价值和广阔应用前景的多功能新型生物制品。
本文从超高压灭菌的哈密瓜汁中筛选得到一株聚γ-谷氨酸高产菌株，对其进行初步鉴定，确定为Bacillus sp.B-53。
通过单因子试验及正交试验分析，得到Bacillus sp.B-53发酵生产聚γ-谷氨酸的最佳培养基组成和培养条件。培养基组成为(g／L)：柠檬酸12，甘油80，L-谷氨酸20，硫酸铵7，K_2HPO_4 0.7，MgSO_4·7H_2O 0.5，MnSO_4·H_2O 0.3，CaCl_20.25，FeCl_3·6H_2O 0.02。Bacillus sp.B-53的适宜培养条件为：培养温度为37℃，初始pH值为6.5，种子液培养48h，接种量4％，装液量为50mL／300mL三角瓶，摇床振荡培养时间为96h，转速150rpm。在优化发酵条件下，Bacillus sp．B-53合成聚γ-谷氨酸可达19.12g／L，产量比优化前的12.56g／L提高52.23％。
通过乙醇、丙酮沉淀及透析处理得到纯化聚γ-谷氨酸，在硅胶薄层层析上显色为单点。对聚γ-谷氨酸的性质研究表明：该物质仅由谷氨酸组成，在212nm处有最大吸收峰；电泳结果表明Bacillus sp．B-53所产聚γ-谷氨酸并非是单一分子量的物质，而是集中在440kDa-669kDa之间的多分子量聚集体；热稳定性试验表明，随着处理温度的升高，聚γ-谷氨酸水溶液在一定的范围内粘度变化不大，聚合物结构比较稳定，在高温下，粘度下降快，聚γ-谷氨酸水解也快，分子量逐渐变小，聚γ-谷氨酸的水解是由谷氨酸链的随机切割引起的；pH值不同，聚γ-谷氨酸的粘度也有差异，在从pH中性调至pH5.0及pH3.0时，粘度分别降低24.58％和97.46％，而从中性调至偏碱性时，粘度变化不明显。
聚γ-谷氨酸能有效的絮凝多种无机和有机介质。在无Ca~(2+)存在时，以高岭土为絮凝介质其絮凝活性可达67.31％，加入Ca~(2+)(2g／L，以CaCl_2计)时絮凝活性最大，达到90％以上，最佳絮凝pH值为中性。聚γ-谷氨酸作为生物絮凝剂由于其高效、用途广、可生物降解且降解产物对环境无害，而具有远大的应用前景和商业价值。
Poly γ-glutamic acid (γ-PGA) is a water-soluble, biodegradable, elible and non-toxic toward humans and the environment. Theirefore, potential applications of y-PGA and its derivatives have been of interest in the past few years in a broad range of industrial fields such as food, cosmetics, medicine and water treatment. y-PGA and its derivatives offer a wide range of unique applications including being used as biopolymer flocculants, thickener, humectant, sustained-release material, drug carrier, highly water absorbable hydrogels, heavy metal absorber.
A bacterium with high γ-PGA productivity was isolated from the cantaloupe juice,This bacterium could be classified as Bacillus sp. B-53.
The production of y-PGA was greatly influenced by the composition of medium and culture condition. A number of experiments were done to evaluate the culture conditions, such as carbon, nitrogen, mineral salts, medium pH, temperature, time, inoculation amount, seed age and aeration. The results shew the optimum culture conditions were as follows (g/L) : Acetic acid 12, Glycerol 80, L-Glutamate 20, (NH4)2SO4 7, K2HPO4 0.7, MgSO4-7H2O 0.5, MnSO4-H2O 0.3, CaCl2 0.25, FeCl3.6H2O 0.02, initial pH 6.5, temperature 37℃, seed age 48h, inoculation amount 4%, culture time 96h.γ-PGA was purified by ethanol precipitation, acetone precipitation and dialysis. The purified material showed a absorption peak at 212nm, and was only composed of Glu. The Mw could be detected through SDS-PAGE, and its Mw was about a molecular mass between 440kDa with 669kDa. Study on the isolated y-PGA 's property shew that the y-PGA had good stability under 90 癈 and was rapidly hydrolyzed by heating at 100℃,and the hydrolysis of γ-PGA in the aqueous solution by heating was a random chain scission. Maximum viscosity was observed under conditions of neutral pH.
This bioflocculant y-PGA efficiently flocculated various organic and inorganic suspensions. It flocculated a suspended kaolin suspension without cations, although its flocculating activity was synergistically stimulated by the addition of ca2+ (0.2%) and rose from 67.31% to above 90%.The sunergistic effect of ca2+ was most effective at neutral pH ranges. The high productivity and versatile application of γ-PGA make its development as a new biodegradable harmless bioflocculant economical and advantageous.
本文从超高压灭菌的哈密瓜汁中筛选得到一株聚γ-谷氨酸高产菌株，对其进行初步鉴定，确定为Bacillus sp.B-53。
通过单因子试验及正交试验分析，得到Bacillus sp.B-53发酵生产聚γ-谷氨酸的最佳培养基组成和培养条件。培养基组成为(g／L)：柠檬酸12，甘油80，L-谷氨酸20，硫酸铵7，K_2HPO_4 0.7，MgSO_4·7H_2O 0.5，MnSO_4·H_2O 0.3，CaCl_20.25，FeCl_3·6H_2O 0.02。Bacillus sp.B-53的适宜培养条件为：培养温度为37℃，初始pH值为6.5，种子液培养48h，接种量4％，装液量为50mL／300mL三角瓶，摇床振荡培养时间为96h，转速150rpm。在优化发酵条件下，Bacillus sp．B-53合成聚γ-谷氨酸可达19.12g／L，产量比优化前的12.56g／L提高52.23％。
通过乙醇、丙酮沉淀及透析处理得到纯化聚γ-谷氨酸，在硅胶薄层层析上显色为单点。对聚γ-谷氨酸的性质研究表明：该物质仅由谷氨酸组成，在212nm处有最大吸收峰；电泳结果表明Bacillus sp．B-53所产聚γ-谷氨酸并非是单一分子量的物质，而是集中在440kDa-669kDa之间的多分子量聚集体；热稳定性试验表明，随着处理温度的升高，聚γ-谷氨酸水溶液在一定的范围内粘度变化不大，聚合物结构比较稳定，在高温下，粘度下降快，聚γ-谷氨酸水解也快，分子量逐渐变小，聚γ-谷氨酸的水解是由谷氨酸链的随机切割引起的；pH值不同，聚γ-谷氨酸的粘度也有差异，在从pH中性调至pH5.0及pH3.0时，粘度分别降低24.58％和97.46％，而从中性调至偏碱性时，粘度变化不明显。
聚γ-谷氨酸能有效的絮凝多种无机和有机介质。在无Ca~(2+)存在时，以高岭土为絮凝介质其絮凝活性可达67.31％，加入Ca~(2+)(2g／L，以CaCl_2计)时絮凝活性最大，达到90％以上，最佳絮凝pH值为中性。聚γ-谷氨酸作为生物絮凝剂由于其高效、用途广、可生物降解且降解产物对环境无害，而具有远大的应用前景和商业价值。
Poly γ-glutamic acid (γ-PGA) is a water-soluble, biodegradable, elible and non-toxic toward humans and the environment. Theirefore, potential applications of y-PGA and its derivatives have been of interest in the past few years in a broad range of industrial fields such as food, cosmetics, medicine and water treatment. y-PGA and its derivatives offer a wide range of unique applications including being used as biopolymer flocculants, thickener, humectant, sustained-release material, drug carrier, highly water absorbable hydrogels, heavy metal absorber.
A bacterium with high γ-PGA productivity was isolated from the cantaloupe juice,This bacterium could be classified as Bacillus sp. B-53.
The production of y-PGA was greatly influenced by the composition of medium and culture condition. A number of experiments were done to evaluate the culture conditions, such as carbon, nitrogen, mineral salts, medium pH, temperature, time, inoculation amount, seed age and aeration. The results shew the optimum culture conditions were as follows (g/L) : Acetic acid 12, Glycerol 80, L-Glutamate 20, (NH4)2SO4 7, K2HPO4 0.7, MgSO4-7H2O 0.5, MnSO4-H2O 0.3, CaCl2 0.25, FeCl3.6H2O 0.02, initial pH 6.5, temperature 37℃, seed age 48h, inoculation amount 4%, culture time 96h.γ-PGA was purified by ethanol precipitation, acetone precipitation and dialysis. The purified material showed a absorption peak at 212nm, and was only composed of Glu. The Mw could be detected through SDS-PAGE, and its Mw was about a molecular mass between 440kDa with 669kDa. Study on the isolated y-PGA 's property shew that the y-PGA had good stability under 90 癈 and was rapidly hydrolyzed by heating at 100℃,and the hydrolysis of γ-PGA in the aqueous solution by heating was a random chain scission. Maximum viscosity was observed under conditions of neutral pH.
This bioflocculant y-PGA efficiently flocculated various organic and inorganic suspensions. It flocculated a suspended kaolin suspension without cations, although its flocculating activity was synergistically stimulated by the addition of ca2+ (0.2%) and rose from 67.31% to above 90%.The sunergistic effect of ca2+ was most effective at neutral pH ranges. The high productivity and versatile application of γ-PGA make its development as a new biodegradable harmless bioflocculant economical and advantageous.
引文
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[2] Ashiuchi M., Tani K., Soda K. A poly-γ-glutamate synthetic system of Bacillus subtilis IFO 3336 gene cloning and biochemical analysis of poly-γ-glutamate produced by E. coli clone cells. Biochem. Biophys. Res. Commun., 1999, 263: 6～12.
[3] Astrid H., Fred B.O., Alexander S. Conversion of the nitrogen content in liquid manure into biomass and poly-glutamic acid by a newly isolated strain of Bacillus licheniformis. FEMS Microbiology Letters, 2003, 218: 39～45.
[4] Bovamick M. J. The formation of extracellular D(-)glutamic acid polypeptide by Bacillus subtilis. J. Biol. Chem, 1942, 145: 415～424.
[5] Bitter G A., Cromwick A-M., Gross R A. γ-poly(glutamic acid) formation by Bacillus licheniformis ATCC9945a: physiological and biochemical studies. J. Biol. Macromol., 1994, 16: 265～275.
[6] Borbély M., Nagasaki Y., Borbély J., et al. lBiosynthesis and chemical modification of poly (γ-glutamic acid). Polymer Bull, 1994, 32: 127～132.
[7] Cheng C., Yoshihiro A., Tokujiro A. Production of γ-polyglutamic acid by Bacillus subtilis A35 under denitrifying conditions. Agric. Biol. Chem., 1989, 53: 2369～2375.
[8] Cromwick A.M., Gross R A. Investigation by NMR of metabolic routes to bacterial γ-poly (glutamic acid) using 13C labeled citrate and glutamate as media carbon sources. Can. J. Microbio l., 1995 b, 41: 902～909.
[9] Cromwick A.M., Gross R A. Effect of manganese (Ⅱ) on Bacillus licheniformis ATCC9945A physiology and γ-poly(glutamic acid) formation. Int. J. Biol. Macromol., 1995, 16: 265～275.
[10] Cromwick A-M., Bitter GA., Gross RA. Effects of pH and aeration on poly(γ-glutamic acid) formation by Bacillus licheniformis in controlled batch fermentor cultures. Biotechnol. Bioeng., 1996, 50: 222～227.
[11] Gardner J M., TroyFA. Chemistry and biosynthesis of the poly(γ-D-glutamate) capsule in Bacillus licheniformis. J. Bio. Chem., 1979, 254: 6262～6269.
[12] Giannos S A., Shah D., Gross R A., et al. Poly(glutamic acid) produced by bacterial fermentation. In: Novel Biodegradable Microbial polymers (ed.Dawes, E.A.), Kluwer, Netherlands, 1990, 457～460.
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