S11菌体酶工业培养条件的研究和优化
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摘要
L-半胱氨酸是一种重要的含硫氨基酸,广泛应用于医药、食品、化妆品以及饲料工业。本课题组在前期工作中已从富含DL-ATC(DL-2-氨基-△2-噻唑啉-4-羧酸)的环境中筛选到一株可转化DL-ATC合成L-半胱氨酸的菌株S11,并且已经利用形态学特征、生理生化特征对11号菌株进行了初步分类鉴定,结果表明该菌株可能属于微球菌属能够利用DL-ATC转化合成L-半胱氨酸的一个亚种。
本论文主要从产物的鉴别、菌株生长及产酶条件的优化等方面进行了研究,研究结果如下:
建立L-半胱氨酸产物定性、定量检测方法(纸层析法、DTNB显色法)和菌株酶活力的检测方法(酸式茚三酮法、结晶法)。通过考察优化了菌株的培养条件和产酶条件。
通过对菌株的培养发现,11号菌株生长最适碳源为葡萄糖,最适氮源为尿素,并采用minitab软件的Placket-Burman设计法和二次响应面分析法对种子培养基进行优化,得到了主要影响因素及其最佳水平值:葡萄糖19.216 g/L,尿素4.009 g/L,转速133.910 r/min,在优化条件下经摇瓶培养实验表明细胞生长量比优化前提高了16.9%。
通过对摇瓶培养产酶条件的研究发现,DL-ATC对酶的产生具有诱导作用,11号菌株以葡萄糖为碳源,尿素为氮源时生长量和酶活力最高,产酶最适温度为30℃,最佳初始pH为7.5。采用Placket-Burman设计法和二次响应面分析法对产酶培养基进行优化,得到了主要影响因素及其最佳水平值:葡萄糖27.6g/L,DL-ATC 7.27 g/L,转速144 r/min。在优化条件下经摇瓶培养实验表明细胞酶活力比优化前提高了7.8%。
L-cysteine is an elementary S-containing amino acid, which has been found utility as medicine, raw materials for medicine, food additive, additive for cosmetics and fodder. In our lab, a germ 11 that can convert DL-ATC to L-cysteine was selected from the soil containing high concentration DL-ATC. Analyzed the phenotypic, physiological and morphological information to identify the taxonomy status of the germ 11 by using polyphasic taxonomy, the germ 11 probably was a subspecies of micrococcus, which can use DL-ATC as substrate to synthesize L-cysteine.
The identification of germ, verification of products, conditions of growth and enzyme producing were studied in detail. The results were as follow.
The qualitative and quantitative detecting method of L-cysteine(the paper developing color method and the DTNB) and enzyme activity are established. Conditions of growth and enzyme producing were studied in detail.
Through investigating of the growth condition we found that glucose is the most suitable carbon source of growth and urea is most optimum nitrogen source. Two level factorials designs of Placket-Burman were constructed to select culture medium components of 11. Three important components, which were glucose, urea and rotate speed were screened. The optimized level of these factors was determined by response surface analysis. The shake flask cultivations showed the cell concentration increased 16.9% at optimized condition.
We also found three important components of the enzymatic production seed, which were glucose, DL-ATC and rotate speed. The shake flask cultivations showed that and enzyme activity increased 7.8% at optimized condition. The optimized enzyme yield conditions were: initial pH 7.0-7.5, 30℃.
本论文主要从产物的鉴别、菌株生长及产酶条件的优化等方面进行了研究,研究结果如下:
建立L-半胱氨酸产物定性、定量检测方法(纸层析法、DTNB显色法)和菌株酶活力的检测方法(酸式茚三酮法、结晶法)。通过考察优化了菌株的培养条件和产酶条件。
通过对菌株的培养发现,11号菌株生长最适碳源为葡萄糖,最适氮源为尿素,并采用minitab软件的Placket-Burman设计法和二次响应面分析法对种子培养基进行优化,得到了主要影响因素及其最佳水平值:葡萄糖19.216 g/L,尿素4.009 g/L,转速133.910 r/min,在优化条件下经摇瓶培养实验表明细胞生长量比优化前提高了16.9%。
通过对摇瓶培养产酶条件的研究发现,DL-ATC对酶的产生具有诱导作用,11号菌株以葡萄糖为碳源,尿素为氮源时生长量和酶活力最高,产酶最适温度为30℃,最佳初始pH为7.5。采用Placket-Burman设计法和二次响应面分析法对产酶培养基进行优化,得到了主要影响因素及其最佳水平值:葡萄糖27.6g/L,DL-ATC 7.27 g/L,转速144 r/min。在优化条件下经摇瓶培养实验表明细胞酶活力比优化前提高了7.8%。
L-cysteine is an elementary S-containing amino acid, which has been found utility as medicine, raw materials for medicine, food additive, additive for cosmetics and fodder. In our lab, a germ 11 that can convert DL-ATC to L-cysteine was selected from the soil containing high concentration DL-ATC. Analyzed the phenotypic, physiological and morphological information to identify the taxonomy status of the germ 11 by using polyphasic taxonomy, the germ 11 probably was a subspecies of micrococcus, which can use DL-ATC as substrate to synthesize L-cysteine.
The identification of germ, verification of products, conditions of growth and enzyme producing were studied in detail. The results were as follow.
The qualitative and quantitative detecting method of L-cysteine(the paper developing color method and the DTNB) and enzyme activity are established. Conditions of growth and enzyme producing were studied in detail.
Through investigating of the growth condition we found that glucose is the most suitable carbon source of growth and urea is most optimum nitrogen source. Two level factorials designs of Placket-Burman were constructed to select culture medium components of 11. Three important components, which were glucose, urea and rotate speed were screened. The optimized level of these factors was determined by response surface analysis. The shake flask cultivations showed the cell concentration increased 16.9% at optimized condition.
We also found three important components of the enzymatic production seed, which were glucose, DL-ATC and rotate speed. The shake flask cultivations showed that and enzyme activity increased 7.8% at optimized condition. The optimized enzyme yield conditions were: initial pH 7.0-7.5, 30℃.
引文
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[2]陈新谦主编。新编药物学,人民卫生出版社。1982, 483。
[3]陈敏元。L-半胱氨酸药物及其合成,化学工程师。1991, 23 (5), 39～41。
[4]雷绍青。氨基酸在临床上的应用,氨基酸杂志4,26-29,1988。
[5]林文修,陈丹,王友泼。电解合成L-半胱氨酸的研究,福建化工,1991, 2, 25。
[6]Niroshini M.Giles,Gregory1 .Giles and Claus Jacob. Multiple roles of cysteine in biocatalysis Biochem. and Biophy. Research Commun., 2003, 300(l), 1-4。
[7]黄宜基,周承文,L-半胱氨酸系列衍生物的合成及其在医药上的应用。氨基酸杂志,1985,2,37～42。
[8]徐衡,张群,孔学军。N-乙酰-L-半胱氨酸合成新工艺。氨基酸和生物资源,2000, 22(1),23～26。
[9]李毅敏,赵树仪。N-乙酰半胱氨酸的研究进展。天津药学,2003,15 (2),50-53。
[10]张静,翁玲玲。O-酰基-L-丝氨酸衍生物和S-酰基-L-半胱氨酸衍生物的合成研究,中国药物化学杂志,2003,13(2),93～96。
[11]马程军,姚斐,张关永。S-甲基-L-半胱氨酸的合成,化学世界,2003,7, 370-372。
[12]陈敏元。应该重视L-半胱氨酸的应用,现代化工,1989,9,3234。
[13]陈勉哉。半胱氨酸性能极其应用,化工时刊,1989, 1, 2528。
[14]赵建国。氨基酸在食品工业中的应用现状及进展,氨基酸杂志,1984, 3, 3237。
[15]宋正选,许晶,郭继兴等。维生素C注射液抗氧剂的选择,西北药学杂志,2003,18。
[16]陈敏元。半胱氨酸美白化妆品,日用化学工业,1992,167( 3),4 9-50。
[17]E.G.Gerwe.Studies on the spontaneous oxidation of cysteine.II I.The method of action of cyanides and cystine on cysteine oxidation.J.Biol.Chem., 1931, 92。
[18]陈庭树。利用胱氨酸生产母液开发蛋白质饲料的饲养效应,农业环境保护,1995,14(6),267-2600。
[19]蒲富永,陈玉成,皮广杰等。利用胱氨酸生产废水开发氨基酸复合(混)固体废料的研究,重庆环境科学,1994,16(5),3-70。
[20]许森清。胱氨酸生产现状和前景探讨,泉州师专学报(自然科学版),1998,16 (3),21-220。
[21]郭生贵。利用毛发生产胱氨酸的实践,黔东南民族师专学报(自然科学版),1994,11(3,4),34-360。
[22]王贵峰。胱氨酸生产中水解工艺设计的改进,化工设计,1997,1,21-22。
[23]刘笃琏。胱氨酸生产中中和过程的数字描述,氨基酸杂志,1994,3,25-270。
[24]朱广军。从羽毛梗中制取胱氨酸的工艺,江苏化工,1994,22 (2),18-20。
[25]刘勋,陈时洪。用氧化亚铜从人发水解液中沉淀胱氨酸的研究,氨基酸和生物资源,2003,25(1),55-57。
[26]杨林。L-半胱氨酸生产存在的问题及解决办法,化工矿山技术,1998,27 (1), 37-39。
[27]陈松茂,顾宏邦。L-半胱氨酸盐酸盐的电解合成,山西食品工业,1996,3,18-200。
[28]Gaitonde,M.K.A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acid.Biochem,J.,1967,104,62 7-633
[29]黄英利,陆维玮,盛永丽。电解还原法合成L-半胱氨酸盐酸盐,山东医药工业,1997,16 (6), 6-8.
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