Bacillus subtilis ZJUTZY发酵生产γ-聚谷氨酸及其分离提纯研究
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摘要
γ-PGA(γ-聚谷氨酸)是一种通过微生物合成的均聚氨基酸化合物,它由谷氨酸单体以γ-羧基与氨基相缩合而成,是一种水溶性和可生物降解的新型生物高分子材料,近年来由于其被作为增稠剂,保湿剂,药物载体等而一直被广泛应用于工业领域,是一种有极大开发价值和前景的多功能性生物制品。
Bacillus subtilis ZJUTZY为本实验室从纳豆中分离出的γ-聚谷氨酸(γ-PGA)高产菌株,本文对其产γ-PGA的发酵条件进行优化。在单因素实验基础上,用响应面法进行了培养基组成优化,用正交法进行了培养条件优化。实验得到最适培养基组成(g/L):蔗糖50,蛋白胨60.9,谷氨酸钠81.8,MgSO_4 1.45,NaCl 7.5,K_2HPO_4 0.75,最适培养条件:培养基初始pH值为7,装液量30 ml,接种量3.3%,优化后γ-PGA产量由22.24 g/L提高到49.90 g/L,是初始γ-PGA产量的2.24倍。
由于经响应面法优化的发酵培养基组成中,所需的有机氮源蛋白胨含量较高,因此发酵成本就相对较高。为了降低生产成本,本文应用转化的方法来发酵生产γ-PGA,在不添加任何碳氮源的培养基中,只添加谷氨酸钠作为底物,利用Bacillus subtilis ZJUTZY将其转化为γ-PGA并对转化条件进行了单因素研究。实验得到的最适转化条件为:谷氨酸钠底物80 g/L,培养基初始pH值为7,装液量27%,转化液与二级种子液的体积比4:1,转化温度30℃,转化时间16 h。
利用Bacillus subtilis ZJUTZY发酵合成γ-聚谷氨酸(γ-PGA)。由1L发酵液制备γ-聚谷氨酸粗品31.761 g,经过液相检测,γ-聚谷氨酸含量为50.5%。经过考马斯亮兰G-250法检测表明,粗品中不含杂蛋白。粗品再经过强碱性阴离子树脂纯化,去除大部分多糖杂质,得到纯度为80.9%的γ-PGA。
Poly(γ-glutamic acid)(γ-PGA) is an unusual anionic,water-soluble and high viscous polymer in which glutamate is polymerized by amide linkages between a-amino andγ-carboxylic acid groups.For its excellent characters,potential applications ofγ-PGA have been of interest in the past few years in a broad range of industrial fields.It has been used as thickeners,humectant and drug carrier.
In this study a novel isolate named Bacillus subtilis ZJUTZY was used to produce Poly-gamma-glumatic acid(γ-PGA).Then the effects of the fermentation conditions were studied by response surface and orthogonal test on the basis of one factor test.The result showed that The optimum medium was sucrose 50 g/L,peptone 60.9 g/L,glutamic acid 81.8 g/L, NaCl 7.5 g/L and K_2HPO_4 0.75 g/L;the optimum initial pH,the liquid volume and inoculated ratio were 7.0,30 ml and 3.3%respectively.The largest product was increased from 22.24 g/L to 49.90 g/L,which was 2.24 times of the initial yield.
For the medium composition contains a high content of peptone,which is a high cost organic nitrogen source after optimization,it must be necessary to reduce cost of production.In this study,the method of transformation was utilized to produceγ-PGA.With no other carbon and nitrogen sources in the medium,Bacillus subtilis ZJUTZY was used to transformγ-PGA from glutamic acid.The optimum transformation condition was as follows:glutamic acid 80 g/L,initial pH 7,liquid volume in flask 27%,transformation liquid:second level seed liquid=4:1(v/v), temperature 30℃,time 16h.
Bacillus subtilis ZJUTZY was utilized to produce poly-gamma-glutamic acid.The yield of crude product was 31.76 g in 1L broth.Based on the determination of HPLC,50.5%of the crude product wasγ-PGA.On the basis of method of Coomassie brilliant blue G250,no protein was found in the crude product.The crude product was then purified by strong basic anion exchange resin,most unwanted poly-saccharide was moved,and 80.9%γ-PGA was yield.
Bacillus subtilis ZJUTZY为本实验室从纳豆中分离出的γ-聚谷氨酸(γ-PGA)高产菌株,本文对其产γ-PGA的发酵条件进行优化。在单因素实验基础上,用响应面法进行了培养基组成优化,用正交法进行了培养条件优化。实验得到最适培养基组成(g/L):蔗糖50,蛋白胨60.9,谷氨酸钠81.8,MgSO_4 1.45,NaCl 7.5,K_2HPO_4 0.75,最适培养条件:培养基初始pH值为7,装液量30 ml,接种量3.3%,优化后γ-PGA产量由22.24 g/L提高到49.90 g/L,是初始γ-PGA产量的2.24倍。
由于经响应面法优化的发酵培养基组成中,所需的有机氮源蛋白胨含量较高,因此发酵成本就相对较高。为了降低生产成本,本文应用转化的方法来发酵生产γ-PGA,在不添加任何碳氮源的培养基中,只添加谷氨酸钠作为底物,利用Bacillus subtilis ZJUTZY将其转化为γ-PGA并对转化条件进行了单因素研究。实验得到的最适转化条件为:谷氨酸钠底物80 g/L,培养基初始pH值为7,装液量27%,转化液与二级种子液的体积比4:1,转化温度30℃,转化时间16 h。
利用Bacillus subtilis ZJUTZY发酵合成γ-聚谷氨酸(γ-PGA)。由1L发酵液制备γ-聚谷氨酸粗品31.761 g,经过液相检测,γ-聚谷氨酸含量为50.5%。经过考马斯亮兰G-250法检测表明,粗品中不含杂蛋白。粗品再经过强碱性阴离子树脂纯化,去除大部分多糖杂质,得到纯度为80.9%的γ-PGA。
Poly(γ-glutamic acid)(γ-PGA) is an unusual anionic,water-soluble and high viscous polymer in which glutamate is polymerized by amide linkages between a-amino andγ-carboxylic acid groups.For its excellent characters,potential applications ofγ-PGA have been of interest in the past few years in a broad range of industrial fields.It has been used as thickeners,humectant and drug carrier.
In this study a novel isolate named Bacillus subtilis ZJUTZY was used to produce Poly-gamma-glumatic acid(γ-PGA).Then the effects of the fermentation conditions were studied by response surface and orthogonal test on the basis of one factor test.The result showed that The optimum medium was sucrose 50 g/L,peptone 60.9 g/L,glutamic acid 81.8 g/L, NaCl 7.5 g/L and K_2HPO_4 0.75 g/L;the optimum initial pH,the liquid volume and inoculated ratio were 7.0,30 ml and 3.3%respectively.The largest product was increased from 22.24 g/L to 49.90 g/L,which was 2.24 times of the initial yield.
For the medium composition contains a high content of peptone,which is a high cost organic nitrogen source after optimization,it must be necessary to reduce cost of production.In this study,the method of transformation was utilized to produceγ-PGA.With no other carbon and nitrogen sources in the medium,Bacillus subtilis ZJUTZY was used to transformγ-PGA from glutamic acid.The optimum transformation condition was as follows:glutamic acid 80 g/L,initial pH 7,liquid volume in flask 27%,transformation liquid:second level seed liquid=4:1(v/v), temperature 30℃,time 16h.
Bacillus subtilis ZJUTZY was utilized to produce poly-gamma-glutamic acid.The yield of crude product was 31.76 g in 1L broth.Based on the determination of HPLC,50.5%of the crude product wasγ-PGA.On the basis of method of Coomassie brilliant blue G250,no protein was found in the crude product.The crude product was then purified by strong basic anion exchange resin,most unwanted poly-saccharide was moved,and 80.9%γ-PGA was yield.
引文
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