L-草铵膦的制备方法
阅读说明:本技术 L-草铵膦的制备方法 (Preparation method of L-glufosinate-ammonium ) 是由 胡磊 曾超 奚超群 于 2019-03-29 设计创作,主要内容包括:本发明公开了一种式I化合物的制备方法,由式V化合物反应得到,反应式如下:<Image he="314" wi="700" file="DDA0002011329780000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image> R<Sub>1</Sub>选自烃基、芳基、杂芳基、取代烃基、取代芳基、取代杂芳基;R<Sub>2</Sub>选自氨基或-OR<Sub>0</Sub>;R<Sub>0</Sub>选自烃基、硅烷基、苄基、烷基甲基、烷基取代烷氧基、3-8元脂环族基、芳基、杂芳基、取代芳基、取代杂芳基、Ar(CH<Sub>2</Sub>)<Sub>n</Sub>O-基团中的一种,Ar代表芳基、杂芳基,n取1-6。本发明工艺流程简单,对设备无特殊要求,适用于工业化生产,对环境友好,极大地降低了成本。(The invention discloses a preparation method of a compound shown in formula I, which is obtained by reacting a compound shown in formula V, wherein the reaction formula is as follows: R 1 selected from the group consisting of alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl, substituted heteroaryl; r 2 Selected from amino OR-OR 0 ;R 0 Selected from the group consisting of hydrocarbyl, silyl, benzyl, alkyl methyl, alkyl-substituted alkoxy, 3-8 membered cycloaliphatic, aryl, heteroaryl, substituted aryl, substituted heteroaryl, Ar (CH) 2 ) n One of O-groups, Ar represents aryl or heteroaryl, and n is 1-6. The method has the advantages of simple process flow, no special requirements on equipment, suitability for industrial production, environmental friendliness and great reduction of cost.)
技术领域
本发明涉及农药的制备方法,具体地指一种L-草铵膦的制备方法。
背景技术
草铵膦,化学名称为4-[羟基(甲基)膦酰基]-DL-高丙氨酸,由赫斯特公司(现德国拜耳公司)于上个世纪80年代开发生产,属膦酸类除草剂,是谷氨酰胺合成抑制剂,非选择性触杀型除草剂,其作为除草剂获得登记使用是1984年。2004年我国才有草铵膦原药登记,2005年我国才有产品登记。
草甘膦自广泛应用以来,抗草甘膦杂草不断增加,危害逐步加重。百草枯是一种强烈的杀灭杂草除莠剂,对人畜有很强的毒性作用。2014年7月1日,我国撤销百草枯水剂登记和生产许可、停止生产;2016年7月1日停止水剂在国内销售和使用。草铵膦是世界大吨位农药品种,也是世界第二大转基因作物耐受除草剂。草铵膦毒性低,较为安全,在土壤中易于降解,对作物安全,不易飘移,除草谱广,活性高,用量少,环境压力小,杀草迅速,能快速杀死100种以上的禾本科和阔叶杂草,可用水做基剂,使用安全方便,这些是该品优于其他除草剂的特点,所以这个产品在出现了众多高效超高效的产品后,依然可以畅销。
目前国内外草铵膦的合成技术路线较多,严海昌等(《农药》,2002,41(9),46-48)做过综述报道,但各路线普遍存在反应步骤多,生产成本高的问题。拜尔公司在专利US4521348及US6359162中提出了以合成二氯甲基膦再合成甲基亚磷酸酯,经过一系列反应合成草铵膦的方法。该路线收率高、成本低,但起始路线二氯甲基膦合成原料、产物物化性质活泼,易燃易爆。合成过程处于500~600℃,如控制不稳定,容易产生极易自然的黄磷和磷化氢,危险性很大。另外,物料具有强腐蚀性,对反应设备材质选择及其加工制造工艺要求苛刻,目前国内加工制造水平难以满足生产要求。
这些方法都是制备草铵膦的方法,草铵膦为L/D混合型,其中起主要作用的是L型;L-草铵膦可在土壤中经微生物降解,而D-型草铵膦较难降解,最终可导致土壤板结。因此,L-草铵膦比较传统草铵膦更加高效、更低成本、更加安全。
因此,研发一种能提高原料利用率、降低生产成本同时避免工业化过程中的危险性的L-草铵膦的生物合成制备方法显得十分必要。
发明内容
本发明所要解决的问题就是要克服背景技术的不足,提供一种L-草铵膦的制备方法,能提高原料利用率、降低生产成本,并且适合工业化生产。
本发明提供了式I化合物的制备方法,由式V化合物反应得到,反应式如下:
R2选自氨基或-OR0;
R0选自氢、烃基、硅烷基、苄基、烷基甲基、烷基取代烷氧基、3-8元脂环族基、芳基、杂芳基、取代芳基、取代杂芳基、Ar(CH2)nO-基团中的一种,Ar代表芳基、杂芳基,n取1-6;
R1选自烃基、芳基、杂芳基、取代烃基、取代芳基、取代杂芳基。
进一步地,L-草铵膦的制备方法,由式IV化合物反应制备式II化合物,反应式如下,脱保护基即得到L-草铵膦,
其中,R3选自氢、烷基、硅烷基、硅醚保护基、苄醚保护基、烷氧基甲基、烷氧基取代甲基、C2-C10直链或支链烯烷基、C2-C10直链或支链炔烷基、3-8元脂环族基团、芳基、杂芳基、氨基、取代氨基、Ar(CH2)a-基团中的一种,Ar代表芳基、杂芳基,a取1-6;
进一步地,式V化合物在酶E1和酶E2的作用下反应制备得到式I化合物,反应式如下:
所述E1为能够将式V化合物水解为式I化合物的酶;
所述E2为能够将式VI化合物消旋的酶;
反应过程中需添加辅酶。
进一步地,L-草铵膦的制备方法,包括式IV化合物在酶E1和酶E2的作用下反应制备得到式II化合物,反应式如下,脱保护基即得到L-草铵膦,
进一步地,L-草铵膦的制备方法,R3为氢,R2为乙基或丙基或异丙基,控制反应体系的pH为5-8.5,所述辅酶为磷酸吡哆醛。
进一步地,酶E1基因来源包括Streptomyces ramulosus、Pseudomonasazotoformans、Mycobacterium tuberculosis、猪肝、Candida antartica、Pseudomonaswadenswilerensis、Pseudomonas sp、Brevundimonas diminuta。
进一步地,L-草铵膦的制备方法,酶E2基因来源包括Bacillaceae、Trypanosomacruzi、Oenococcus oeni、Amycolatopsis orientalis、Streptomyces lavendulae、Arthrobacter nicotianae、Ochrobactrum anthropi、Achromobacter obae。
进一步地,L-草铵膦的制备方法,酶E1和酶E2为基因工程菌全细胞、破碎酶液、冻干粉或固定化酶或固定化细胞。
进一步地,L-草铵膦的制备方法,酶E1为氨基酸序列为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、或SEQ ID NO:9所示的蛋白质,或者是SEQ ID NO:1、SEQ IDNO:2、SEQ ID NO:3、SEQ ID NO:4、或SEQ ID NO:9经过一个或几个氨基酸残基取代、缺失或添加后具有将式V化合物水解为式I化合物活性的蛋白质,或者是SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、或SEQ ID NO:9所示的氨基酸序列具有80%以上同源性具有将式V化合物水解为式I化合物活性的蛋白质。
进一步地,L-草铵膦的制备方法,酶E2为氨基酸序列为SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:10所示的蛋白质,或者是SEQ ID NO:5、SEQ IDNO:6、SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:10经过一个或几个氨基酸残基取代、缺失或添加后具有将式VI化合物消旋活性的蛋白质,或与SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:10所示的氨基酸序列具有80%以上同源性且具有将式VI化合物消旋活性的蛋白质。
本发明的优点主要体现在如下几方面:
其一,本发明工艺流程简单,对设备无特殊要求,适用于工业化生产;
其二,本发明底物利用率高,反应条件温和,对环境友好;
其三,本发明制备工艺是对传统化学合成方法的升级换代,极大地降低了成本。
具体实施方式
以下结合具体实施例对本发明作进一步的详细描述,但该实施例不应该理解为对本发明的限制。
实施例1
1、酶E1在大肠杆菌中的表达
分别选择来源于Streptomyces ramulosus、Pseudomonas azotoformans、Mycobacterium tuberculosis、猪肝、Candida antartica、Pseudomonaswadenswilerensis、Pseudomonas sp、Brevundimonas diminuta等的酶,多种序列,所述基因序列经密码子优化在大肠杆菌中表达,通过人工合成,克隆到表达载体pET28a中并放入表达株宿主菌E.coli BL21(DE3)中,挑取阳性转化子并测序鉴定后,得到重组表达载体;将重组表达载体转入E.coli BL21(DE3)菌株中;
含有克隆基因的各大肠杆菌E.coli BL21(DE3)菌株在具有适当抗生素的LB培养基中,于37℃过夜培养,得到种子培养液;将种子培养液接种到含适当抗生素的TB培养基中,接种量为含卡那霉素的TB培养基体积的1%;然后置于37℃下培养2-5h,加入无菌的IPTG诱导,使IPTG终浓度达到0.1mM,置于25℃下继续培养20h。分别得到氨基酸序列为SEQID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:9的酶E1。
2、酶E2在大肠杆菌中的表达
分别选择来源于Bacillaceae、Trypanosoma cruzi、Oenococcus oeni、Amycolatopsis orientalis、Streptomyces lavendulae、Arthrobacter nicotianae、Ochrobactrum anthropi、Achromobacter obae等的消旋酶,多种序列,所述基因序列经密码子优化在大肠杆菌中表达,通过人工合成,克隆到表达载体pET28a中并放入表达株宿主菌E.coli BL21(DE3)中,挑取阳性转化子并测序鉴定后,得到重组表达载体;将重组表达载体转入E.coli BL21(DE3)菌株中;
含有克隆的基因的各大肠杆菌E.coli BL21(DE3)菌株在具有适当抗生素的LB培养基中,于37℃过夜培养,得到种子培养液;将种子培养液接种到含适当抗生素的TB培养基中,接种量为含卡那霉素的TB培养基体积的1%;然后置于37℃下培养2-5h,加入无菌的IPTG诱导,使IPTG终浓度达到0.1mM,置于25℃下继续培养20h。分别得到氨基酸序列为SEQID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:10的酶E2。
实施例2
消旋的2-氨基丙酰胺在酶E1和酶E2的作用下反应制备得到单一手性的S-2-氨基丙酸。反应式如下:
酶E1和酶E2表达如实施例1所述,反应(5mL)使用酶E1(50g湿细胞/L)、酶E2(60g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.5mM)和消旋的2-氨基丙酰胺(30g/L)在柠檬酸-柠檬酸钠缓冲溶液(100mM,pH5.0)中进行,转化温度(37℃),转化12h后,得到单一手性的S-2-氨基丙酸。衍生检测转化率和EE值如下表所示:
序号
酶E1来源
酶E1氨基酸序列
酶E2来源
酶E2氨基酸序列
转化率
EE值
1
Streptomyces ramulosus
SEQ ID NO:1
Bacillaceae
SEQ ID NO:5
86.1%
84.9%
2
Pseudomonas azotoformans
SEQ ID NO:2
Trypanosoma cruzi
SEQ ID NO:6
89.3%
88.4%
3
Mycobacterium tuberculosis
SEQ ID NO:3
Ochrobactrum anthropi
SEQ ID NO:7
81.1%
82.1%
4
Brevundimonas diminuta
SEQ ID NO:4
Achromobacter obae
SEQ ID NO:8
78.7%
82.5%
实施例3
消旋的2-氨基丁酸甲酯在酶E1和酶E2的作用下反应制备得到单一手性的S-2-氨基丁酸。反应式如下:
酶E1和酶E2表达如实施例1所述,反应(5mL)使用酶E1(45g湿细胞/L)、酶E2(70g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.5mM)和消旋的2-氨基丁酸甲酯(30g/L)在磷酸氢二钠-磷酸二氢钠缓冲溶液(100mM,pH7.0)中进行,转化温度(30℃),转化12h后,得到单一手性的S-2-氨基丁酸。衍生检测转化率和EE值如下表所示:
序号
酶E1来源
酶E1氨基酸序列
酶E2来源
酶E2氨基酸序列
转化率
EE值
1
Streptomyces ramulosus
SEQ ID NO:1
Bacillaceae
SEQ ID NO:5
93.6%
88.2%
2
Pseudomonas azotoformans
SEQ ID NO:2
Trypanosoma cruzi
SEQ ID NO:6
83.2%
81.6%
3
Mycobacterium tuberculosis
SEQ ID NO:3
Ochrobactrum anthropi
SEQ ID NO:7
81.5%
82.4%
4
Brevundimonas diminuta
SEQ ID NO:4
Achromobacter obae
SEQ ID NO:8
74.9%
73.6%
实施例4
消旋的2-氨基-4-甲基-戊酸苯甲酯在酶E1和酶E2的作用下反应制备得到单一手性的S-2-氨基-4-甲基-戊酸。反应式如下:
酶E1和酶E2表达如实施例1所述,反应(10mL)使用酶E1(40g湿细胞/L)、酶E2(80g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.5mM)和消旋的2-氨基-4-甲基-戊酸苯甲酯(10g/L)在Tris-盐酸缓冲溶液(100mM,pH7.5)中进行,转化温度(25℃),转化12h后,得到单一手性的S-2-氨基-4-甲基-戊酸,衍生检测转化率和EE值如下表所示:
序号
酶E1来源
酶E1氨基酸序列
酶E2来源
酶E2氨基酸序列
转化率
EE值
1
Streptomyces ramulosus
SEQ ID NO:1
Bacillaceae
SEQ ID NO:5
89.2%
79.1%
2
Pseudomonas azotoformans
SEQ ID NO:2
Trypanosoma cruzi
SEQ ID NO:6
89.8%
73.2%
3
Mycobacterium tuberculosis
SEQ ID NO:3
Ochrobactrum anthropi
SEQ ID NO:7
78.7%
75.3%
4
Brevundimonas diminuta
SEQ ID NO:4
Achromobacter obae
SEQ ID NO:8
81.5%
82.8%
实施例5
消旋的2-氨基-3-苯基-丙酸甲酯在酶E1和酶E2的作用下反应制备得到单一手性的S-2-氨基-3-苯基-丙酸。反应式如下:
酶E1和酶E2表达如实施例1所述,反应(10mL)使用酶E1(40g湿细胞/L)、酶E2(80g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.5mM)和消旋的2-氨基-3-苯基-丙酸甲酯(10g/L)在磷酸二氢钾-氢氧化钠缓冲溶液(100mM,pH7.5)中进行,转化温度(25℃),转化12h后,得到单一手性的S-2-氨基-3-苯基-丙酸,衍生检测转化率和EE值如下表所示:
序号
酶E1来源
酶E1氨基酸序列
酶E2来源
酶E2氨基酸序列
转化率
EE值
1
Streptomyces ramulosus
SEQ ID NO:1
Bacillaceae
SEQ ID NO:5
88.1%
89.4%
2
Pseudomonas azotoformans
SEQ ID NO:2
Trypanosoma cruzi
SEQ ID NO:6
85.9%
86.8%
3
Mycobacterium tuberculosis
SEQ ID NO:3
Ochrobactrum anthropi
SEQ ID NO:7
89.6%
88.4%
4
Brevundimonas diminuta
SEQ ID NO:4
Achromobacter obae
SEQ ID NO:8
88.5%
86.7%
实施例6
消旋的2-氨基-3-氯-丁酸甲酯在酶E1和酶E2的作用下反应制备得到单一手性的S-2-氨基-3-氯-丁酸。反应式如下:
酶E1和酶E2表达如实施例1所述,反应(50mL)使用酶E1(60g湿细胞/L)、酶E2(50g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.5mM)和消旋的2-氨基-4-甲基-戊酸甲酯(20g/L)在柠檬酸-柠檬酸钠缓冲溶液(100mM,pH6.0)中进行,转化温度(28℃),转化12h后,得到单一手性的S-2-氨基-3-氯-丁酸,衍生检测转化率和EE值如下表所示:
序号
酶E1来源
酶E1氨基酸序列
酶E2来源
酶E2氨基酸序列
转化率
EE值
1
Streptomyces ramulosus
SEQ ID NO:1
Bacillaceae
SEQ ID NO:5
91.6%
88.1%
2
Pseudomonas azotoformans
SEQ ID NO:2
Trypanosoma cruzi
SEQ ID NO:6
88.3%
85.7%
3
Mycobacterium tuberculosis
SEQ ID NO:3
Ochrobactrum anthropi
SEQ ID NO:7
89.8%
82.4%
4
Brevundimonas diminuta
SEQ ID NO:4
Achromobacter obae
SEQ ID NO:8
86.7%
86.5%
实施例7
消旋的2-氨基-4-(羟基甲基膦酰基)-丁酰胺在酶E1和酶E2的作用下反应制备得到单一手性的L-2-氨基-4-(甲氧基甲基膦酰基)-丁酸。反应式如下:
酶E1和酶E2表达如实施例1所述,反应(50mL)使用酶E1(35g湿细胞/L)、酶E2(85g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.3mM)和消旋的2-氨基-4-(羟基甲基膦酰基)-丁酰胺(50g/L)在柠檬酸-柠檬酸钠缓冲溶液(100mM,pH6.5)中进行,转化温度(37℃),转化12h后,得到单一手性的L-2-氨基-4-(羟基甲基膦酰基)-丁酸,即L-草铵膦,衍生检测转化率和EE值如下表所示:
序号
酶E1来源
酶E1氨基酸序列
酶E2来源
酶E2氨基酸序列
转化率
EE值
1
Streptomyces ramulosus
SEQ ID NO:1
Bacillaceae
SEQ ID NO:5
97.8%
94.1%
2
Pseudomonas azotoformans
SEQ ID NO:2
Trypanosoma cruzi
SEQ ID NO:6
95.3%
92.6%
3
Mycobacterium tuberculosis
SEQ ID NO:3
Ochrobactrum anthropi
SEQ ID NO:7
93.4%
91.5%
4
Brevundimonas diminuta
SEQ ID NO:4
Achromobacter obae
SEQ ID NO:8
86.9%
97.4%
实施例8
消旋的2-氨基-4-(异丙氧基甲基膦酰基)-丁酸叔丁酯在酶E1和酶E2的作用下反应制备得到单一手性的L-2-氨基-4-(异丙氧基甲基膦酰基)-丁酸。反应式如下:
酶E1和酶E2表达如实施例1所述,反应(100mL)使用酶E1(80g湿细胞/L)、酶E2(40g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.5mM)和消旋的2-氨基-4-(异丙氧基甲基膦酰基)-丁酸叔丁酯(40g/L)在磷酸氢二钠-磷酸二氢钾缓冲溶液(100mM,pH8.0)中进行,转化温度(35℃),转化12h后,向转化液中加入浓度为10%-37%的盐酸,去保护基,得到单一手性的L-2-氨基-4-(异丙氧基甲基膦酰基)-丁酸,化学法去保护基,得到单一手性的L-2-氨基-4-(羟基甲基膦酰基)-丁酸,即L-草铵膦,衍生检测转化率和EE值如下表所示:
序号
酶E1来源
酶E1氨基酸序列
酶E2来源
酶E2氨基酸序列
转化率
EE值
1
Streptomyces ramulosus
SEQ ID NO:1
Bacillaceae
SEQ ID NO:5
97.8%
95.7%
2
Pseudomonas azotoformans
SEQ ID NO:2
Trypanosoma cruzi
SEQ ID NO:6
93.7%
92.8%
3
Mycobacterium tuberculosis
SEQ ID NO:3
Ochrobactrum anthropi
SEQ ID NO:7
97.9%
94.6%
4
Brevundimonas diminuta
SEQ ID NO:4
Achromobacter obae
SEQ ID NO:8
93.2%
92.1%
实施例9
消旋的2-氨基-4-(甲氧基甲基膦酰基)-丁酸甲酯在酶E1和酶E2的作用下反应制备得到单一手性的L-2-氨基-4-(甲氧基甲基膦酰基)-丁酸。反应式如下:
酶E1和酶E2表达如实施例1所述,反应(50mL)使用酶E1(30g湿细胞/L)、酶E2(90g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.3mM)和消旋的2-氨基-4-(甲氧基甲基膦酰基)-丁酸甲酯(50g/L)在柠檬酸-柠檬酸钠缓冲溶液(100mM,pH6.5)中进行,转化温度(37℃),转化12h后,得到单一手性的L-2-氨基-4-(甲氧基甲基膦酰基)-丁酸,化学法去保护基,得到单一手性的L-2-氨基-4-(羟基甲基膦酰基)-丁酸,即L-草铵膦,衍生检测转化率和EE值如下表所示:
序号
酶E1来源
酶E1氨基酸序列
酶E2来源
酶E2氨基酸序列
转化率
EE值
1
Streptomyces ramulosus
SEQ ID NO:1
Bacillaceae
SEQ ID NO:5
97.6%
96.2%
2
Pseudomonas azotoformans
SEQ ID NO:2
Trypanosoma cruzi
SEQ ID NO:6
94.8%
91.6%
3
Mycobacterium tuberculosis
SEQ ID NO:3
Ochrobactrum anthropi
SEQ ID NO:7
97.2%
90.8%
4
Brevundimonas diminuta
SEQ ID NO:4
Achromobacter obae
SEQ ID NO:8
89.7%
98.2%
实施例10
消旋的2-氨基-4-(乙氧基甲基膦酰基)-丁酸异丙酯在酶E1和酶E2的作用下反应制备得到单一手性的L-2-氨基-4-(乙氧基甲基膦酰基)-丁酸。反应式如下:
酶E1和酶E2表达如实施例1所述,反应(200mL)使用酶E1(90g湿细胞/L)、酶E2(60g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.2mM)和消旋的2-氨基-4-(乙氧基甲基膦酰基)-丁酸异丙酯(70g/L)在Tris-盐酸缓冲溶液(100mM,pH8.5)中进行,转化温度(40℃),转化12h后,得到单一手性的L-2-氨基-4-(乙氧基甲基膦酰基)-丁酸,化学法去保护基,得到单一手性的L-2-氨基-4-(羟基甲基膦酰基)-丁酸,即L-草铵膦,衍生检测转化率和EE值如下表所示:
序号
酶E1来源
酶E1氨基酸序列
酶E2来源
酶E2氨基酸序列
转化率
EE值
1
Streptomyces ramulosus
SEQ ID NO:1
Bacillaceae
SEQ ID NO:5
98.7%
96.8%
2
Pseudomonas azotoformans
SEQ ID NO:2
Trypanosoma cruzi
SEQ ID NO:6
97.5%
95.9%
3
Mycobacterium tuberculosis
SEQ ID NO:3
Ochrobactrum anthropi
SEQ ID NO:7
99.1%
91.4%
4
Brevundimonas diminuta
SEQ ID NO:4
Achromobacter obae
SEQ ID NO:8
95.4%
96.3%
实施例11
消旋的2-氨基-4-(羟基甲基膦酰基)-丁酸苯甲酯在酶E1和酶E2的作用下反应制备得到单一手性的L-2-氨基-4-(羟基甲基膦酰基)-丁酸。反应式如下:
酶E1和酶E2表达如实施例1所述,反应(100mL)使用酶E1(55g湿细胞/L)、酶E2(65g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.4mM)和消旋的2-氨基-4-(羟基甲基膦酰基)-丁酸苯甲酯(60g/L)在磷酸氢二钠-磷酸二氢钠缓冲溶液(100mM,pH7.0)中进行,转化温度(35℃),转化12h后,得到单一手性的L-2-氨基-4-(羟基甲基膦酰基)-丁酸,衍生检测转化率和EE值如下表所示:
序号
酶E1来源
酶E1氨基酸序列
酶E2来源
酶E2氨基酸序列
转化率
EE值
1
Streptomyces ramulosus
SEQ ID NO:1
Bacillaceae
SEQ ID NO:5
99.3%
96.4%
2
Pseudomonas azotoformans
SEQ ID NO:2
Trypanosoma cruzi
SEQ ID NO:6
96.8%
93.4%
3
Mycobacterium tuberculosis
SEQ ID NO:3
Ochrobactrum anthropi
SEQ ID NO:7
96.7%
98.2%
4
Brevundimonas diminuta
SEQ ID NO:4
Achromobacter obae
SEQ ID NO:8
97.3%
93.5%
实施例12
消旋的2-氨基-4-(羟基甲基膦酰基)-丁酸乙酯在酶E1变体和酶E2变体的作用下反应制备得到单一手性的L-2-氨基-4-(羟基甲基膦酰基)-丁酸。反应式如下:
酶E1变体和酶E2变体表达如实施例1所述,反应(50mL)在30℃下使用酶E1(70g湿细胞/L)、酶E2(45g湿细胞/L)、磷酸吡哆醛(5mM)、CoCl2(0.5mM)和消旋的2-氨基-4-(羟基甲基膦酰基)-丁酸乙酯(80g/L)在磷酸二氢钾-氢氧化钠缓冲溶液(100mM,pH7.5)中进行,转化温度(37℃),转化12h后,得到单一手性的L-2-氨基-4-(羟基甲基膦酰基)-丁酸乙酯,衍生检测转化率和EE值如下表所示:
序号
酶E1变体氨基酸序列
酶E2变体氨基酸序列
转化率
EE值
1
SEQ ID NO:9
SEQ ID NO:10
99.7%
99.5%
其中,实施例12中酶E1变体为再SEQ ID NO:1基础上突变6个氨基酸所得,氨基酸序列如SEQ ID NO:9所示,酶E2变体为再SEQ ID NO:5基础上突变5个氨基酸所得,氨基酸序列如SEQ ID NO:10所示。
本说明书中未作详细描述的内容,属于本专业技术人员公知的现有技术。
序列表
<110> 武汉茵茂特生物技术有限公司
<120> L-草铵膦的制备方法
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 268
<212> PRT
<213> Streptomyces ramulosus
<400> 1
Met Arg Leu Ser Arg Arg Ala Ala Thr Ala Ser Ala Leu Leu Leu Thr
1 5 10 15
Pro Ala Leu Ala Leu Phe Gly Ala Ser Ala Ala Val Arg Ala Pro Arg
20 25 30
Ile Gln Ala Thr Asp Tyr Val Ala Leu Gly Asp Ser Tyr Ser Ser Gly
35 40 45
Val Gly Ala Gly Ser Tyr Asp Ser Ser Ser Gly Ser Cys Lys Arg Ser
50 55 60
Thr Lys Ser Tyr Pro Ala Leu Trp Ala Ala Ser His Thr Gly Thr Arg
65 70 75 80
Phe Asn Phe Thr Ala Cys Ser Gly Ala Arg Thr Gly Asp Val Leu Ala
85 90 95
Lys Gln Leu Thr Pro Val Asn Ser Gly Thr Asp Leu Val Ser Ile Thr
100 105 110
Ile Gly Gly Asn Asp Ala Gly Phe Ala Asp Thr Met Thr Thr Cys Asn
115 120 125
Asp Gln Gly Glu Ser Ala Cys Leu Ala Arg Ile Ala Lys Ala Arg Ala
130 135 140
Tyr Ile Gln Gln Thr Leu Pro Ala Gln Leu Asp Gln Val Tyr Asp Ala
145 150 155 160
Ile Asp Ser Arg Ala Pro Ala Ala Gln Val Val Val Leu Gly Tyr Pro
165 170 175
Arg Phe Tyr Lys Leu Gly Gly Ser Cys Ala Val Gly Leu Ser Glu Lys
180 185 190
Ser Arg Ala Ala Ile Asn Ala Ala Ala Asp Asp Ile Asn Ala Val Thr
195 200 205
Ala Lys Arg Ala Ala Asp His Gly Phe Ala Phe Gly Asp Val Asn Thr
210 215 220
Thr Phe Ala Gly His Glu Leu Cys Ser Gly Ala Pro Trp Leu His Ser
225 230 235 240
Val Thr Leu Pro Val Glu Asn Ser Tyr His Val Thr Ala Asn Gly Gln
245 250 255
Ser Lys Gly Tyr Leu Pro Val Leu Asn Ser Ala Thr
260 265
<210> 2
<211> 310
<212> PRT
<213> Pseudomonas azotoformans
<400> 2
Met Glu Phe Ile Glu Lys Ile Arg Glu Gly Tyr Ala Ala Phe Gly Ala
1 5 10 15
Tyr Gln Thr Trp Tyr Arg Val Thr Gly Asp Leu Ser Ser Gly Arg Thr
20 25 30
Pro Leu Val Val Ile His Gly Gly Pro Gly Cys Thr His Asp Tyr Val
35 40 45
Asp Ala Phe Lys Asp Val Ala Ala Ser Gly His Ala Val Ile His Tyr
50 55 60
Asp Gln Leu Gly Asn Gly Arg Ser Arg His Leu Pro Asp Lys Asp Pro
65 70 75 80
Ser Phe Trp Thr Val Gly Leu Phe Leu Glu Glu Leu Asn Asn Leu Leu
85 90 95
Asp His Leu Gln Ile Ser Asp Asn Tyr Ala Ile Leu Gly Gln Ser Trp
100 105 110
Gly Gly Met Leu Gly Ser Glu His Ala Ile Leu Gln Pro Lys Gly Leu
115 120 125
Arg Ala Phe Ile Pro Ala Asn Ser Pro Thr Cys Met Arg Thr Trp Val
130 135 140
Ser Glu Ala Asn Arg Leu Arg Lys Leu Leu Pro Glu Gly Val His Glu
145 150 155 160
Thr Leu Leu Lys His Glu Thr Ala Gly Thr Tyr Gln Asp Pro Glu Tyr
165 170 175
Leu Ala Ala Ser Arg Val Phe Tyr Asp His Asn Val Cys Arg Val Ile
180 185 190
Pro Trp Pro Glu Glu Val Ala Arg Thr Phe Ala Ala Val Asp Ala Asp
195 200 205
Pro Thr Val Tyr His Ala Met Ser Gly Pro Thr Glu Phe His Val Ile
210 215 220
Gly Ser Leu Lys Asp Trp Lys Ser Thr Gly Arg Leu Ser Ala Ile Asn
225 230 235 240
Val Pro Thr Leu Val Ile Ser Gly Arg His Asp Glu Ala Thr Pro Leu
245 250 255
Val Val Lys Pro Phe Leu Asp Glu Ile Ala Asp Val Arg Trp Ala Leu
260 265 270
Phe Glu Asp Ser Ser His Met Pro His Val Glu Glu Arg Gln Ala Cys
275 280 285
Met Gly Thr Val Val Lys Phe Leu Asp Glu Val Cys Ser Ala Lys Tyr
290 295 300
Lys Val Leu Lys Ala Ser
305 310
<210> 3
<211> 277
<212> PRT
<213> Mycobacterium tuberculosis
<400> 3
Met Arg Ala Pro Gly Val Arg Ala Ala Asp Gly Ala Gly Arg Val Val
1 5 10 15
Leu Tyr Leu His Gly Gly Ala Phe Val Met Cys Gly Pro Asn Ser His
20 25 30
Ser Arg Ile Val Asn Ala Leu Ser Gly Phe Ala Glu Ser Pro Val Leu
35 40 45
Ile Val Asp Tyr Arg Leu Ile Pro Lys His Ser Leu Gly Met Ala Leu
50 55 60
Asp Asp Cys His Asp Ala Tyr Gln Trp Leu Arg Ala Arg Gly Tyr Arg
65 70 75 80
Pro Glu Gln Ile Val Leu Pro Gly Asp Ser Ala Gly Gly Tyr Leu Ala
85 90 95
Leu Ala Leu Ala Gln Arg Leu Gln Cys Asp Asp Glu Lys Pro Ala Ala
100 105 110
Ile Val Ala Ile Ser Pro Leu Leu Gln Leu Ala Lys Gly Pro Lys Gln
115 120 125
Asp His Pro Asn Ile Gly Thr Asp Ala Met Phe Pro Ala Arg Ala Phe
130 135 140
Asp Ala Leu Ala Ala Trp Val Arg Ala Ala Ala Ala Lys Asn Met Val
145 150 155 160
Asp Gly Arg Pro Glu Asp Leu Tyr Glu Pro Leu Asp His Ile Glu Ser
165 170 175
Ser Leu Pro Pro Thr Leu Ile His Val Ser Gly Ser Glu Val Leu Leu
180 185 190
His Asp Ala Gln Leu Gly Ala Val Lys Leu Ala Ala Ala Gly Val Cys
195 200 205
Ala Glu Val Arg Val Trp Pro Gly Gln Ala His Leu Phe Gln Leu Ala
210 215 220
Thr Pro Leu Val Pro Glu Ala Thr Arg Ser Leu Arg Gln Ile Gly Gln
225 230 235 240
Phe Ile Arg Asp Ala Thr Ala Asp Ser Ser Leu Ser Ile Val His Arg
245 250 255
Ser Arg Tyr Val Ala Gly Ser Pro Arg Ala Ala Ser Arg Gly Ala Phe
260 265 270
Gly Gln Ser Pro Ile
275
<210> 4
<211> 493
<212> PRT
<213> Brevundimonas diminuta
<400> 4
Met Gly Met Lys Ile Glu Phe Val Ala Ala Ser Gly Ala Ala Glu Ile
1 5 10 15
Leu Ala Leu Leu Val His Glu Asp Arg Ala Leu Ala Gly Thr Gly Pro
20 25 30
Ala Leu Asp Ala Ala Ala Ser Gly Ala Leu Val Lys Ala Met Lys Lys
35 40 45
Ser Arg Phe Val Gly Ala Ala Ser Ser Ser Leu Asn Val Ala Ala Pro
50 55 60
Ser Gly Ile Asp Ala Asn Ala Val Leu Leu Val Gly Ala Gly Ala Ala
65 70 75 80
Asp Lys Leu Asp Asp Leu Ala Val Glu Thr Phe Gly Ala Ala Ala Val
85 90 95
Gln Ala Thr Lys Leu Ser Gly Ala Glu Val Leu Thr Ile Asp Val Ser
100 105 110
Gly Leu Ser Pro Glu Leu Ala Ala Arg Ala Gly Phe Ala Ala Arg Leu
115 120 125
Ala Ala Tyr Arg Phe Ala Lys Tyr Leu Thr Lys Gln Lys Ala Asp Lys
130 135 140
Ile Pro Ser Val Thr Ala Val Arg Val Val Thr Ser Asp Val Lys Ala
145 150 155 160
Ala Glu Ala Ala Leu Gln Pro Leu Ser Ala Val Ala Asp Ala Val Leu
165 170 175
Phe Ala Arg Asp Leu Val Ser Glu Pro Ala Asn Ile Leu Tyr Pro Ala
180 185 190
Glu Phe Ala Arg Arg Val Lys Glu Leu Glu Ala Leu Gly Ala Lys Val
195 200 205
Glu Ile Leu Gly Glu Ala Glu Met Gln Lys Leu Gly Met Gly Ser Leu
210 215 220
Leu Gly Val Gly Gln Gly Ser Val Arg Glu Ser Gln Leu Ala Val Ile
225 230 235 240
Gln Trp Asn Gly Gly Glu Glu Gly Glu Ala Pro Ile Ala Phe Val Gly
245 250 255
Lys Gly Val Cys Phe Asp Thr Gly Gly Ile Ser Leu Lys Ala Ala Asp
260 265 270
Gly Met Glu Glu Met Lys Trp Asp Met Gly Gly Ala Ala Ala Val Thr
275 280 285
Gly Leu Met His Ala Leu Val Gly Arg Lys Ala Lys Val Asn Val Val
290 295 300
Gly Val Leu Gly Leu Val Glu Asn Met Pro Asp Gly Asn Ala Gln Arg
305 310 315 320
Pro Gly Asp Val Val Thr Ser Met Ser Gly Gln Thr Val Glu Val Leu
325 330 335
Asn Thr Asp Ala Glu Gly Arg Leu Val Leu Ala Asp Ala Leu Trp Tyr
340 345 350
Thr Gln Gln Arg Phe Lys Pro Lys Phe Met Ile Asp Leu Ala Thr Leu
355 360 365
Thr Gly Ala Met Ile Ile Ser Leu Gly Leu Glu Tyr Ala Gly Val Phe
370 375 380
Thr Asn Ser Asp Ala Leu Ala Ala Asn Ile Ala Asp Ala Gly Pro Lys
385 390 395 400
Val Gly Glu Asn Ser Trp Arg Met Pro Ile Pro Ala Glu Tyr Asp Gln
405 410 415
His Ile Asp Ser Pro Ile Ala Asp Val Lys Asn Met Gly Asn Gly Arg
420 425 430
Ala Gly Gly Ser Ile Thr Ala Ala Leu Phe Leu Gln Arg Phe Thr Asn
435 440 445
Gly Thr Pro Trp Ala His Ile Asp Ile Ala Pro Thr Ala Trp Val Lys
450 455 460
Asp Ser Lys Asn Pro Thr Val Pro Asp Gly Gly Val Gly Tyr Gly Val
465 470 475 480
Arg Leu Leu Asp Arg Met Val Ala Asp His Tyr Glu Gly
485 490
<210> 5
<211> 264
<212> PRT
<213> Bacillaceae
<400> 5
Met Glu Arg Ala Ile Gly Val Ile Asp Ser Gly Glu Gly Gly Leu Thr
1 5 10 15
Val Ala Lys Glu Ile Met Arg Gln Leu Pro Lys Glu Arg Ile Val Tyr
20 25 30
Leu Gly Asp Thr Ala Arg Cys Pro Tyr Gly Pro Arg Pro Lys Glu Glu
35 40 45
Ile Arg Gln Phe Thr Trp Glu Met Thr Asn Tyr Leu Leu Arg Tyr Asn
50 55 60
Ile Lys Met Leu Val Ile Ala Cys Asn Thr Ala Thr Ala Val Val Leu
65 70 75 80
Asp Glu Ile Arg Glu Gln Leu Asp Ile Pro Val Leu Gly Val Ile His
85 90 95
Pro Gly Ala Arg Ala Ala Leu Lys Ala Thr Lys Asn Gly His Ile Gly
100 105 110
Val Ile Gly Thr Ile Gly Thr Val Lys Ser Gly Ala Tyr Glu Lys Ala
115 120 125
Leu Lys Ser Ile Asn His Arg Val Arg Val Glu Ser Leu Ala Cys Pro
130 135 140
Lys Phe Val Pro Leu Val Glu Ser Gly Asn Phe Glu Gly Glu Glu Ala
145 150 155 160
Arg Lys Ile Val Ala Glu Ser Leu Glu Pro Leu Lys Ser Ser Asn Met
165 170 175
Asp Val Leu Ile Leu Gly Cys Thr His Tyr Pro Leu Leu Ser Pro Leu
180 185 190
Ile Lys Glu Tyr Met Gly Lys Arg Val Lys Leu Ile Cys Ser Gly Asp
195 200 205
Glu Thr Ala Arg Glu Val Ser Ala Ile Leu His His Ser His Leu Leu
210 215 220
Tyr Thr Gly Gln Arg Glu Ala Glu His Leu Phe Phe Thr Thr Gly Ser
225 230 235 240
Lys Glu Leu Phe Gln Lys Ile Ala Ser Lys Trp Phe Gly Lys Pro Ile
245 250 255
Glu Asn Val Gln Thr Ile Glu Leu
260
<210> 6
<211> 423
<212> PRT
<213> Trypanosoma cruzi
<400> 6
Met Arg Lys Ser Val Cys Pro Lys Gln Lys Phe Phe Phe Ser Ala Phe
1 5 10 15
Pro Phe Phe Phe Phe Phe Cys Val Phe Pro Leu Ile Ser Arg Thr Gly
20 25 30
Gln Glu Lys Leu Leu Phe Asp Gln Lys Tyr Lys Ile Ile Lys Gly Glu
35 40 45
Lys Lys Glu Lys Lys Lys Asn Gln Arg Ala Asn Arg Arg Glu His Gln
50 55 60
Gln Lys Arg Glu Ile Met Arg Phe Lys Lys Ser Phe Thr Cys Ile Asp
65 70 75 80
Met His Thr Glu Gly Glu Ala Ala Arg Ile Val Thr Ser Gly Leu Pro
85 90 95
His Ile Pro Gly Ser Asn Met Ala Glu Lys Lys Ala Tyr Leu Gln Glu
100 105 110
Asn Met Asp Tyr Leu Arg Arg Gly Ile Met Leu Glu Pro Arg Gly His
115 120 125
Asp Asp Met Phe Gly Ala Phe Leu Phe Arg Pro Ile Glu Glu Gly Ala
130 135 140
Asp Leu Gly Met Val Phe Met Asp Thr Gly Gly Tyr Leu Asn Met Cys
145 150 155 160
Gly His Asn Ser Ile Ala Ala Val Thr Ala Ala Val Glu Thr Gly Ile
165 170 175
Val Ser Val Pro Ala Phe Ala Thr Asn Val Pro Val Val Leu Asp Thr
180 185 190
Pro Ala Gly Leu Val Arg Gly Thr Ala His Leu Gln Ser Gly Thr Glu
195 200 205
Ser Glu Val Ser Asn Ala Ser Ile Ile Asn Val Pro Ser Phe Leu Tyr
210 215 220
Gln Gln Asp Val Val Val Val Leu Pro Lys Pro Tyr Gly Glu Val Arg
225 230 235 240
Val Asp Ile Ala Phe Gly Gly Asn Phe Phe Ala His Val Pro Ala Glu
245 250 255
Gln Leu Gly Ile Asp Ile Ser Val Gln Asn Leu Ser Arg Leu Gln Glu
260 265 270
Ala Gly Glu Leu Leu Arg Thr Glu Ile Asn Arg Ser Val Lys Val Gln
275 280 285
His Pro Gln Leu Pro His Ile Asn Thr Val Asp Cys Val Glu Ile Tyr
290 295 300
Gly Pro Pro Thr Asn Pro Glu Ala Asn Tyr Lys Asn Val Val Ile Phe
305 310 315 320
Gly Asn Arg Gln Ala Asp Arg Ser Pro Cys Gly Thr Gly Thr Ser Ala
325 330 335
Lys Met Ala Thr Leu Tyr Ala Lys Gly Gln Leu Arg Ile Gly Glu Thr
340 345 350
Phe Val Tyr Glu Ser Ile Leu Gly Ser Leu Phe Gln Gly Arg Val Leu
355 360 365
Gly Glu Glu Arg Ile Pro Gly Val Lys Val Pro Val Thr Lys Asp Ala
370 375 380
Glu Glu Gly Met Leu Val Val Thr Ala Glu Ile Thr Gly Lys Ala Phe
385 390 395 400
Ile Met Gly Phe Asn Thr Met Leu Phe Asp Pro Thr Asp Pro Phe Lys
405 410 415
Asn Gly Phe Thr Leu Lys Gln
420
<210> 7
<211> 439
<212> PRT
<213> Ochrobactrum anthropi
<400> 7
Met Gln Thr Pro Leu Ser Leu Arg Glu Arg Asp Ala Arg Val Ile Ala
1 5 10 15
Glu Ile Gly Arg Leu Arg Phe Ser Pro Leu Ser Leu Ile Gly Gly Lys
20 25 30
Gly Asn Arg Leu Ile Glu Glu Gly Gly Arg Ser Ile Leu Asp Leu Ser
35 40 45
Gly Ser Ala Gly Pro Ala Ala Leu Gly Tyr Gly His Pro Ala Ile Val
50 55 60
Glu Ala Val Glu Lys Ser Val Arg Asp Met Ala Gly Ala Ser Leu Leu
65 70 75 80
Leu Tyr Pro Asn Glu Ala Ala Val Ser Leu Ala Glu Asp Leu Leu Arg
85 90 95
Ile Thr Pro Gly Asn Gly Glu Arg Arg Val Trp Phe Gly His Ser Gly
100 105 110
Ser Asp Ala Asn Asp Cys Ala Val Arg Val Leu Thr Ala Ala Thr Lys
115 120 125
Arg Ser Arg Ile Ile Ser Ala Ile Gly Ser Tyr His Gly Asn Leu Thr
130 135 140
Gly Ser Met Gly Ile Ser Gly His Thr Ala Met Thr His Thr Leu Pro
145 150 155 160
Arg Pro Gly Val Leu Leu Leu Pro Tyr Pro Asp Pro Phe Arg Pro Arg
165 170 175
Phe Ser Ala Glu Ala Val Leu Glu Leu Leu Asp Tyr His Phe Ala Thr
180 185 190
Ser Cys Pro Pro Glu Gln Val Ala Ala Val Phe Ile Glu Pro Ile Leu
195 200 205
Ser Asp Gly Gly Leu Val Val Pro Pro Pro Ala Phe Leu Glu Ala Leu
210 215 220
Gln Asp Arg Cys Arg Lys His Gly Gln Leu Val Val Val Asp Glu Val
225 230 235 240
Lys Val Gly Leu Gly Arg Thr Gly Leu Met His Cys Phe Gln His Glu
245 250 255
Gly Leu Glu Pro Asp Met Val Val Phe Gly Lys Gly Leu Gly Gly Gly
260 265 270
Leu Pro Leu Ser Ala Val Val Gly Pro Gln Trp Val Met Asp His Ala
275 280 285
Pro Ala Phe Val Leu Gln Thr Thr Ala Gly Asn Pro Val Ala Thr Ala
290 295 300
Ala Gly Arg Ala Val Leu Asn Thr Ile Glu Arg Gln Gly Leu Ala Gln
305 310 315 320
Arg Ser Glu Arg Val Gly Gly Ile Phe Ala Asp Arg Leu Arg Arg Leu
325 330 335
Ser Asp Lys His Ser Ile Ile Gly Asp Val Arg Gly Arg Gly Leu Ala
340 345 350
Ile Gly Val Asp Leu Val Ser Asp Arg Gly Ser Arg Glu Pro Ala Pro
355 360 365
Val Thr Thr Thr Ala Lys Ile Ile Tyr Arg Gly Tyr Gln Leu Gly Ala
370 375 380
Ala Phe Thr Tyr Val Gly Leu Asn Ala Asn Val Leu Glu Phe Met Pro
385 390 395 400
Pro Leu Thr Leu Thr Glu Pro Glu Ile Asp Lys Ala Ala Asp Ile Val
405 410 415
Asp Gln Ala Ile Gly Asp Val Leu Asp Gly Lys Val Ala Asp Ser Asp
420 425 430
Val Ala His Phe Met Met Trp
435
<210> 8
<211> 436
<212> PRT
<213> Achromobacter obae
<400> 8
Met Thr Lys Ala Leu Tyr Asp Arg Asp Gly Ala Ala Ile Gly Asn Leu
1 5 10 15
Gln Lys Leu Arg Phe Phe Pro Leu Ala Ile Ser Gly Gly Arg Gly Ala
20 25 30
Arg Leu Ile Glu Glu Asn Gly Arg Glu Leu Ile Asp Leu Ser Gly Ala
35 40 45
Trp Gly Ala Ala Ser Leu Gly Tyr Gly His Pro Ala Ile Val Ala Ala
50 55 60
Val Ser Ala Ala Ala Ala Asn Pro Ala Gly Ala Thr Ile Leu Ser Ala
65 70 75 80
Ser Asn Ala Pro Ala Val Thr Leu Ala Glu Arg Leu Leu Ala Ser Phe
85 90 95
Pro Gly Glu Gly Thr His Lys Ile Trp Phe Gly His Ser Gly Ser Asp
100 105 110
Ala Asn Glu Ala Ala Tyr Arg Ala Ile Val Lys Ala Thr Gly Arg Ser
115 120 125
Gly Val Ile Ala Phe Ala Gly Ala Tyr His Gly Cys Thr Val Gly Ser
130 135 140
Met Ala Phe Ser Gly His Ser Val Gln Ala Asp Ala Ala Lys Ala Asp
145 150 155 160
Gly Leu Ile Leu Leu Pro Tyr Pro Asp Glu Tyr Arg Pro Tyr Arg Asn
165 170 175
Asp Pro Thr Gly Asp Ala Ile Leu Thr Leu Leu Thr Glu Lys Leu Ala
180 185 190
Ala Val Pro Ala Gly Ser Ile Gly Ala Ala Phe Ile Glu Pro Ile Gln
195 200 205
Ser Asp Gly Gly Leu Ile Val Pro Pro Asp Gly Phe Leu Arg Lys Phe
210 215 220
Ala Asp Ile Cys Arg Ala His Gly Ile Leu Val Val Cys Asp Glu Val
225 230 235 240
Lys Val Gly Leu Ala Arg Ser Gly Arg Gly His Cys Phe Glu His Glu
245 250 255
Gly Phe Val Pro Asp Ile Leu Val Leu Gly Lys Gly Leu Gly Gly Gly
260 265 270
Leu Pro Leu Ser Ala Val Ile Ala Pro Ala Glu Ile Leu Asp Cys Ala
275 280 285
Ser Ala Phe Ala Met Gln Thr Leu His Gly Asn Pro Ile Ser Ala Ala
290 295 300
Ala Gly Leu Ala Val Leu Glu Thr Ile Asp Arg Asp Asp Leu Pro Ala
305 310 315 320
Met Ala Glu Arg Lys Gly Arg Leu Leu Arg Asp Gly Leu Ser Glu Leu
325 330 335
Ala Lys Arg His Pro Leu Ile Gly Asp Ile Arg Gly Arg Gly Leu Ala
340 345 350
Cys Gly Met Glu Leu Val Cys Asp Arg Gln Ser Arg Glu Pro Ala Arg
355 360 365
Ala Glu Thr Ala Lys Leu Ile Tyr Arg Ala Tyr Gln Leu Gly Leu Val
370 375 380
Val Tyr Tyr Val Gly Met Asn Gly Asn Val Leu Glu Phe Thr Pro Pro
385 390 395 400
Leu Thr Ile Thr Glu Thr Asp Ile His Lys Ala Leu Asp Leu Leu Asp
405 410 415
Arg Ala Phe Ser Glu Leu Ser Ala Val Ser Asn Glu Glu Ile Ala Gln
420 425 430
Phe Ala Gly Trp
435
<210> 9
<211> 268
<212> PRT
<213> Streptomyces ramulosus
<400> 9
Met Arg Leu Ser Arg Arg Ala Ala Thr Ala Ser Ala Leu Leu Leu Thr
1 5 10 15
Pro Ala Leu Ala Leu Phe Gly Ala Ser Leu Ala Val Arg Ala Pro Arg
20 25 30
Ile Gln Ala Thr Asp Tyr Val Ala Leu Gly Asp Ser Tyr Ser Ser Gly
35 40 45
Val Gly Ala Gly Ser Tyr Asp Ser Ser Ser Gly Ser Cys Lys Arg Ser
50 55 60
Thr Ile Ser Tyr Pro Ala Leu Trp Ala Ala Ser His Thr Gly Thr Arg
65 70 75 80
Phe Asn Phe Thr Ala Cys Ser Gly Ala Arg Thr Gly Asp Val Leu Ala
85 90 95
Lys Gln Leu Thr Pro Val Asn Asp Gly Thr Asp Leu Val Ser Ile Thr
100 105 110
Ile Gly Gly Asn Asp Ala Gly Phe Ala Asp Thr Met Thr Thr Cys Asn
115 120 125
Asp Gln Gly Glu Ser Ala Cys Leu Ala Arg Ile Ala Lys Ala Arg Ala
130 135 140
Tyr Ile Gln Gln Thr Leu Pro Ala Gln Leu Asp Gln Val Tyr Asp Ala
145 150 155 160
Ile Asp Ser Asn Ala Pro Ala Ala Gln Val Val Val Leu Gly Tyr Pro
165 170 175
Arg Phe Tyr Lys Leu Gly Gly Ser Cys Ala Val Gly Leu Ser Glu Lys
180 185 190
Ser Arg Ala Ala Ile Asn Ala Ala Ala Asp Val Ile Asn Ala Val Thr
195 200 205
Ala Lys Arg Ala Ala Asp His Gly Phe Ala Phe Gly Asp Val Asn Thr
210 215 220
Thr Phe Ala Gly His Glu Leu Cys Phe Gly Ala Pro Trp Leu His Ser
225 230 235 240
Val Thr Leu Pro Val Glu Asn Ser Tyr His Val Thr Ala Asn Gly Gln
245 250 255
Ser Lys Gly Tyr Leu Pro Val Leu Asn Ser Ala Thr
260 265
<210> 10
<211> 264
<212> PRT
<213> Bacillaceae
<400> 10
Met Glu Arg Ala Ile Gly Val Ile Asp Ser Gly Glu Gly Gly Leu Thr
1 5 10 15
Val Ala Lys Glu Ile Met Arg Gln Leu Pro Lys Glu Arg Ile Val Tyr
20 25 30
Leu Gly Asp Thr Ala Arg Cys Pro Tyr Gly Pro Arg Pro Lys Glu Glu
35 40 45
Ile Arg Gln Phe Thr Trp Glu Met Thr Asn Tyr Leu Leu Arg Tyr Asn
50 55 60
Arg Lys Met Leu Val Ile Ala Cys Asn Thr Ala Thr Ala Val Val Leu
65 70 75 80
Asp Glu Ile Met Glu Gln Leu Asp Ile Pro Val Leu Gly Val Ile His
85 90 95
Pro Gly Ala Arg Ala Ala Leu Lys Ala Thr Lys Asn Gly His Ile Gly
100 105 110
Val Ile Gly Thr Ile Gly Thr Val Lys Ser Gly Ala Tyr Glu Lys Ala
115 120 125
Leu Lys Ser Ile Asn His Arg Val Arg Val Glu Ser Leu Ala Cys Pro
130 135 140
Lys Phe Val Pro Leu Val Glu Ser Gly Asn Phe Glu Gly Glu Glu Ala
145 150 155 160
Arg Lys Ile Val Ala Pro Ser Leu Glu Pro Leu Lys Ser Ser Asn Met
165 170 175
Asp Val Leu Ile Leu Gly Cys Thr His Tyr Pro Leu Leu Ser Pro Leu
180 185 190
Ile Lys Glu Tyr Met Gly Lys Arg Val Lys Leu Ile Cys Ser Cys Asp
195 200 205
Glu Thr Ala Arg Glu Val Ser Ala Ile Leu His His Ser His Leu Leu
210 215 220
Tyr Thr Gly Gln Arg Glu Ala Glu His Leu Phe Phe Thr Thr Gly Ser
225 230 235 240
Lys Glu Leu Phe Ser Lys Ile Ala Ser Lys Trp Phe Gly Lys Pro Ile
245 250 255
Glu Asn Val Gln Thr Ile Glu Leu
260
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