L-缬氨酸高产菌株的选育及其特性的初步研究
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摘要
L-缬氨酸不仅是支链氨基酸,而且是人体八种必需氨基酸之一,在生命体各种重要的生理活动中起着不可替代的作用。L-缬氨酸应用领域非常广阔,在医药、食品强化剂、饲料添加剂、调味品和农药等方面都有重要的应用。现阶段国内的L-缬氨酸发酵水平不高,全国年产量远远不能满足国内市场日益增长的需求。为了解决上述问题,本课题依据代谢工程原理,选育可以高效生产L-缬氨酸的菌种,具体结果如下:
⑴用硫酸二乙酯(DES)和亚硝胍(NTG)处理出发菌株黄色短杆菌(Brevibacterium flavum) ZGH6128,经过筛选最终获得菌株NVT1103(Leu~l,α-AB~(hr), 2-TA~(hr),SG~r)与菌株JVHK597(Leu~-,Ile~-,Met~l,α-AB~r,2-TA~r)。在未优化的条件下,两株菌的摇瓶产酸量分别为37.6g/L和41.2g/L。
⑵利用原生质融合技术进行氨基酸菌种选育的研究引起国内外技术人员的关注,本课题以菌株NV1103和JVHK597为亲本菌株,采用原生质体融合方法进行选育L-缬氨酸高产菌株。用0.5U/mL与1U/mL的青霉素钠盐溶液分别预处理双亲本菌株(NV1103与JVHK597)2.5h和3h;溶菌酶处理亲本株的最适浓度为1g/L与2g/L,分别处理9h和11h;再生培养基中渗透压稳定剂浓度为0.6mol/L;在34°C、pH10的缓冲体系中,400g/L的PEG介导25min,可以获得理想的融合率。最终选育出高产菌株NJv61,遗传标记为(Leu~-,Ile~-,Met~l, 2-TA~(hr), SG~r, Glc~(hr),α-AB~(hr)),未优化条件下,摇瓶产酸量为45.6g/L。
⑶利用正交表进行实验设计确定菌株NJv61种子培养基的组成:葡萄糖24g/L,玉米浆35g/L,(NH_4)_2SO_4 5g/L,KH_2PO_4 0.6g/L,MgSO_4·7H_2O 0.5g/L,CaCO_3 10g/L。对菌株发酵过程中所需的碳(氮)源、无机盐、生长因子等进行了单因素实验,在此基础上通过响应面方法确定了该菌株发酵培养基的最佳组成:葡萄糖149g/L,玉米浆19.4g/L,(NH_4)_2SO_4 48.4g/L,KH_2PO_4 1.5g/L,MgSO_4·7H_2O 0.6g/L,VB1 150μg/L,VH 60μg/L。在7L发酵罐中,优化条件下NJv61经过72h发酵,在发酵液中积累L-缬氨酸达到51.8g/L。
⑷对L-缬氨酸产生菌筛选过程中所获得的代表菌株(ZGH6128、NVT1103、JVHK597和NJv61),在7L发酵罐产酸过程的中后期进行取样分析,明确了该时期相关菌株细胞内与合成L-缬氨酸相关的代谢途径网络分布。与出发菌株ZGH6128相应途径上的流量相比,目的菌株NJv61在节点G6P处流向EMP途径的碳架流量有了显著的提高,同时,在HMP途径中仍然保持较大流量。在丙酮酸节点处,目的菌株流向TCA循环的代谢流量较出发菌株大大减少,使更多的碳架流进入L-缬氨酸合成途径。目的菌株中丙氨酸生物合成途径中的碳架流量较出发菌株没有明显的下降。
⑸乙酰羟基酸合成酶(AHAS)是半理性化育种工作的靶酶,目的菌株NJv61对高浓度α-AB和2-TA具有抗性,研究发现该菌株的AHAS基因发生了突变,更彻底地解除L-缬氨酸反馈抑制与阻遏作用,并且酶的相对活性也有改变。本课题对AHAS基因进行了克隆,对测序的结果进行了比较分析发现,调节亚基所对应的基因区段,仅分布着整个基因突变位点总数大约10%的突变碱基,而编码催化亚基的基因区段布着的突变位点数约为整个基因所有突变位总数的90%。最后把菌株NJv61的AHAS基因与特定的表达载体相连接,导入模式菌株,经IPTG诱导,所表达的酶蛋白分子在4℃条件下不稳定,22h内该酶相对酶活迅速下降,仅保持大约20%相对活性。
L-Valine is not only one of three branched-chain amino acids but also a member of eight essential amino acids which can’t be synthesized by humans themselves. It plays an important role in many kinds of physiological functions in vertebrates’bodies. The applications of L-valine are very broad: precursor of pharmaceuticals, additives to food or feedstuff for livestocks, material for pesticides, nutrition infusion for patients, health care product, and condiment and so on. Nowadays, scales of the industrial plants for L-valine in China are small, and the actual production level of L-valine prodution isn’t high. The annual production amount of L-valine can’t meet the growing demand of domestic market. In order to solve problems mentioned above, it is necessary to breed highly efficient L-valine-producing strains based on metabolic engineering principles.
(1) The origin strain is strain ZGH6128 (Brevibacterium flavum) which was treated with DES and NTG in the screening process. Strain NVT1103 (Leu~l,α-AB~(hr), 2-TA~(hr),SG~r) and strain JVHK597(Leu~-, Ile~-, Met~l,α-AB~r, 2-TA~r) were obtained in the early stages. Under unoptimized conditions, the L-valine productions in flasks of the two strains were 37.6g/L and 41.2g/L respectively.
(2) Scientists at home and abroad were drawn attention to the application of protoplast fusion technology to screen L type amino acids producing strains. Strain NV1103 and strain JVHK597 was used as parent strains during the process to beed L-valine hyper-producer by protoplast fusion technology in this thesis. Parent strains (NV1103 and JVHK597) were pretreated for 2.5 hours and 3 hours with 0.5 U/mL and 1U/mL penicillin sodium salt solution. The two parent strains were treated by lysozyme in the optimal concentration of 1g/L and 2g/L for 9 hours and 11hours repectively. The optimal concentration of osmotic pressure stabilizer in the regeneration medium is 0.6 mol/L. A good fusion rate was got when the two parent stains was mediated by PEG for 25 minutes in pH 10 buffer system at 34°C. Eventually, L-valine hyper-producer NJv61 was obtained, and genetic markers of strain NJv61 were (Leu~-, Ile~-, Met~l, 2-TA~(hr), SG~r, Glc~(hr),α-AB~(hr)). Under unoptimized conditions, the L–valine production in flasks of the strain was 45.6 g/L.
(3) The composition of seed medium of strain NJv61 was fixed through orthogonal table experiment design: Glucose 24g/L, (NH_4)_2SO_4 5g/L, KH_2PO_4 0.6 g/L, MgSO_4·7H_2O 0.5g/L, corn-steep 35g/L, CaCO_3 10g/L. Single-factor experiments were conducted, including carbon or nitrogen source, inorganic salts, growth factors and other necessary factors. The optimal composition of the fermentation medium was fixed by response surface methodology. The culture medium used for the fermentation of NJv61 contained: Gulcose 149g/L, (NH_4)_2SO_4 48.4g/L, KH_2PO_4 1.5g/L, MgSO_4·7H_2O 0.6g/L, VB1 150μg/L, biotin 60μg/L, corn-steep 19.4g/L. The L-valine production of strain NJv61 in 7L fermentor was 51.8 g/L under unoptimized conditions after 72 hours fermentation.
(4) Representative strains (ZGH6128, NVT1103, JVHK597 and NJv61) were obtained in the screening process. And broth samlpes of the representative strains were analysed when these strains accumulated in the 7 fermentor during the late metaphase of the fermentation process. Metabolic flux distribution network diagram from glucose to L-valine was figured out based on results of analysis of the samples. Diagrams of frames of carbon distribution in metabolic network were compared between the initial strain ZGH6128 and the target strain NJv61. In the screened strain NJv61, Carbon flows into the EMP pathway was enhanced at the G6P node, and there was still sufficient metabolic fluxes in the HMP pathway. Compared with the original strain, metabolic fluxes into the TCA cycle were decreasing sharply at pyruvate node in order that more carbon frame fluxes flowed into the L-valine biosynthetic pathway. Compared with the starting stain, Carbon frame fluxes in the L-alanine biosynthetic pathway didn’t decline markedly in the strain NJv61.
(5) Target strain NJv61 could resist the high concentration ofα-AB and 2-TA because of the mutations in acetohydroxyacid synthase gene. In cells of strain NJv61, L-valine feedback inhibition and repression effect were released drastically. Meanwhile, relative activity of the enzyme was changed. Strain NJv61’s AHAS gene was cloned and sequenced in the thesis, and the result was analysed with biological softwares. There are nearly 10% of the total uumber mutations in the whole gene distributed in the section encoding regulatory subunit; while there are 90% of mutations in the entire gene distributed in the AHAS gene segment encoding the catalytic subunit. Finally, the NJv61’s AHAS gene was connected with a given expression vector and exported into a mode strain. Induced by IPTG, the enzymatic activity of enzyme protein molecules expressed by the mode strain was unstable at 4℃, and the relative activity of the enzyme decreased rapidly in the first 22 hours, just keeping about 20% of relative activity only.
⑴用硫酸二乙酯(DES)和亚硝胍(NTG)处理出发菌株黄色短杆菌(Brevibacterium flavum) ZGH6128,经过筛选最终获得菌株NVT1103(Leu~l,α-AB~(hr), 2-TA~(hr),SG~r)与菌株JVHK597(Leu~-,Ile~-,Met~l,α-AB~r,2-TA~r)。在未优化的条件下,两株菌的摇瓶产酸量分别为37.6g/L和41.2g/L。
⑵利用原生质融合技术进行氨基酸菌种选育的研究引起国内外技术人员的关注,本课题以菌株NV1103和JVHK597为亲本菌株,采用原生质体融合方法进行选育L-缬氨酸高产菌株。用0.5U/mL与1U/mL的青霉素钠盐溶液分别预处理双亲本菌株(NV1103与JVHK597)2.5h和3h;溶菌酶处理亲本株的最适浓度为1g/L与2g/L,分别处理9h和11h;再生培养基中渗透压稳定剂浓度为0.6mol/L;在34°C、pH10的缓冲体系中,400g/L的PEG介导25min,可以获得理想的融合率。最终选育出高产菌株NJv61,遗传标记为(Leu~-,Ile~-,Met~l, 2-TA~(hr), SG~r, Glc~(hr),α-AB~(hr)),未优化条件下,摇瓶产酸量为45.6g/L。
⑶利用正交表进行实验设计确定菌株NJv61种子培养基的组成:葡萄糖24g/L,玉米浆35g/L,(NH_4)_2SO_4 5g/L,KH_2PO_4 0.6g/L,MgSO_4·7H_2O 0.5g/L,CaCO_3 10g/L。对菌株发酵过程中所需的碳(氮)源、无机盐、生长因子等进行了单因素实验,在此基础上通过响应面方法确定了该菌株发酵培养基的最佳组成:葡萄糖149g/L,玉米浆19.4g/L,(NH_4)_2SO_4 48.4g/L,KH_2PO_4 1.5g/L,MgSO_4·7H_2O 0.6g/L,VB1 150μg/L,VH 60μg/L。在7L发酵罐中,优化条件下NJv61经过72h发酵,在发酵液中积累L-缬氨酸达到51.8g/L。
⑷对L-缬氨酸产生菌筛选过程中所获得的代表菌株(ZGH6128、NVT1103、JVHK597和NJv61),在7L发酵罐产酸过程的中后期进行取样分析,明确了该时期相关菌株细胞内与合成L-缬氨酸相关的代谢途径网络分布。与出发菌株ZGH6128相应途径上的流量相比,目的菌株NJv61在节点G6P处流向EMP途径的碳架流量有了显著的提高,同时,在HMP途径中仍然保持较大流量。在丙酮酸节点处,目的菌株流向TCA循环的代谢流量较出发菌株大大减少,使更多的碳架流进入L-缬氨酸合成途径。目的菌株中丙氨酸生物合成途径中的碳架流量较出发菌株没有明显的下降。
⑸乙酰羟基酸合成酶(AHAS)是半理性化育种工作的靶酶,目的菌株NJv61对高浓度α-AB和2-TA具有抗性,研究发现该菌株的AHAS基因发生了突变,更彻底地解除L-缬氨酸反馈抑制与阻遏作用,并且酶的相对活性也有改变。本课题对AHAS基因进行了克隆,对测序的结果进行了比较分析发现,调节亚基所对应的基因区段,仅分布着整个基因突变位点总数大约10%的突变碱基,而编码催化亚基的基因区段布着的突变位点数约为整个基因所有突变位总数的90%。最后把菌株NJv61的AHAS基因与特定的表达载体相连接,导入模式菌株,经IPTG诱导,所表达的酶蛋白分子在4℃条件下不稳定,22h内该酶相对酶活迅速下降,仅保持大约20%相对活性。
L-Valine is not only one of three branched-chain amino acids but also a member of eight essential amino acids which can’t be synthesized by humans themselves. It plays an important role in many kinds of physiological functions in vertebrates’bodies. The applications of L-valine are very broad: precursor of pharmaceuticals, additives to food or feedstuff for livestocks, material for pesticides, nutrition infusion for patients, health care product, and condiment and so on. Nowadays, scales of the industrial plants for L-valine in China are small, and the actual production level of L-valine prodution isn’t high. The annual production amount of L-valine can’t meet the growing demand of domestic market. In order to solve problems mentioned above, it is necessary to breed highly efficient L-valine-producing strains based on metabolic engineering principles.
(1) The origin strain is strain ZGH6128 (Brevibacterium flavum) which was treated with DES and NTG in the screening process. Strain NVT1103 (Leu~l,α-AB~(hr), 2-TA~(hr),SG~r) and strain JVHK597(Leu~-, Ile~-, Met~l,α-AB~r, 2-TA~r) were obtained in the early stages. Under unoptimized conditions, the L-valine productions in flasks of the two strains were 37.6g/L and 41.2g/L respectively.
(2) Scientists at home and abroad were drawn attention to the application of protoplast fusion technology to screen L type amino acids producing strains. Strain NV1103 and strain JVHK597 was used as parent strains during the process to beed L-valine hyper-producer by protoplast fusion technology in this thesis. Parent strains (NV1103 and JVHK597) were pretreated for 2.5 hours and 3 hours with 0.5 U/mL and 1U/mL penicillin sodium salt solution. The two parent strains were treated by lysozyme in the optimal concentration of 1g/L and 2g/L for 9 hours and 11hours repectively. The optimal concentration of osmotic pressure stabilizer in the regeneration medium is 0.6 mol/L. A good fusion rate was got when the two parent stains was mediated by PEG for 25 minutes in pH 10 buffer system at 34°C. Eventually, L-valine hyper-producer NJv61 was obtained, and genetic markers of strain NJv61 were (Leu~-, Ile~-, Met~l, 2-TA~(hr), SG~r, Glc~(hr),α-AB~(hr)). Under unoptimized conditions, the L–valine production in flasks of the strain was 45.6 g/L.
(3) The composition of seed medium of strain NJv61 was fixed through orthogonal table experiment design: Glucose 24g/L, (NH_4)_2SO_4 5g/L, KH_2PO_4 0.6 g/L, MgSO_4·7H_2O 0.5g/L, corn-steep 35g/L, CaCO_3 10g/L. Single-factor experiments were conducted, including carbon or nitrogen source, inorganic salts, growth factors and other necessary factors. The optimal composition of the fermentation medium was fixed by response surface methodology. The culture medium used for the fermentation of NJv61 contained: Gulcose 149g/L, (NH_4)_2SO_4 48.4g/L, KH_2PO_4 1.5g/L, MgSO_4·7H_2O 0.6g/L, VB1 150μg/L, biotin 60μg/L, corn-steep 19.4g/L. The L-valine production of strain NJv61 in 7L fermentor was 51.8 g/L under unoptimized conditions after 72 hours fermentation.
(4) Representative strains (ZGH6128, NVT1103, JVHK597 and NJv61) were obtained in the screening process. And broth samlpes of the representative strains were analysed when these strains accumulated in the 7 fermentor during the late metaphase of the fermentation process. Metabolic flux distribution network diagram from glucose to L-valine was figured out based on results of analysis of the samples. Diagrams of frames of carbon distribution in metabolic network were compared between the initial strain ZGH6128 and the target strain NJv61. In the screened strain NJv61, Carbon flows into the EMP pathway was enhanced at the G6P node, and there was still sufficient metabolic fluxes in the HMP pathway. Compared with the original strain, metabolic fluxes into the TCA cycle were decreasing sharply at pyruvate node in order that more carbon frame fluxes flowed into the L-valine biosynthetic pathway. Compared with the starting stain, Carbon frame fluxes in the L-alanine biosynthetic pathway didn’t decline markedly in the strain NJv61.
(5) Target strain NJv61 could resist the high concentration ofα-AB and 2-TA because of the mutations in acetohydroxyacid synthase gene. In cells of strain NJv61, L-valine feedback inhibition and repression effect were released drastically. Meanwhile, relative activity of the enzyme was changed. Strain NJv61’s AHAS gene was cloned and sequenced in the thesis, and the result was analysed with biological softwares. There are nearly 10% of the total uumber mutations in the whole gene distributed in the section encoding regulatory subunit; while there are 90% of mutations in the entire gene distributed in the AHAS gene segment encoding the catalytic subunit. Finally, the NJv61’s AHAS gene was connected with a given expression vector and exported into a mode strain. Induced by IPTG, the enzymatic activity of enzyme protein molecules expressed by the mode strain was unstable at 4℃, and the relative activity of the enzyme decreased rapidly in the first 22 hours, just keeping about 20% of relative activity only.
引文
[1] Sahm H, Eggeling L. D-Pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction[J]. Appl Environ Microbiol,1999,65:1973-1979.
[2]贾庆忠,马桂林,黎文志.抗高血压药缬沙坦的合成[J].中国医药工业杂志,2001,32(9): 385-387.
[3]张伟国,钱和.氨基酸生产技术及其应用[M].北京:中国轻工业出社,1997,3-22.
[4]赵丽丽. L-缬氨酸高产菌的选育及其发酵条件研究[D].天津科技大学,2003.
[5] Sahm H, Eggeling L, Morbach S, et al. Construction of L-isoleucine overproducing strains of Corynebacterium glutamicum[J]. Naturwissenschaften,1999,86:33-38.
[6]黄红英,贺建华,范志勇等.添加缬氨酸和异亮氨酸对哺乳母猪及其仔猪生产性能的影响[J].动物营养学报,2008,20(3):281-287.
[7]董晓慧.支链氨基酸营养作用与拮抗[J].饲料博览,1999,11(10):17-18.
[8] Katsurada N, Uchibori H, Tsuchida T. Process for Producing L-valine by fermentation [P].UP5188948,1993.
[9]欧阳平凯.生物化工产品[M].北京:化学工业出版社,1999.
[10]张伟国,钱和. L-缬氨酸发酵条件的研究[J].氨基酸和生物资源,2001, 23(3):28-31.
[11] Iborra JL. Analysis of alaminated enzyme membrane reactor for continuous resolution of amino acids-L-amino acid e.g. L-valine production by immobilized aminoacylase[J]. Biotechnol. Appl. Biochem.,1992,15(l):22-30.
[12] Bódalo-Santoyo A, Gómez-Carrasco JL, Gómez-Gómez E, et al. Production of optically pure L-valine influided and packed bed reactors with irnrnobilized L-aminoacylase[J]. Journal of Chemical Technology and Biotechnology, 1999,74(5):403-408.
[13]李良铸,李明华.最新生化药物制备技术[M].中国药科技术出版社,2001.
[14] Katsumata, Ryoichi, Hashimoto, et al. Proeess for producing L-valine[P]. United States Patent,UP5521074,1996-05-25.
[15]刘忠,赵丽丽.二次响应面分析法用于L-缬氨酸发酵培养基的优化.南开大学学报(自然科学版),2009, 42(4): 24-28.
[16] Kinoshita S,Udaka S,Shimono M. Studies on the amino acid fermentation[J].J Gen Appl Microbiol,1957,3(3):193-205.
[17] Nakayama K, Kitada S, Kinoshita S. Studies on lysine fermentation I. The controlmechanism on lysine accumulation by homoserine and threonine[J].J Gen Appl Microbiol, 1961, 7(3):145-154.
[18] Sikyta B.Methods in industrial microbiology[M].Ellis Horwood,Chichester,1983.
[19] Buckland B C, Lilly M D, Fermentation: an overview[M]. In: Stephanopoulos G(ed) Biotechnology, 2nd edition, vol.3: Bioprocessing. VCH Verlagsgesellschaft mbH,Weinheim, 1993:p7.
[20]张嗣良.发酵过程多水平问题及其生物反应器装置技术研究——基于过程参数相关的发酵过程优化与放大技术[J].中国工程科学, 2001,3(8):37-45.
[21] Yamane T,Shimizu S. Fed-batch techniques in microbial processes[M].In: Fiechter A(ed) Advances in biochemical engineering/biotechnology. Springer, Berlin Heidelberg New York, 1984:p147.
[22]浦军平,肖泉,林学军.一次性高糖发酵L-缬氨酸菌种的选育及发酵条件[J].大连轻工业学院学报, 2001,20(4):264-266.
[23]张伟国,钱和.L-缬氨酸高产菌选育及营养需求研究[J].沈阳药科大学学报,2001,18(6): 443-446.
[24] Liebl W, Ehrmann M, Ludwig W, et al . Int J Syst Bacteriol,1991,41:225.
[25] Kinoshita S.Taxonomic position of glutamic acid producing bacteria. In: Flickinger M C, Drew S W (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation.Wiley, 1999:p1330.
[26]刘苗,郑璞,杜巧燕等.耐温性L-谷氨酸发酵菌种的选育[J].生物加工过程, 2009, 7(6): 74-78.
[27]陈泽钧,江泳.谷氨酸高温发酵的探讨[J].发酵科技通讯,1984,13(2):210-213.
[28] Kelle R,Hermann T,Weuster-Botz D, et al. Glucose-controlled L-isoleucine fed-batch production with recombinant strains of Corynebacterium glutamicum[J]. J Biotechnol, 1996, 50:123-136.
[29] Ono Y, Sato K. Production of L-leucine[P].Japan Patent,JP8 266 295 A, 1996.
[30] Changeux JP. The feedback control mechanisms of biosynthetic L-threonine deaminase by L-isoleucine[J]. Cold Spring Harb Symp Quant Biol,1961,26:313-318.
[31] Eggeling I, Cordes C, Eggeling L, et al. Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of alfa-ketobutyrate to L-isoleucine[J]. Appl Microbiol Biotechnol,1987,25:346-35.
[32] ElisákováV, Pátek M, Holátko J, et al. Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum[J]. Appl Environ Microbiol,2005, 71:207-213.
[33] McCourt JA, Duggleby RG. Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids[J]. Amino Acids,2006,31:173-210.
[34] Lawther R P, Wek R C, Lopes J M, et al. The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12[J]. Nucleic Acids Res, 1987, 15:2137-2155.
[35] Vinogradov V, Vyazmensky M, Engel S, et al. Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties[J]. Biochim Biophys Acta, 2006, 1760:356-363.
[36] Leyval D, Uy D, Delaunay S, et al. Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum[J]. J Biotechnol,2003,104:241-252.
[37] McHardy A C, Tauch A, Ruckert C, et al. Genome-based analysis of biosynthetic amino transferase genes of Corynebacterium glutamicum[J]. J Biotechnol,2003,104:229-240.
[38] Marienhagen J, Kennerknecht N, Sahm H, et al. Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum[J]. J Bacteriol, 2005,187:7639-7646.
[39] Goto M, Miyahara I, Hayashi H, et al. Crystal structures of branched-chain amino acid aminotransferase complexed with glutamate and glutarate: true reaction intermediate and double substrate recognition of the enzyme[J].Biochemistry,2003,42:3725-3733.
[40] Gusberti L, Cantoni R, De Rossi E, et al. Cloning and sequencing of the ilvBNC gene cluster from Mycobacterium avium[J].Gene,1996,177:83-85.
[41] Umbarger H E. Cell Mol Biol[M]. Washington, D C: Neidhardt F C, Ingraham J L, Low B L, Magasanik B, Schaechter M, Umbarger H E (eds), 1996, pp 442-457.
[42] Lawther R P, Wek R C, Lopes J M, et al. The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12[J]. Nucleic Acids Res, 1987, 15:2137-2155.
[43] Friden P, Newman T, Freundlich M. Nucleotide sequence of the ilvB promoter regulatory region: a biosynthetic operon controlled by attenuation and cyclic AMP[J]. Proc Natl Acad Sci USA,1982,79:6156-6160.
[44] Dailey F E, Cronan J E Jr. Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source[J]. J Bacteriol,1986, 165:453-460.
[45] Tedin K, Norel F. Comparison of ?relA strains of Escherichia coli and Salmonella enterica serovar Typhimurium suggests a role for ppGpp in attenuation regulation of branched-chain amino acid biosynthesis[J]. J Bacteriol, 2001, 183: 6184 -6196.
[46] Morbach S, Junger C, Sahm H, et al. Attenuation control of ilvBNC in Corynebacterium glutamicum: evidence of leader peptide formation without the presence of a ribosome binding site[J]. J Biosci Bioeng,2000, 90:501-507.
[47] Keilhauer C, Eggeling L, Sahm H. Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon[J]. J Bacteriol, 1993, 175: 5595-5603.
[48]中国科学院微生物研究所氨基酸组. L-缬氨酸产生菌Asl.586的选育[J].微生物学报, 1975,15(4):325-329.
[49]唐任天,郭永复,陈琦. L-缬氨酸生物合成的研究.氨基酸杂志,1986,1:12-17.
[50]王秀岭,屈明波.多重抗性突变株L-缬氨酸发酵[J].微生物学通报,1990,17(5):276-279.
[51]黄海华,李福德.发酵法生产L-缬氨酸的研究[J].微生物学杂志,1989, (2):l-8.
[52]来彩霞,刘党生,陈炜. L-缬氨酸产生菌菌种选育和发酵条件考察[J].沈阳药科大学学报, 1997,14(2):107-110.
[53]徐旭东,肖灿鹏,尹鸿萍. L-缬氨酸产生菌的选育[J].药物生物技术,1999,6(l):24-27.
[54]陈宁,熊明勇,赵丽丽等. L-缬氨酸生产菌的选育及基于遗传算法的发酵培养基优化[J].天津轻工业学院学报,2002, (4):18-22.
[55]马雷,秦永峰,徐庆阳等.溶氧对L-缬氨酸发酵过程的影响[J].生物技术通讯, 2010, 21(4): 509-514.
[56]王平,何英,贺小伟. L-缬氨酸菌种的选育及其发酵条件[J].大连轻工业学院学报, 2002,21(2): 116-119.
[57]赵丽丽,刘忠. L-缬氨酸产生菌的选育及发酵条件的优化[J].齐鲁药事, 2008, 27(12):745-747.
[58] Nakayama K, Kitada S and Kinoshita S. L-valine production using microbial auxotroph[J]. J.Gen. Appl. Microbiol.Tokyo,1961,7(1):52-69.
[59]木下祝郎编.发酵工业[M].新版.东京:大日本图书, 1975.
[60] Maryadele JO, Ann S, Patricia E, et al. The Merck Index[M].Merck & Co., INC., Whitehouse Station,NJ,2001.
[61] Tsuchida. Preparation of L-valine by fermentation method[P]. JP:56051989, 1981.
[62] Katsumata, Ryoichi, Hashimoto et al. Proeess for producing L-valine[P].UP:5521074,1996.
[63] Hoechst AG, Marquardt R. New extrachromosomal element-containing aliphatic- transaminase gene-from Escherichia coli; alanine,valine and isoleucine over production. EP265852, 1986.
[64] Mitsubishi. Acetohydroxy-acid-isomeroreductasegene-ketol-acid-reductoiso merase genecloning and expression in e.g. Brevibacterium flavum for improved L-leucine and L-valine production[P]. JP: 06277067,1993.
[65] V·A·里夫希特斯, V·G·多罗申科,N·V·戈斯科瓦等.突变体ilvH基因和制备L-缬氨酸的方法[P].CN:01103331.2.
[66]伊莱纳·V·西彻瓦,弗斯沃洛德·A·塞里布赖尼,塔蒂亚纳·A·扬波尔斯卡亚等.突变乙酰乳酸合酶和用于生产支链L-氨基酸的方法[P].CN:101386841.
[67] Xu DQ, Wang XY. Construction of an Ilva mutant of Brevibacterium ?avum that can over-produce L-valine [J] . Journal of Biotechnology 136S, 2008, S22–S71.
[68] Terasawamasto. Produetionof L-valine[P]. JP:2072888,1990.
[69] Araki K. Preparation of L-valine[P]. JP:61028398,1986.
[70] Tomita F, Yokota Atsushi, Hashiguchi Kenichi, et al. Methods for producing L-valine and L-leucine[P].UP: 6214591,2001.
[71] Matsui Hiroshi,Tsuchida Takayasu,Nakamori Shigeru.Method for producing L-valine by fermentation[P]. UP:4391907,1983.
[72] Nakazawa Eiji, Seki Mitsuyoshi, Akutsu Eiichi. Preparation of L-valine by fermentation method[P]. JP:56039792, 1981.
[73] Thrkawa k. Fermentative preparation of L-valine by mixed culture[P]. JP:57208993,1982.
[74] Bodalo-santoyo A. Production of optically pure L-valine influided and packed bed reactors with irnobilized L-aminoacylase. Journal of Chemical Technology and Biotechnology, 1999,75(5):403-408.
[75] Spackman D H, Stein W H, Moore S. Automatic recording apparatus for use in the chromatography of amino acids[J]. Anal. Chem.,1958,30(7):1190-1206.
[76] Macchi F D, Shen F J, Keck R G, et al. In: Cooper C,Packer N,Williams K.Methods in Molecular Biology,Vol.159: AminoAcid Analysis Protocols. TotowaNJ:Humana Press Inc,2001: 9~30.
[77]谢保源.我国氨基酸制剂的研究和开发概况[J].中国医药杂志.1990,21(1):37-41.
[78] Koniekova R M, Smeakal F. Preparation of Brevibacterium sp. M27 mutant strain producing Valine-strain improved by chemical mutagenesis (conference abstract)[J]. Folia Microbiol, 1990,35(6):496.
[79]黄和容,李玲阁,王秀玲等. L-苏氨酸产生菌的选育[J].微生物学报,1982,22(3):276-283.
[80]张伟国. L-异亮氨酸产生菌选育的研究.氨基酸与生物资源[J],1996,18(3):1-5.
[81]徐旭东,肖灿鹏. L-缬氨酸产生菌的选育[J].药物生物技术,1999,6(1):24-27.
[82]林殿海,黄和容.产L-异亮氨酸新菌株选育及其发酵产物提纯和鉴定[J].氨基酸杂志,1990, 4:l-6.
[83]杨志坤,徐晓颖L-亮氨酸产生菌的获得[J].黑龙江医药, 2008,21(4):51-52.
[84]张克旭,陈宁,张蓓等.代谢控制发酵[M].中国轻工业出版社,1998.
[85]殷树昌. L-精氨酸产生菌的选育及其代谢调控的初步研究[D].江南大学,2009:13.
[86]李晓华,陈宁,张克旭.化学比色法测定发酵液中L-缬氨酸的研究[J].氨基酸和生物资源, 2003, 25(4):55-57.
[87] Mesbah M, Premachandran U, Whitman W B. Precise measurement of the G+C content of deoxyribonucleic acid by high performance liquid chromatography[J]. Int. J. Syst. Bacteriol. 1989,39:159-167.
[88]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994,145-178.
[89]齐秀兰,王军丽.硫酸二乙酯诱变原生质体选育赖氨酸高产菌株的研究[J].药物生物技术, 1998,5(1):17-20.
[90]汪世华,王维平,吴思方. L-谷氨酰胺高产菌株的硫酸二乙酯诱变育种[J].冷饮与速冻食品工业,2001,7(1):4-5.
[91]施巧琴,吴松刚.工业微生物育种学(第二版)[M].北京:科学出版社, 2003.
[92]王红霞,刘红彦,刘玉霞等.枯草芽孢杆菌B-903菌株的诱变选育[J].微生物通报, 2005,32(3):10-13.
[93]张卫民,张琦行.蔬菜、干鲜果品和饮料中总维生素C的荧光测定法[J].食品与发酵工业. 1986(6):50-56.
[94] Joseph B,Ramteke P W,Thomas G, et al. Cold-active microbial lipases: a versatile tool for industrial applications[J]. Biotechnol. Mol. Bio. Rev.2:39-48.
[95]路志强,龚建华,丁久元等.L-精氨酸产生菌诱变育种的研究[J].微生物学报,1988, 28(2):131-135.
[96]汪世华,白文钊,吴思方等. L-谷氨酰胺高产菌的定向选育及产胺条件的研究[J].微生物学报. 2002,42(2):751-754.
[97]张伟国,陈坚,伦世仪. L-异亮氨酸高产菌育种机理初步研究[J].无锡轻工大学学报. 1998,17(2):19-23.
[98]周德庆.微生物学教程[M].高等教育出版社,1993:212-214.
[99]沈同,王镜岩.生物化学(第二版)[M].北京:高等教育出版社,1991:371-373.
[100]陈三风,刘德虎.现代微生物遗传学[M].北京:化学工业出版社,2003:308-316.
[101]谭周进,肖启明,肖克宇等.原生质体融合技术在微生物菌种选育中的应用[J].生物技术, 2003,2(35):36-39.
[102]杜连祥,路福平.微生物学实验技术[M].北京:中国轻工业出版社,2005:210-211.
[103] Ferencezy L, Kevei F, Zsolt J. Fusion of fungal protoplast[J]. Nature, 1974, 248: 793-794.
[104] Harumi Kaneko, Kenji Sakaguchi.Fusion of Protoplasts and Genetic Recombination of Brevibacterium flavum[J]. Agric.Biol.Chem,1979,43(5) :1007-1013.
[105]王菲,张克旭,宋文军等.应用原生质体融合技术选育L-异亮氨酸生产菌[J].天津科技大学学报,2004,19(1):9-16.
[106] Karasawa M, Tosaka O, Ikeda S, et al.Application of Protoplast Fusion to the Development of L-Threonine and L-Lysine Producers[J]. Agric.Biol.Chem,1986,50(2):339-346.
[107]乔宝义,徐浩.棒状杆菌原生质体的形成、再生以及融合的初步探讨[J].微生物学报, 1983,23(l):33-34.
[108]洪益国,郑幼霞,杨颐康等.通过热灭活棒状杆菌原生质体融合选育谷氨酸高产菌株[J].生物工程学报,1988,4(4):327-332.
[109]罗明典.氨基酸发酵生产及其产生菌研究进展[J].氨基酸杂志,1985,1:39-42.
[110]周东坡,文平祥,贾树彪等.原生质体融合选育赖氨酸高产菌种的研究[J].微生物学报, 1991,31(4):287-292.
[111]张蓓,张克旭,陈宁.谷氨酸高产菌FTNg108的细胞融合育种及其发酵条件的研究[J].食品与发酵工业,1992,5:21-25.
[112]郭成金,赵润,朱文碧.冬虫夏草与蛹虫草原生质体融合初探[J].食品科学, 2010, 31(1): 165–171.
[113]张婧芳,汪江波,黄金明,等. L-鸟氨酸高产菌的原生质体融合育种[J].氨基酸和生物资源,2009,31(1):53-57.
[114]莫静燕,陈献忠,王正祥.地衣芽孢杆菌原生质体的制备、再生及转化研究[J].生物技术, 2009, 19(5): 75-77.
[115]李再资.生物化学工程基础[M].北京:化学工业出版社,1999:18-19.
[116]芦敬华.利用复合诱变与原生质体融合提高中性纤维素酶活力的研究[D].中国农业大学, 2006.
[117]郑应福,阚振荣,邸胜苗.α-ALDC产生菌的抗性标记与融合条件初探[J].中国酿造,2006,2,16-20.
[118]俞俊棠,唐孝宣.新编生物工艺学[M].北京:化学工业出版社,2003:99-119.
[119]熊宗贵.发酵工艺原理[M].北京:中国医药科技出版社.
[120]沈同,王镜岩.生物化学(第二版)[M].北京,高等教育出版社,1999.
[121] Hermann S, Lothar E, Bernd E, et al. Metabolic design in amino acid producing bacterium Corynebacterium glutamicum[J]. FEMS Microbiology Reviews, 1995, 16:243-252.
[122]李琼.以纯生物素代玉米浆、糖蜜的研究[J].广西轻工业.2000, 4:22-27.
[123]秦海斌. L-谷氨酸产生菌选育、发酵条件研究及其代谢流分析[D].江南大学, 2009.
[124] Giovanni M. Response surface methodology and product optimization[J].Food Technology, 1983, 37(11):41?45.
[125]杨文雄,高炎祥.响应面法及其在食品工业中的应用[J].中国食品添加剂, 2005, 2(2): 68?71.
[126] Kari KN. Metabolic engineering of Lactobacillus helveticus CNRZ32 for production of pure L-(+)-Lactic acid[J]. Appl Environ Microbiol, 2000, 66(9) : 3835-3841.
[127]刘春辉. L-谷氨酰胺产生菌选育及其代谢调控的初步研究[D].江南大学,2009.
[128]伍时华. L-亮氨酸高产菌的选育及其发酵条件的研究[D].天津轻工业学院, 2001.
[129] Bailey J E. Towards a science of metabolic engineering[J]. Science. 1991, 252 :1668-1674.
[130] Schilling C H, Letscher D, Palsson B O. Theory for the systemic defination of metabolic pathways and their use in interpreting metabolic function from a pathway-oriented perspective [J]. J.Theor. Biol. 2000, 2: 229-248.
[131]李秀敏. L-缬氨酸育种及其代谢流分析[D].江南大学,2003.
[132] Shiio I. Production of lysine by pyrute dehydrogenase mutants of Brevibacterium flavum[J]. Agric Biol Chem, 1984, 48:3091-3098.
[133] Hua Q, Fu PC and Yang C. Microaerobic lysine fermentations and metabolic analysis[J]. Biochem Eng J, 1998, (2):89-100.
[134] Otsuka S and Miyajima R. Comparative studies on the mechanisum of microbial glutamic formation from glucose in Brevibacterium flavum and in Micrococcus glutamicus[J]. J Gen Appl Microbiol, 1965, 11:285-294.
[135] Sugimoto S. Fructose metabolism and regulation of 1-phosphofructokinase and 6-phospho- fructokinase in Brevibacterium flavum[J]. Agric Biol Chem, 1989, 53:2081-2087.
[136] Tosaka o and Morioka. The role of biotin dependent pyrute carboxylase in L-lysine production[J]. Agric Biol Chem, 1979,43:1513-1519.
[137] Isamu Shio and Shin-ichi. Effect of carbon source surgars on the yield of amino acid production and sucrose metabolism in Brevibacterium flavum[J]. Agri Biol Chem, 1990,54(6): 1513-1519.
[138] Joseph J. Metabolic flux distributions in Corynebactrium glutamicum during growth andlysine overproduction[J]. Biotechnology and Bioengineering, 1992,41(6):633-646.
[139] Stephanopoulos G N, Aristidou A A, Nielsen J. Metabolic Engineering. New York: Academic Press, 1998. 105-132.
[140]杜军,陈宁,刘辉等.基于代谢流量分析的L-谷氨酸发酵过程优化[J].中国食品学报, 2008, 8(2): 30-34.
[141] Umbarger H E. Amino acid biosynthesis and its regulation[J]. Annu. Rev. Biochem, 1978, 47: 533-606.
[142] Umbarger H E.Biosynthesis of the branched-chain amino acids. Escherichia coli and Salmonella typhimurium. Cell Mol Biol[M]. ASM Press, Washington, DC, 1996, pp 442-457.
[143] Eggeling I, Cordes C, Eggeling L, et al. Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of alfa-ketobutyrate to L-isoleucine[J]. Appl Microbiol Biotechnol,1987, 25:346-351.
[144] McCourt J A, Duggleby R G. Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids[J]. Amino Acids, 2006, 31(2):173-210.
[145] Thomas B. Ray. Site of action of Cholorsulfuron[J]. Plantphysiol, 1984, 75: 827-831.
[146] Chio J D, Moon H, Chang S I, et al. Inhibition of acetolactate synthase by pyrimidyl- oxy-benzoate and pyrimidyl-thio-benzenes. Karean Biochem J[J]. 1993, 26(7): 638-643.
[147] Daqing Xu, Yanzhen Tan, Xiaojing Huan, et al. Construction of a novel shuttle vector for use in Brevibacterium flavum, an industrial amino acid producer. Journal of Microbiological Methods[J]. 2010, 80:86-92.
[148] Umbarger H E and Brown B. Isoleucine and Valine Metabolism in Escherichia coli[J]. The Journal of Biological of Chemistry[J]. 1958, Vol. 233,No.5:1156-1160.
[149] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. [J]. 1976,72:248-254.
[150] Ibdah M, Bar-Ilan A, Livnah O, et al. Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis. Biochemistry[J]. 1996, 35(50):16282-16291.
[2]贾庆忠,马桂林,黎文志.抗高血压药缬沙坦的合成[J].中国医药工业杂志,2001,32(9): 385-387.
[3]张伟国,钱和.氨基酸生产技术及其应用[M].北京:中国轻工业出社,1997,3-22.
[4]赵丽丽. L-缬氨酸高产菌的选育及其发酵条件研究[D].天津科技大学,2003.
[5] Sahm H, Eggeling L, Morbach S, et al. Construction of L-isoleucine overproducing strains of Corynebacterium glutamicum[J]. Naturwissenschaften,1999,86:33-38.
[6]黄红英,贺建华,范志勇等.添加缬氨酸和异亮氨酸对哺乳母猪及其仔猪生产性能的影响[J].动物营养学报,2008,20(3):281-287.
[7]董晓慧.支链氨基酸营养作用与拮抗[J].饲料博览,1999,11(10):17-18.
[8] Katsurada N, Uchibori H, Tsuchida T. Process for Producing L-valine by fermentation [P].UP5188948,1993.
[9]欧阳平凯.生物化工产品[M].北京:化学工业出版社,1999.
[10]张伟国,钱和. L-缬氨酸发酵条件的研究[J].氨基酸和生物资源,2001, 23(3):28-31.
[11] Iborra JL. Analysis of alaminated enzyme membrane reactor for continuous resolution of amino acids-L-amino acid e.g. L-valine production by immobilized aminoacylase[J]. Biotechnol. Appl. Biochem.,1992,15(l):22-30.
[12] Bódalo-Santoyo A, Gómez-Carrasco JL, Gómez-Gómez E, et al. Production of optically pure L-valine influided and packed bed reactors with irnrnobilized L-aminoacylase[J]. Journal of Chemical Technology and Biotechnology, 1999,74(5):403-408.
[13]李良铸,李明华.最新生化药物制备技术[M].中国药科技术出版社,2001.
[14] Katsumata, Ryoichi, Hashimoto, et al. Proeess for producing L-valine[P]. United States Patent,UP5521074,1996-05-25.
[15]刘忠,赵丽丽.二次响应面分析法用于L-缬氨酸发酵培养基的优化.南开大学学报(自然科学版),2009, 42(4): 24-28.
[16] Kinoshita S,Udaka S,Shimono M. Studies on the amino acid fermentation[J].J Gen Appl Microbiol,1957,3(3):193-205.
[17] Nakayama K, Kitada S, Kinoshita S. Studies on lysine fermentation I. The controlmechanism on lysine accumulation by homoserine and threonine[J].J Gen Appl Microbiol, 1961, 7(3):145-154.
[18] Sikyta B.Methods in industrial microbiology[M].Ellis Horwood,Chichester,1983.
[19] Buckland B C, Lilly M D, Fermentation: an overview[M]. In: Stephanopoulos G(ed) Biotechnology, 2nd edition, vol.3: Bioprocessing. VCH Verlagsgesellschaft mbH,Weinheim, 1993:p7.
[20]张嗣良.发酵过程多水平问题及其生物反应器装置技术研究——基于过程参数相关的发酵过程优化与放大技术[J].中国工程科学, 2001,3(8):37-45.
[21] Yamane T,Shimizu S. Fed-batch techniques in microbial processes[M].In: Fiechter A(ed) Advances in biochemical engineering/biotechnology. Springer, Berlin Heidelberg New York, 1984:p147.
[22]浦军平,肖泉,林学军.一次性高糖发酵L-缬氨酸菌种的选育及发酵条件[J].大连轻工业学院学报, 2001,20(4):264-266.
[23]张伟国,钱和.L-缬氨酸高产菌选育及营养需求研究[J].沈阳药科大学学报,2001,18(6): 443-446.
[24] Liebl W, Ehrmann M, Ludwig W, et al . Int J Syst Bacteriol,1991,41:225.
[25] Kinoshita S.Taxonomic position of glutamic acid producing bacteria. In: Flickinger M C, Drew S W (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation.Wiley, 1999:p1330.
[26]刘苗,郑璞,杜巧燕等.耐温性L-谷氨酸发酵菌种的选育[J].生物加工过程, 2009, 7(6): 74-78.
[27]陈泽钧,江泳.谷氨酸高温发酵的探讨[J].发酵科技通讯,1984,13(2):210-213.
[28] Kelle R,Hermann T,Weuster-Botz D, et al. Glucose-controlled L-isoleucine fed-batch production with recombinant strains of Corynebacterium glutamicum[J]. J Biotechnol, 1996, 50:123-136.
[29] Ono Y, Sato K. Production of L-leucine[P].Japan Patent,JP8 266 295 A, 1996.
[30] Changeux JP. The feedback control mechanisms of biosynthetic L-threonine deaminase by L-isoleucine[J]. Cold Spring Harb Symp Quant Biol,1961,26:313-318.
[31] Eggeling I, Cordes C, Eggeling L, et al. Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of alfa-ketobutyrate to L-isoleucine[J]. Appl Microbiol Biotechnol,1987,25:346-35.
[32] ElisákováV, Pátek M, Holátko J, et al. Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum[J]. Appl Environ Microbiol,2005, 71:207-213.
[33] McCourt JA, Duggleby RG. Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids[J]. Amino Acids,2006,31:173-210.
[34] Lawther R P, Wek R C, Lopes J M, et al. The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12[J]. Nucleic Acids Res, 1987, 15:2137-2155.
[35] Vinogradov V, Vyazmensky M, Engel S, et al. Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties[J]. Biochim Biophys Acta, 2006, 1760:356-363.
[36] Leyval D, Uy D, Delaunay S, et al. Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum[J]. J Biotechnol,2003,104:241-252.
[37] McHardy A C, Tauch A, Ruckert C, et al. Genome-based analysis of biosynthetic amino transferase genes of Corynebacterium glutamicum[J]. J Biotechnol,2003,104:229-240.
[38] Marienhagen J, Kennerknecht N, Sahm H, et al. Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum[J]. J Bacteriol, 2005,187:7639-7646.
[39] Goto M, Miyahara I, Hayashi H, et al. Crystal structures of branched-chain amino acid aminotransferase complexed with glutamate and glutarate: true reaction intermediate and double substrate recognition of the enzyme[J].Biochemistry,2003,42:3725-3733.
[40] Gusberti L, Cantoni R, De Rossi E, et al. Cloning and sequencing of the ilvBNC gene cluster from Mycobacterium avium[J].Gene,1996,177:83-85.
[41] Umbarger H E. Cell Mol Biol[M]. Washington, D C: Neidhardt F C, Ingraham J L, Low B L, Magasanik B, Schaechter M, Umbarger H E (eds), 1996, pp 442-457.
[42] Lawther R P, Wek R C, Lopes J M, et al. The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12[J]. Nucleic Acids Res, 1987, 15:2137-2155.
[43] Friden P, Newman T, Freundlich M. Nucleotide sequence of the ilvB promoter regulatory region: a biosynthetic operon controlled by attenuation and cyclic AMP[J]. Proc Natl Acad Sci USA,1982,79:6156-6160.
[44] Dailey F E, Cronan J E Jr. Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source[J]. J Bacteriol,1986, 165:453-460.
[45] Tedin K, Norel F. Comparison of ?relA strains of Escherichia coli and Salmonella enterica serovar Typhimurium suggests a role for ppGpp in attenuation regulation of branched-chain amino acid biosynthesis[J]. J Bacteriol, 2001, 183: 6184 -6196.
[46] Morbach S, Junger C, Sahm H, et al. Attenuation control of ilvBNC in Corynebacterium glutamicum: evidence of leader peptide formation without the presence of a ribosome binding site[J]. J Biosci Bioeng,2000, 90:501-507.
[47] Keilhauer C, Eggeling L, Sahm H. Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon[J]. J Bacteriol, 1993, 175: 5595-5603.
[48]中国科学院微生物研究所氨基酸组. L-缬氨酸产生菌Asl.586的选育[J].微生物学报, 1975,15(4):325-329.
[49]唐任天,郭永复,陈琦. L-缬氨酸生物合成的研究.氨基酸杂志,1986,1:12-17.
[50]王秀岭,屈明波.多重抗性突变株L-缬氨酸发酵[J].微生物学通报,1990,17(5):276-279.
[51]黄海华,李福德.发酵法生产L-缬氨酸的研究[J].微生物学杂志,1989, (2):l-8.
[52]来彩霞,刘党生,陈炜. L-缬氨酸产生菌菌种选育和发酵条件考察[J].沈阳药科大学学报, 1997,14(2):107-110.
[53]徐旭东,肖灿鹏,尹鸿萍. L-缬氨酸产生菌的选育[J].药物生物技术,1999,6(l):24-27.
[54]陈宁,熊明勇,赵丽丽等. L-缬氨酸生产菌的选育及基于遗传算法的发酵培养基优化[J].天津轻工业学院学报,2002, (4):18-22.
[55]马雷,秦永峰,徐庆阳等.溶氧对L-缬氨酸发酵过程的影响[J].生物技术通讯, 2010, 21(4): 509-514.
[56]王平,何英,贺小伟. L-缬氨酸菌种的选育及其发酵条件[J].大连轻工业学院学报, 2002,21(2): 116-119.
[57]赵丽丽,刘忠. L-缬氨酸产生菌的选育及发酵条件的优化[J].齐鲁药事, 2008, 27(12):745-747.
[58] Nakayama K, Kitada S and Kinoshita S. L-valine production using microbial auxotroph[J]. J.Gen. Appl. Microbiol.Tokyo,1961,7(1):52-69.
[59]木下祝郎编.发酵工业[M].新版.东京:大日本图书, 1975.
[60] Maryadele JO, Ann S, Patricia E, et al. The Merck Index[M].Merck & Co., INC., Whitehouse Station,NJ,2001.
[61] Tsuchida. Preparation of L-valine by fermentation method[P]. JP:56051989, 1981.
[62] Katsumata, Ryoichi, Hashimoto et al. Proeess for producing L-valine[P].UP:5521074,1996.
[63] Hoechst AG, Marquardt R. New extrachromosomal element-containing aliphatic- transaminase gene-from Escherichia coli; alanine,valine and isoleucine over production. EP265852, 1986.
[64] Mitsubishi. Acetohydroxy-acid-isomeroreductasegene-ketol-acid-reductoiso merase genecloning and expression in e.g. Brevibacterium flavum for improved L-leucine and L-valine production[P]. JP: 06277067,1993.
[65] V·A·里夫希特斯, V·G·多罗申科,N·V·戈斯科瓦等.突变体ilvH基因和制备L-缬氨酸的方法[P].CN:01103331.2.
[66]伊莱纳·V·西彻瓦,弗斯沃洛德·A·塞里布赖尼,塔蒂亚纳·A·扬波尔斯卡亚等.突变乙酰乳酸合酶和用于生产支链L-氨基酸的方法[P].CN:101386841.
[67] Xu DQ, Wang XY. Construction of an Ilva mutant of Brevibacterium ?avum that can over-produce L-valine [J] . Journal of Biotechnology 136S, 2008, S22–S71.
[68] Terasawamasto. Produetionof L-valine[P]. JP:2072888,1990.
[69] Araki K. Preparation of L-valine[P]. JP:61028398,1986.
[70] Tomita F, Yokota Atsushi, Hashiguchi Kenichi, et al. Methods for producing L-valine and L-leucine[P].UP: 6214591,2001.
[71] Matsui Hiroshi,Tsuchida Takayasu,Nakamori Shigeru.Method for producing L-valine by fermentation[P]. UP:4391907,1983.
[72] Nakazawa Eiji, Seki Mitsuyoshi, Akutsu Eiichi. Preparation of L-valine by fermentation method[P]. JP:56039792, 1981.
[73] Thrkawa k. Fermentative preparation of L-valine by mixed culture[P]. JP:57208993,1982.
[74] Bodalo-santoyo A. Production of optically pure L-valine influided and packed bed reactors with irnobilized L-aminoacylase. Journal of Chemical Technology and Biotechnology, 1999,75(5):403-408.
[75] Spackman D H, Stein W H, Moore S. Automatic recording apparatus for use in the chromatography of amino acids[J]. Anal. Chem.,1958,30(7):1190-1206.
[76] Macchi F D, Shen F J, Keck R G, et al. In: Cooper C,Packer N,Williams K.Methods in Molecular Biology,Vol.159: AminoAcid Analysis Protocols. TotowaNJ:Humana Press Inc,2001: 9~30.
[77]谢保源.我国氨基酸制剂的研究和开发概况[J].中国医药杂志.1990,21(1):37-41.
[78] Koniekova R M, Smeakal F. Preparation of Brevibacterium sp. M27 mutant strain producing Valine-strain improved by chemical mutagenesis (conference abstract)[J]. Folia Microbiol, 1990,35(6):496.
[79]黄和容,李玲阁,王秀玲等. L-苏氨酸产生菌的选育[J].微生物学报,1982,22(3):276-283.
[80]张伟国. L-异亮氨酸产生菌选育的研究.氨基酸与生物资源[J],1996,18(3):1-5.
[81]徐旭东,肖灿鹏. L-缬氨酸产生菌的选育[J].药物生物技术,1999,6(1):24-27.
[82]林殿海,黄和容.产L-异亮氨酸新菌株选育及其发酵产物提纯和鉴定[J].氨基酸杂志,1990, 4:l-6.
[83]杨志坤,徐晓颖L-亮氨酸产生菌的获得[J].黑龙江医药, 2008,21(4):51-52.
[84]张克旭,陈宁,张蓓等.代谢控制发酵[M].中国轻工业出版社,1998.
[85]殷树昌. L-精氨酸产生菌的选育及其代谢调控的初步研究[D].江南大学,2009:13.
[86]李晓华,陈宁,张克旭.化学比色法测定发酵液中L-缬氨酸的研究[J].氨基酸和生物资源, 2003, 25(4):55-57.
[87] Mesbah M, Premachandran U, Whitman W B. Precise measurement of the G+C content of deoxyribonucleic acid by high performance liquid chromatography[J]. Int. J. Syst. Bacteriol. 1989,39:159-167.
[88]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994,145-178.
[89]齐秀兰,王军丽.硫酸二乙酯诱变原生质体选育赖氨酸高产菌株的研究[J].药物生物技术, 1998,5(1):17-20.
[90]汪世华,王维平,吴思方. L-谷氨酰胺高产菌株的硫酸二乙酯诱变育种[J].冷饮与速冻食品工业,2001,7(1):4-5.
[91]施巧琴,吴松刚.工业微生物育种学(第二版)[M].北京:科学出版社, 2003.
[92]王红霞,刘红彦,刘玉霞等.枯草芽孢杆菌B-903菌株的诱变选育[J].微生物通报, 2005,32(3):10-13.
[93]张卫民,张琦行.蔬菜、干鲜果品和饮料中总维生素C的荧光测定法[J].食品与发酵工业. 1986(6):50-56.
[94] Joseph B,Ramteke P W,Thomas G, et al. Cold-active microbial lipases: a versatile tool for industrial applications[J]. Biotechnol. Mol. Bio. Rev.2:39-48.
[95]路志强,龚建华,丁久元等.L-精氨酸产生菌诱变育种的研究[J].微生物学报,1988, 28(2):131-135.
[96]汪世华,白文钊,吴思方等. L-谷氨酰胺高产菌的定向选育及产胺条件的研究[J].微生物学报. 2002,42(2):751-754.
[97]张伟国,陈坚,伦世仪. L-异亮氨酸高产菌育种机理初步研究[J].无锡轻工大学学报. 1998,17(2):19-23.
[98]周德庆.微生物学教程[M].高等教育出版社,1993:212-214.
[99]沈同,王镜岩.生物化学(第二版)[M].北京:高等教育出版社,1991:371-373.
[100]陈三风,刘德虎.现代微生物遗传学[M].北京:化学工业出版社,2003:308-316.
[101]谭周进,肖启明,肖克宇等.原生质体融合技术在微生物菌种选育中的应用[J].生物技术, 2003,2(35):36-39.
[102]杜连祥,路福平.微生物学实验技术[M].北京:中国轻工业出版社,2005:210-211.
[103] Ferencezy L, Kevei F, Zsolt J. Fusion of fungal protoplast[J]. Nature, 1974, 248: 793-794.
[104] Harumi Kaneko, Kenji Sakaguchi.Fusion of Protoplasts and Genetic Recombination of Brevibacterium flavum[J]. Agric.Biol.Chem,1979,43(5) :1007-1013.
[105]王菲,张克旭,宋文军等.应用原生质体融合技术选育L-异亮氨酸生产菌[J].天津科技大学学报,2004,19(1):9-16.
[106] Karasawa M, Tosaka O, Ikeda S, et al.Application of Protoplast Fusion to the Development of L-Threonine and L-Lysine Producers[J]. Agric.Biol.Chem,1986,50(2):339-346.
[107]乔宝义,徐浩.棒状杆菌原生质体的形成、再生以及融合的初步探讨[J].微生物学报, 1983,23(l):33-34.
[108]洪益国,郑幼霞,杨颐康等.通过热灭活棒状杆菌原生质体融合选育谷氨酸高产菌株[J].生物工程学报,1988,4(4):327-332.
[109]罗明典.氨基酸发酵生产及其产生菌研究进展[J].氨基酸杂志,1985,1:39-42.
[110]周东坡,文平祥,贾树彪等.原生质体融合选育赖氨酸高产菌种的研究[J].微生物学报, 1991,31(4):287-292.
[111]张蓓,张克旭,陈宁.谷氨酸高产菌FTNg108的细胞融合育种及其发酵条件的研究[J].食品与发酵工业,1992,5:21-25.
[112]郭成金,赵润,朱文碧.冬虫夏草与蛹虫草原生质体融合初探[J].食品科学, 2010, 31(1): 165–171.
[113]张婧芳,汪江波,黄金明,等. L-鸟氨酸高产菌的原生质体融合育种[J].氨基酸和生物资源,2009,31(1):53-57.
[114]莫静燕,陈献忠,王正祥.地衣芽孢杆菌原生质体的制备、再生及转化研究[J].生物技术, 2009, 19(5): 75-77.
[115]李再资.生物化学工程基础[M].北京:化学工业出版社,1999:18-19.
[116]芦敬华.利用复合诱变与原生质体融合提高中性纤维素酶活力的研究[D].中国农业大学, 2006.
[117]郑应福,阚振荣,邸胜苗.α-ALDC产生菌的抗性标记与融合条件初探[J].中国酿造,2006,2,16-20.
[118]俞俊棠,唐孝宣.新编生物工艺学[M].北京:化学工业出版社,2003:99-119.
[119]熊宗贵.发酵工艺原理[M].北京:中国医药科技出版社.
[120]沈同,王镜岩.生物化学(第二版)[M].北京,高等教育出版社,1999.
[121] Hermann S, Lothar E, Bernd E, et al. Metabolic design in amino acid producing bacterium Corynebacterium glutamicum[J]. FEMS Microbiology Reviews, 1995, 16:243-252.
[122]李琼.以纯生物素代玉米浆、糖蜜的研究[J].广西轻工业.2000, 4:22-27.
[123]秦海斌. L-谷氨酸产生菌选育、发酵条件研究及其代谢流分析[D].江南大学, 2009.
[124] Giovanni M. Response surface methodology and product optimization[J].Food Technology, 1983, 37(11):41?45.
[125]杨文雄,高炎祥.响应面法及其在食品工业中的应用[J].中国食品添加剂, 2005, 2(2): 68?71.
[126] Kari KN. Metabolic engineering of Lactobacillus helveticus CNRZ32 for production of pure L-(+)-Lactic acid[J]. Appl Environ Microbiol, 2000, 66(9) : 3835-3841.
[127]刘春辉. L-谷氨酰胺产生菌选育及其代谢调控的初步研究[D].江南大学,2009.
[128]伍时华. L-亮氨酸高产菌的选育及其发酵条件的研究[D].天津轻工业学院, 2001.
[129] Bailey J E. Towards a science of metabolic engineering[J]. Science. 1991, 252 :1668-1674.
[130] Schilling C H, Letscher D, Palsson B O. Theory for the systemic defination of metabolic pathways and their use in interpreting metabolic function from a pathway-oriented perspective [J]. J.Theor. Biol. 2000, 2: 229-248.
[131]李秀敏. L-缬氨酸育种及其代谢流分析[D].江南大学,2003.
[132] Shiio I. Production of lysine by pyrute dehydrogenase mutants of Brevibacterium flavum[J]. Agric Biol Chem, 1984, 48:3091-3098.
[133] Hua Q, Fu PC and Yang C. Microaerobic lysine fermentations and metabolic analysis[J]. Biochem Eng J, 1998, (2):89-100.
[134] Otsuka S and Miyajima R. Comparative studies on the mechanisum of microbial glutamic formation from glucose in Brevibacterium flavum and in Micrococcus glutamicus[J]. J Gen Appl Microbiol, 1965, 11:285-294.
[135] Sugimoto S. Fructose metabolism and regulation of 1-phosphofructokinase and 6-phospho- fructokinase in Brevibacterium flavum[J]. Agric Biol Chem, 1989, 53:2081-2087.
[136] Tosaka o and Morioka. The role of biotin dependent pyrute carboxylase in L-lysine production[J]. Agric Biol Chem, 1979,43:1513-1519.
[137] Isamu Shio and Shin-ichi. Effect of carbon source surgars on the yield of amino acid production and sucrose metabolism in Brevibacterium flavum[J]. Agri Biol Chem, 1990,54(6): 1513-1519.
[138] Joseph J. Metabolic flux distributions in Corynebactrium glutamicum during growth andlysine overproduction[J]. Biotechnology and Bioengineering, 1992,41(6):633-646.
[139] Stephanopoulos G N, Aristidou A A, Nielsen J. Metabolic Engineering. New York: Academic Press, 1998. 105-132.
[140]杜军,陈宁,刘辉等.基于代谢流量分析的L-谷氨酸发酵过程优化[J].中国食品学报, 2008, 8(2): 30-34.
[141] Umbarger H E. Amino acid biosynthesis and its regulation[J]. Annu. Rev. Biochem, 1978, 47: 533-606.
[142] Umbarger H E.Biosynthesis of the branched-chain amino acids. Escherichia coli and Salmonella typhimurium. Cell Mol Biol[M]. ASM Press, Washington, DC, 1996, pp 442-457.
[143] Eggeling I, Cordes C, Eggeling L, et al. Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of alfa-ketobutyrate to L-isoleucine[J]. Appl Microbiol Biotechnol,1987, 25:346-351.
[144] McCourt J A, Duggleby R G. Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids[J]. Amino Acids, 2006, 31(2):173-210.
[145] Thomas B. Ray. Site of action of Cholorsulfuron[J]. Plantphysiol, 1984, 75: 827-831.
[146] Chio J D, Moon H, Chang S I, et al. Inhibition of acetolactate synthase by pyrimidyl- oxy-benzoate and pyrimidyl-thio-benzenes. Karean Biochem J[J]. 1993, 26(7): 638-643.
[147] Daqing Xu, Yanzhen Tan, Xiaojing Huan, et al. Construction of a novel shuttle vector for use in Brevibacterium flavum, an industrial amino acid producer. Journal of Microbiological Methods[J]. 2010, 80:86-92.
[148] Umbarger H E and Brown B. Isoleucine and Valine Metabolism in Escherichia coli[J]. The Journal of Biological of Chemistry[J]. 1958, Vol. 233,No.5:1156-1160.
[149] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. [J]. 1976,72:248-254.
[150] Ibdah M, Bar-Ilan A, Livnah O, et al. Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis. Biochemistry[J]. 1996, 35(50):16282-16291.