CN114317508B - Halohydrin dehalogenase mutant, engineering bacterium and application thereof - Google Patents
Halohydrin dehalogenase mutant, engineering bacterium and application thereof Download PDFInfo
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- CN114317508B CN114317508B CN202210007671.5A CN202210007671A CN114317508B CN 114317508 B CN114317508 B CN 114317508B CN 202210007671 A CN202210007671 A CN 202210007671A CN 114317508 B CN114317508 B CN 114317508B
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- mutant
- halohydrin dehalogenase
- reaction
- halohydrin
- epichlorohydrin
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Abstract
本发明公开了一种卤醇脱卤酶突变体、工程菌及其应用,其特征在于所述突变体是将SEQ ID NO.2所示氨基酸序列第105位、第134位、第136位、第137位、第178位或第186位进行单突变或多突变获得。本发明利用水‑有机双相缓冲溶液体系(乙酸乙酯:磷酸钠缓冲液=6:4)使得卤醇脱卤酶突变体的产物(S)‑环氧氯丙烷的e.e.值超过99%,湿细胞酶活分别从23.56U/mL提高至100‑600U/mL,产率达15.9%‑80.2%。The invention discloses a halohydrin dehalogenase mutant, an engineering bacterium and applications thereof, which are characterized in that the mutant is the 105th, 134th, 136th, 136th, 136th, The 137th, 178th or 186th position is obtained by single or multiple mutations. The present invention utilizes water-organic biphasic buffer solution system (ethyl acetate: sodium phosphate buffer = 6:4) to make the e.e. value of the product (S)-epichlorohydrin of the halohydrin dehalogenase mutant exceed 99%, The wet cell enzyme activity increased from 23.56U/mL to 100-600U/mL, and the yield reached 15.9%-80.2%.
Description
(一)技术领域(1) Technical field
本发明涉及一种卤醇脱卤酶突变体,以及其在制备(S)-环氧氯丙烷中的应用。The invention relates to a halohydrin dehalogenase mutant and its application in preparing (S)-epichlorohydrin.
(二)背景技术(2) Background technology
卤代醇化合物作为医药化工等领域的重要中间体,在有机合成与医药研发等方面具有广泛的应用,然而自然界中大部分卤化物因其降解能力较差、毒性大和潜在的致癌性等缺点,已成为主要的环境污染物之一,因此人们越来越重视对其的降解处理以期减轻该类有机卤化物对自然环境的污染和对人类潜在的威胁。利用传统的物理化学方法不仅反应条件苛刻,而且很有可能带来二次污染,而利用生物降解方法具有环境无污染、反应条件温和;常温、常压、中性、或者接近中性pH等、反应步骤简单、产物易分离并且在操作过程中对设备性能要求不高,投入资金相对较少,生产过程安全等突出优点,已成为目前卤代化合物降解的主要发展趋势。As important intermediates in pharmaceutical and chemical industries, halohydrin compounds are widely used in organic synthesis and pharmaceutical research and development. However, most halides in nature have disadvantages such as poor degradation ability, high toxicity and potential carcinogenicity. It has become one of the main environmental pollutants, so people pay more and more attention to its degradation treatment in order to reduce the pollution of this kind of organic halides to the natural environment and the potential threat to human beings. The use of traditional physical and chemical methods not only has harsh reaction conditions, but also may cause secondary pollution, while the use of biodegradation methods has environmental pollution-free, mild reaction conditions; normal temperature, normal pressure, neutral, or close to neutral pH, etc., The advantages of simple reaction steps, easy separation of products, low requirements on equipment performance during operation, relatively small investment, and safe production process have become the main development trend of the degradation of halogenated compounds.
在微生物中发现了一系列能降解这类有毒物质的酶,其中一种关键的酶就是卤醇脱卤酶。卤醇脱卤酶(Halohydrin dehalogenase,EC 4.5.1.X,简称HHDH)是一类通过分子内亲核取代机制催化邻卤醇转化为环氧化物的脱卤酶,可以高效高选择性的催化环氧化物和邻卤醇之间的转化,因而可以用来合成具有光学纯的环氧化物,具有传统化学合成法无法比拟的优越感。A series of enzymes that can degrade such toxic substances have been found in microorganisms, and one of the key enzymes is halohydrin dehalogenase. Halohydrin dehalogenase (EC 4.5.1.X, HHDH for short) is a class of dehalogenases that catalyze the conversion of ortho-halohydrins into epoxides through an intramolecular nucleophilic substitution mechanism, and can catalyze with high efficiency and high selectivity. The conversion between epoxides and o-halohydrins can therefore be used to synthesize optically pure epoxides, which has a sense of superiority that cannot be compared with traditional chemical synthesis methods.
卤醇脱卤酶不仅可以催化碳-卤键的断裂,形成环氧化物,同时还可以进行相应的逆反应,并且在非自然亲核试剂的存在下,例如N3-、CN-、NO2-、SCN-、OCN-和HCOO-等,催化环氧化物的开环,形成一系列新的C-C、C-O和C-N键等,这为制备各类光学纯的β-取代醇和环氧化物提供了一个高效经济环保的方法,在药物研发和有机合成等方面具有较高的应用价值。自1968年Castro首次在以2,3-二溴丙醇作为唯一的碳源而生存的黄杆菌属(Flavobaterium sp.)菌株中发现后科学家们陆陆续续发现了多种卤醇脱卤酶。到目前为止,部分已知的卤醇脱卤酶已被克隆、测序及进行体外重组表达研究。近年来,随着生物信息学的发展,对卤醇脱卤酶的研究逐渐加深。在卤醇脱卤酶催化合成手性环氧氯丙烷(ECH)合成过程中,因其催化效率低、立体选择性不高,且当前在卤醇脱卤酶催化反应的过程中,由于手性ECH存在消旋或降解,导致产物光学纯度低,从而限制了其应用与工业化生产。Halohydrin dehalogenase can not only catalyze the cleavage of carbon-halogen bonds to form epoxides, but also carry out the corresponding reverse reaction, and in the presence of unnatural nucleophiles, such as N3-, CN-, NO2-, SCN -, OCN- and HCOO-, etc., catalyze the ring opening of epoxides to form a series of new C-C, C-O and C-N bonds, etc., which provides an efficient and economical method for the preparation of various optically pure β-substituted alcohols and epoxides The method is environmentally friendly and has high application value in drug development and organic synthesis. Since Castro was first discovered in 1968 in a strain of Flavobaterium sp. that uses 2,3-dibromopropanol as the sole carbon source, scientists have successively discovered a variety of halohydrin dehalogenases. So far, some known halohydrin dehalogenases have been cloned, sequenced and studied in vitro recombinant expression. In recent years, with the development of bioinformatics, the research on halohydrin dehalogenase has gradually deepened. In the synthesis process of chiral epichlorohydrin (ECH) catalyzed by halohydrin dehalogenase, because of its low catalytic efficiency and low stereoselectivity, and currently in the process of halohydrin dehalogenase catalyzed reaction, due to chiral Racemization or degradation of ECH leads to low optical purity of the product, which limits its application and industrial production.
(三)发明内容(3) Contents of the invention
本发明所要解决的问题是克服现有方法中卤醇脱卤酶催化的e.e.值不高及产物消旋等问题,提供了一种来源于放射性农杆菌的卤醇脱卤酶突变体及以该突变体在双相缓冲溶液中制备高产率和e.e.值的(S)-ECH的应用。为了更好的实现卤醇脱卤酶工业化生产应用,本发明通过对酶活性中心及Loop环附近的关键氨基酸进行改造,获得了能够在双相缓冲溶液中(有机相:水相=6:4)催化底物1,3-DCP得到高立体选择性和高产率的突变体。The problem to be solved by the present invention is to overcome the problems of low e.e. value and product racemization of halohydrin dehalogenase catalysis in the existing method, and provide a halohydrin dehalogenase mutant derived from radioactive Agrobacterium and the Application of mutants to prepare (S)-ECH with high yield and e.e. value in biphasic buffer solution. In order to better realize the industrial production and application of halohydrin dehalogenase, the present invention obtains the enzyme activity center and key amino acids near the Loop ring, which can be used in a biphasic buffer solution (organic phase: aqueous phase = 6:4 ) catalyzed the substrate 1,3-DCP to obtain mutants with high stereoselectivity and high yield.
本发明采用的技术方案是:The technical scheme adopted in the present invention is:
本发明提供一种卤醇脱卤酶突变体,所述突变体是将SEQ ID NO.2所示氨基酸序列第105位、第134位、第136位、第137位、第178位或第186位进行单突变或多突变获得。The present invention provides a halohydrin dehalogenase mutant, the mutant is the 105th, 134th, 136th, 137th, 178th or 186th amino acid sequence shown in SEQ ID NO.2 single or multiple mutations.
进一步,所述卤醇脱卤酶突变体是将SEQ ID NO.2所示氨基酸序列第105位的亮氨酸突变为天冬氨酸(L105N)、第134位丙氨酸突变为亮氨酸(A134L)、第136位脯氨酸突变为天冬酰胺(P136N)、第137位苯丙氨酸突变为丝氨酸(F137S)、第178位酪氨酸突变为蛋氨酸(Y178M)、第186位酪氨酸突变为天冬酰胺(Y186N)、第136位脯氨酸突变为天冬酰胺且第137位苯丙氨酸突变为丝氨酸(P136N/F137S)。Further, the halohydrin dehalogenase mutant is mutated from leucine at position 105 to aspartic acid (L105N) and alanine at position 134 into leucine in the amino acid sequence shown in SEQ ID NO.2 (A134L), the 136th proline mutation to asparagine (P136N), the 137th phenylalanine mutation to serine (F137S), the 178th tyrosine mutation to methionine (Y178M), the 186th tyrosine mutation Amino acid was mutated to asparagine (Y186N), proline at position 136 was mutated to asparagine and phenylalanine at position 137 was mutated to serine (P136N/F137S).
本发明还涉及所述卤醇脱卤酶突变体编码基因构建的重组载体及重组载体转化制备的重组基因工程菌。本发明的重组载体没有限制,只要其可以在原核和/或真核细胞的各种宿主细胞中保持其复制或自主复制,所述载体可为本领域常规的各种载体,如各种质粒、噬菌体或病毒载体等,优选以pET28a(+)质粒为表达载体,以大肠杆菌为表达宿主(大肠杆菌BL21细胞或大肠杆菌DH5α)。The present invention also relates to the recombinant vector constructed by the gene encoding the halohydrin dehalogenase mutant and the recombinant genetically engineered bacteria prepared by transformation of the recombinant vector. The recombinant vector of the present invention is not limited, as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and/or eukaryotic cells, the vector can be various conventional vectors in the art, such as various plasmids, For phage or viral vectors, the pET28a(+) plasmid is preferably used as the expression vector, and Escherichia coli is used as the expression host (Escherichia coli BL21 cells or Escherichia coli DH5α).
本发明还提供一种所述卤醇脱卤酶突变体在催化1,3-二氯-2-丙醇(1,3-DCP)合成(S)-环氧氯丙烷((S)-ECH)中的应用,所述的应用方法为:以含卤醇脱卤酶突变体编码基因的重组基因工程菌经发酵培养获得的湿菌体作为催化剂,以1,3-二氯-2-丙醇为底物,以pH 7.0-10.0(优选pH 8.0)的水-有机溶剂双相体系为反应介质构成反应体系,在300-700rpm(优选为600rpm),37℃条件下进行反应,反应结束后,获得含(S)-环氧氯丙烷的反应液,将反应液分离纯化,获得(S)-环氧氯丙烷;所述水-有机溶剂双相体系是由体积比6:4的乙酸乙酯和200mM、pH 8.0磷酸钠缓冲液组成。The present invention also provides a kind of said halohydrin dehalogenase mutant in catalyzing 1,3-dichloro-2-propanol (1,3-DCP) to synthesize (S)-epichlorohydrin ((S)-ECH ), the application method is as follows: the wet thallus obtained by fermenting and culturing the recombinant genetically engineered bacteria containing the halohydrin dehalogenase mutant coding gene is used as a catalyst, and 1,3-dichloro-2-propane Alcohol is used as the substrate, and the water-organic solvent two-phase system with pH 7.0-10.0 (preferably pH 8.0) is used as the reaction medium to form the reaction system. The reaction is carried out at 300-700rpm (preferably 600rpm) and 37°C. After the reaction , to obtain a reaction solution containing (S)-epichlorohydrin, the reaction solution is separated and purified to obtain (S)-epichlorohydrin; the water-organic solvent two-phase system is composed of ethyl acetate with a volume ratio of 6:4 ester and 200mM sodium phosphate buffer, pH 8.0.
进一步,所述催化剂用量以湿菌体的重量计为10-40g/L缓冲液(优选20g/L),所述底物初始加入浓度为10-80mM(优选20mM)。Further, the amount of the catalyst is 10-40g/L buffer solution (preferably 20g/L) based on the weight of wet bacteria, and the initial concentration of the substrate is 10-80mM (preferably 20mM).
进一步,所述湿菌体按如下方法制备:将含有卤醇脱卤酶突变体编码基因的重组工程菌接种到含有终浓度为50μg/mL卡那霉素的LB培养液中,在37℃条件下培养8h,获得种子液;然后将种子液以体积浓度2%的接种量接种至无菌的含有终浓度50μg/mL卡那霉素的LB液体培养基中,在37℃条件下培养约1.5-2.5h,使得菌体浓度度OD600为0.4-0.8,再向培养液中加入终浓度为0.1-1.0mM(优选0.1mM)的异丙基硫代-β-D-半乳糖苷(IPTG),28℃诱导表达12h后,4℃、4000rpm离心10-20min,收集湿菌体;LB液体培养基:蛋白胨10g/L,酵母膏5g/L,氯化钠10g/L,溶剂为去离子水,pH 8.0。Further, the wet bacterium was prepared as follows: Inoculate the recombinant engineered bacteria containing the gene encoding the halohydrin dehalogenase mutant into the LB culture solution containing the final concentration of 50 μg/mL kanamycin, and inoculate it at 37°C. Cultivate for 8 hours to obtain seed solution; then inoculate the seed solution with an inoculum volume concentration of 2% into sterile LB liquid medium containing a final concentration of 50 μg/mL kanamycin, and cultivate at 37°C for about 1.5 -2.5h, so that the bacterium concentration OD 600 is 0.4-0.8, then add isopropylthio-β-D-galactoside (IPTG) with a final concentration of 0.1-1.0mM (preferably 0.1mM) ), after inducing expression at 28°C for 12h, centrifuge at 4°C and 4000rpm for 10-20min to collect the wet cells; LB liquid medium: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, solvent is deionized water, pH 8.0.
本发明所述的卤醇脱卤酶突变体可以以全细胞形式完成催化,也可以通过细胞破碎的粗酶液或者完全破碎的纯酶进行催化。此外,还可以利用特定的固定化技术将卤醇脱卤酶制备成固定化酶或者固定化细胞形式的酶。The halohydrin dehalogenase mutants of the present invention can be catalyzed in the form of whole cells, or can be catalyzed by the crude enzyme solution of broken cells or completely broken pure enzymes. In addition, the halohydrin dehalogenase can also be prepared as an immobilized enzyme or an enzyme in the form of immobilized cells by using a specific immobilization technique.
与现有技术相比,本发明有益效果主要体现在:Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:
本发明分别利用定向进化和半理性设计方法对来源于放射性农杆菌的卤醇脱卤酶HheC进行改造,发现第105位、第134位、第136位、第137位、第176位、第186位是影响酶活的关键位点:分别对这三个位点利用定点饱和技术,通过96孔板筛选的到6个单突变体,L105N、A134L、P136N、F137S、Y178M、Y186N的比酶活分别为251.19U/mL、176.86U/mL、507.19U/mL,613.45U/mL、478.63U/mL,109.37U/mL,是原始酶的10.7倍、7.5倍、21.5倍、26.0倍、20.3倍、4.6倍。The present invention uses directed evolution and semi-rational design methods to transform the halohydrin dehalogenase HheC derived from radioactive Agrobacterium, and finds the 105th, 134th, 136th, 137th, 176th, and 186th The site is the key site that affects the enzyme activity: the specific enzyme activities of L105N, A134L, P136N, F137S, Y178M, and Y186N were screened through 96-well plates for the three sites using the site-fixed saturation technique. 251.19U/mL, 176.86U/mL, 507.19U/mL, 613.45U/mL, 478.63U/mL, 109.37U/mL, 10.7 times, 7.5 times, 21.5 times, 26.0 times, 20.3 times of the original enzyme , 4.6 times.
本发明还将所获得6个突变位点进行两两随机组合,筛选得到突变体P136N/F137S的比酶活为481.52U/mL,是原始酶活的20.4倍。In the present invention, the obtained 6 mutation sites are randomly combined in pairs, and the specific enzyme activity of the mutant P136N/F137S obtained by screening is 481.52 U/mL, which is 20.4 times of the original enzyme activity.
本发明利用水-有机双相缓冲溶液体系(乙酸乙酯:磷酸钠缓冲液=6:4)使得卤醇脱卤酶突变体的产物(S)-环氧氯丙烷的e.e.值超过99%,湿细胞酶活分别从23.56U/mL提高至100-600U/mL,产率达15.9%-80.2%。The present invention utilizes the water-organic biphasic buffer solution system (ethyl acetate: sodium phosphate buffer = 6:4) to make the e.e. value of the product (S)-epichlorohydrin of the halohydrin dehalogenase mutant exceed 99%, The wet cell enzyme activity increased from 23.56U/mL to 100-600U/mL, and the yield reached 15.9%-80.2%.
本发明卤醇脱卤酶突变体是在SEQ ID NO.2所示的野生型卤醇脱卤酶HheC的氨基酸基础上,通过定点饱和突变的方法进行改造的,从而改变其氨基酸序列,实现蛋白结构和功能的改变,再通过定向筛选的方法,得到其有上述突变的卤醇脱卤酶突变体,本发明的卤醇脱卤酶突变体具有酶活性大幅度提高的优势,其酶活相对于野生型卤醇脱卤酶HhecC提高了多倍,从而更有利于实现工业化生产。The halohydrin dehalogenase mutant of the present invention is modified on the basis of the amino acid of the wild-type halohydrin dehalogenase HheC shown in SEQ ID NO. Changes in structure and function, and then through the method of directional screening, obtain the halohydrin dehalogenase mutant with the above mutation, the halohydrin dehalogenase mutant of the present invention has the advantage of greatly improving the enzyme activity, and its enzyme activity is relatively The wild-type halohydrin dehalogenase HhecC is increased many times, which is more conducive to the realization of industrial production.
(四)附图说明(4) Description of drawings
图1是底物1,3-DCP的三维结构图。Figure 1 is a three-dimensional structure diagram of the substrate 1,3-DCP.
图2是催化产物经气相检测之后的色谱图。Figure 2 is a chromatogram of the catalytic product after gas phase detection.
(五)具体实施方式(5) Specific implementation methods
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:
LB液体培养基:蛋白胨10g/L,酵母膏5g/L,氯化钠10g/L,溶剂为去离子水,pH8.0。LB liquid medium: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, solvent is deionized water, pH 8.0.
LB平板是在LB液体培养基添加15g/L琼脂。LB plate is added 15g/L agar in LB liquid medium.
实施例1:对卤醇脱卤酶进行同源建模和分子动力学模拟Example 1: Homology Modeling and Molecular Dynamics Simulation of Halohydrin Dehalogenases
以GenBank AAK92099中来源于放射性农杆菌(Agrobacteriumradiobacterstrain AD1)的HheC(PDB ID:1ZO8)三维结构为模板,采用ChemDraw 8.1绘制了1,3-dcp的三维结构,通过分子对接程序按照AutoDock 4.6.2程序对HheC和底物1,3-dcp进行对接。以上所有结构均采用PyMOL程序进行可视化分析,发现第105位、第134位、第136位、第137位、第178位、第186位是影响酶活的关键位点(图1)。Using the three-dimensional structure of HheC (PDB ID: 1ZO8) derived from Agrobacterium radiobacterium (Agrobacterium radiobacterstrain AD1) in GenBank AAK92099 as a template, the three-dimensional structure of 1,3-dcp was drawn using ChemDraw 8.1, and the molecular docking program followed the AutoDock 4.6.2 program Docking of HheC and substrate 1,3-dcp. All the above structures were visually analyzed using the PyMOL program, and it was found that the 105th, 134th, 136th, 137th, 178th, and 186th positions are key sites that affect the enzyme activity (Figure 1).
实施例2:卤醇脱卤酶定点饱和文库的构建Example 2: Construction of Halohydrin Dehalogenase Site-Directed Saturation Library
根据实施例1结论,以GenBank中收录的野生型卤醇脱卤酶HheC的基因序列(核苷酸序列为SEQ ID NO.1所示,氨基酸序列为SEQ ID NO.2所示)为基础设计引物(见表1)。分别以引物L105X-F/L105X-R、A134X-F/A134X-R、P136X-F/P136X-R、F137X-F/F137X-R、Y178X-F/Y178X-R、T186X-F/T186X-R对亲本HheC基因(核苷酸序列为SEQ ID NO.1),进行定点饱和突变,以pET-28b(+)为表达载体,分别获得带有目的基因的突变质粒,并将带有目的基因的突变质粒转化至E.coli BL21(DE3)中,分别获得含卤醇脱卤酶突变基因的重组菌的突变体,分别为E.coli BL21(DE3)-L105X(记为突变体L105X)、E.coli BL21(DE3)-A134X(记为突变体A134X)、E.coli BL21(DE3)-P136X(记为突变体P136X)、E.coli BL21(DE3)-F137X(记为突变体F137X)、E.coli BL21(DE3)-Y178X(记为突变体Y178X)、E.coli BL21(DE3)-Y186X(记为突变体Y186X)。According to the conclusion of Example 1, design based on the gene sequence (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO.2) of the wild-type halohydrin dehalogenase HheC included in GenBank Primers (see Table 1). Respectively with primers L105X-F/L105X-R, A134X-F/A134X-R, P136X-F/P136X-R, F137X-F/F137X-R, Y178X-F/Y178X-R, T186X-F/T186X-R The parental HheC gene (nucleotide sequence is SEQ ID NO.1) was subjected to site-directed saturation mutation, and pET-28b(+) was used as the expression vector to obtain mutant plasmids with the target gene, and the target gene The mutant plasmid was transformed into E.coli BL21(DE3), and the mutants of recombinant bacteria containing the halohydrin dehalogenase mutant gene were respectively obtained, which were respectively E.coli BL21(DE3)-L105X (referred to as mutant L105X), E. .coli BL21(DE3)-A134X (referred to as mutant A134X), E.coli BL21(DE3)-P136X (referred to as mutant P136X), E.coli BL21(DE3)-F137X (referred to as mutant F137X), E. coli BL21(DE3)-Y178X (referred to as mutant Y178X), E. coli BL21(DE3)-Y186X (referred to as mutant Y186X).
SEQ ID NO.2:SEQ ID NO.2:
MASTAIVTNVKHFGGMGSALRLSEAGHTVACHDESFKQKDELEAFAETYPQLKPMSEQEPAELIEAVTSAYGQVDVLVSNDIFAPEFQPIDKYAVEDYRGAVEALQIRPFALVNAVASQMKKRKSGHIIFITSATPFGPWKELSTYTSARAGACTLANALSKELGEYNIPVFAIGSNYLHSEDSPYFYPTEPWKTNPEHVAHVKKVTALQRLGTQKELGELVAFLASGSCDYLTGQVFWLAGGFPMIERPPGMPELE。MASTAIVTNVKHFGGMGSALRLSEAGHTVACHDESFKQKDELEAFEATYPQLKPMSEQEPAELIEAVTSAYGQVDVLVSNDIFAPEFQPIDKYAVEDYRGAVEALQIRPFALVNAVASQMKKRKSGHIFITSATPFGPWKELSTYTSARAGACTLANALSKELGEYNIPVFAIGSNYLHSEDSPY FYPTEPWKTNPEHVAHVKKVTALQRLGTQKELGELVAFLASGSCDYLTGQVFWLAGGFPMIERPPGMPELE.
表1:卤醇脱卤酶定点饱和突变文库构建的引物设计表Table 1: Primer design table for construction of halohydrin dehalogenase site-directed saturation mutation library
PCR扩增体系为:50μL反应体系:The PCR amplification system is: 50μL reaction system:
2×phantamax Buffera:25μL;2×phantamax Buffer: 25μL;
dNTP Mix(10Mm each):1μL;dNTP Mix (10Mm each): 1μL;
上游引物(50μM):2μL;Upstream primer (50 μM): 2 μL;
下游引物(50μM):2μL;Downstream primer (50 μM): 2 μL;
Phanta Max super-Fidelity DNA Polymerase:1μL;Phanta Max super-Fidelity DNA Polymerase: 1μL;
模板DNA(质粒):0.5μL;Template DNA (plasmid): 0.5 μL;
ddH2O:18.5μL; ddH2O : 18.5 μL;
PCR反应条件为:预变性95℃10min,随后进入温度循环95℃30s,55℃30s,72℃6min,共30个循环,最后72℃延伸10min,终止温度为4℃。PCR产物经1%琼脂糖凝胶电泳分析验证后,在PCR产物中加入1μL DpnI、5μL buffer,37℃消化2h去除模板质粒DNA,在65℃灭活10min后采用PCR cleanup Kit对产物进行纯化后,转化至E.coli BL21(DE3)感受态细胞,涂布含卡那霉素(50μg/mL)的LB平板,37℃培养过夜,得到卤醇脱卤酶的突变文库,此时LB平板上呈现出许多不同突变的单菌落,这些单菌落用于后续的突变文库的筛选。The PCR reaction conditions were as follows: pre-denaturation at 95°C for 10 min, followed by a temperature cycle of 95°C for 30 s, 55°C for 30 s, and 72°C for 6 min, a total of 30 cycles, and finally an extension at 72°C for 10 min, with a termination temperature of 4°C. After the PCR product was verified by 1% agarose gel electrophoresis analysis, 1 μL DpnI and 5 μL buffer were added to the PCR product, digested at 37°C for 2 hours to remove the template plasmid DNA, and inactivated at 65°C for 10 minutes to purify the product with PCR cleanup Kit , transformed into E.coli BL21(DE3) competent cells, spread on LB plates containing kanamycin (50 μg/mL), and culture overnight at 37°C to obtain a mutant library of halohydrin dehalogenase, at this time on LB plates Single colonies exhibiting many different mutations were used for subsequent screening of mutant libraries.
同样方法构建亲本菌株:E.coli BL21(DE3)-HheC。The same method was used to construct the parental strain: E.coli BL21(DE3)-HheC.
实施例3:卤醇脱卤酶突变文库的筛选Example 3: Screening of Halohydrin Dehalogenase Mutant Library
1、卤醇脱卤酶突变文库的筛选以突变前野生型HheC为参考,挑取单菌落克隆子(由实施例2构建的突变文库)至2mL深96孔板中进行培养,事先加入含终浓度50μg/mL卡那霉素的600μL的LB培养液,同时挑取2个亲本菌株在96孔板的最后2个孔中作为对照。将2mL的96孔板置于37℃条件下培养8h作为种子液,然后取200μL种子液加入新的无菌的含有终浓度50μg/mL卡那霉素及0.1mM的IPTG的600μL的LB培养液中,含有种子液的28℃诱导表达12h后,4000rpm离心20min,弃上清,收集湿菌体,进行下一步的高通量筛选。1. Screening of the halohydrin dehalogenase mutant library Taking wild-type HheC before mutation as a reference, pick a single colony clone (the mutant library constructed in Example 2) and culture it in a 2mL deep 96-well plate. 600 μL of LB culture solution with a concentration of 50 μg/mL kanamycin, and at the same time pick two parental strains in the last two wells of a 96-well plate as controls. Place a 2 mL 96-well plate at 37°C for 8 hours as a seed solution, then add 200 μL of the seed solution to a new sterile 600 μL LB culture solution containing a final concentration of 50 μg/mL kanamycin and 0.1 mM IPTG , after induction of expression at 28°C containing the seed solution for 12 hours, centrifuge at 4000 rpm for 20 minutes, discard the supernatant, collect the wet cells, and carry out the next step of high-throughput screening.
2、根据卤醇脱卤酶催化反应的特点,高通量筛选的方法可通过检测反应体系中H+的浓度来对酶的活性进行测定,卤醇脱卤酶在催化邻卤醇形成环氧化物的同时,会释放1分子的H+,可以在反应缓冲液体系中加入pH指示剂,根据pH指示剂的颜色的变化,因定性或定量卤醇脱卤酶的活性。另外一种方法是对卤醇脱卤酶反应中生成的氯离子进行定量分析。2. According to the characteristics of the catalytic reaction of halohydrin dehalogenase, the high-throughput screening method can measure the activity of the enzyme by detecting the concentration of H+ in the reaction system. At the same time, a molecule of H+ will be released, and a pH indicator can be added to the reaction buffer system. According to the color change of the pH indicator, the activity of halohydrin dehalogenase can be qualitatively or quantitatively determined. Another method is the quantitative analysis of chloride ions generated in the halohydrin dehalogenase reaction.
优选的,本实施例根据pH指标为基础的比色反应在水-有机缓冲体系中更易筛选出高活性和对映体选择性的阳性菌,具体为:Preferably, in this embodiment, according to the colorimetric reaction based on the pH index, it is easier to screen positive bacteria with high activity and enantioselectivity in the water-organic buffer system, specifically:
步骤1中的96孔板离心收集的湿菌体在每孔中加入200μL的PB缓冲液(pH=8.0、200mM)制成细胞悬浮液。比色反应在96孔石英板中进行,反应体系为200μL:80μL的细胞悬浮液,终浓度20mM 1,3-DCP及终浓度0.09mg/ml溴百里香酚蓝,以体积比6:4的乙酸乙酯和磷酸钠缓冲液(200mM、pH=8.0)补足至200μL,混合后,在37℃条件下保温20min,反应相同时间,根据反应液的颜色(由蓝色到黄色)的变化速度确定卤醇脱卤酶的酶活。在步骤1筛菌过程中,每个突变位点构成的突变文库共筛约400个单菌落,在6个突变位点构成的突变文库中通过高通量筛选的方法共筛选2400个单菌落,在相同时间内,根据pH指示剂的颜色变化快慢及深浅,与亲本HheC对照组进行比较发现,酶活比亲本酶活高的反应液的颜色比亲本反应液所呈现的颜色更黄,从而初筛获得7个活性较高的含卤醇脱卤酶突变基因的重组菌的突变体,经过测序得到分别为E.coli BL21(DE3)-L105N(记为突变体L105N,核苷酸序列为SEQ ID NO.3所示)、E.coli BL21(DE3)-A134L(记为突变体A134L,核苷酸序列为SEQID NO.4所示)、E.coli BL21(DE3)-P136N(记为突变体P136N,核苷酸序列为SEQ ID NO.5所示)、E.coli BL21(DE3)-F137S(记为突变体F137S,核苷酸序列为SEQ ID NO.6所示)、E.coli BL21(DE3)-Y178M(记为突变体Y178M,核苷酸序列为SEQ ID NO.7所示)、E.coliBL21(DE3)-Y186N(记为突变体Y186N,核苷酸序列为SEQ ID NO.8所示)。Add 200 μL of PB buffer (pH=8.0, 200 mM) to each well of the wet bacteria collected by centrifugation of the 96-well plate in step 1 to prepare a cell suspension. The colorimetric reaction was carried out in a 96-well quartz plate, and the reaction system was 200 μL: 80 μL of cell suspension, a final concentration of 20 mM 1,3-DCP and a final concentration of 0.09 mg/ml bromothymol blue, and acetic acid with a volume ratio of 6:4 Ethyl ester and sodium phosphate buffer (200mM, pH=8.0) were added to 200μL, after mixing, they were incubated at 37°C for 20min, and reacted for the same time, according to the change speed of the color of the reaction solution (from blue to yellow). Alcohol dehalogenase activity. In step 1, during the bacterial screening process, a total of about 400 single colonies were screened from the mutant library composed of each mutation site, and a total of 2,400 single colonies were screened by high-throughput screening in the mutant library composed of 6 mutation sites. In the same time period, according to the color change speed and depth of the pH indicator, compared with the parental HheC control group, it was found that the color of the reaction solution with higher enzyme activity than the parental reaction solution was more yellow than that of the parental reaction solution, thus initially The mutants of 7 highly active recombinant bacteria containing halohydrin dehalogenase mutant genes were obtained by screening, and obtained by sequencing were respectively E.coli BL21(DE3)-L105N (referred to as mutant L105N, and the nucleotide sequence is SEQ ID NO.3), E.coli BL21(DE3)-A134L (referred to as mutant A134L, nucleotide sequence shown in SEQID NO.4), E.coli BL21(DE3)-P136N (denoted as mutant Body P136N, the nucleotide sequence is shown in SEQ ID NO.5), E.coli BL21(DE3)-F137S (denoted as mutant F137S, the nucleotide sequence is shown in SEQ ID NO.6), E.coli BL21(DE3)-Y178M (referred to as mutant Y178M, the nucleotide sequence is shown in SEQ ID NO.7), E.coliBL21(DE3)-Y186N (referred to as mutant Y186N, the nucleotide sequence is shown in SEQ ID NO. .8).
实施例4:卤醇脱卤酶双突变体的构建Example 4: Construction of Halohydrin Dehalogenase Double Mutants
将通过高通量筛选获得的6个卤醇脱卤酶突变体L105N、突变体A134L、突变体P136N、突变体F137S、突变体Y178M、突变体Y186N进行两两组合构建卤醇脱卤酶双突变文库,两两组合共构建15种可能的双突变文库,PCR反应条件及构建方法同实施例2,双突变文库的初筛方法同实施例3,在实施例3的筛菌过程中,每个双突变文库共筛约400个单菌落,在构建的15种可能的双突变文库中共筛约6000个单菌落,在相同时间内,根据pH指示剂的颜色变化快慢及深浅,与亲本HheC对照组进行比较发现,酶活比亲本酶活高的反应液的颜色比亲本反应液所呈现的颜色更黄,从而获得1个活性较高的双突变体,经过测序得到E.coli BL21(DE3)-P136N/F137S(记为突变体P136N/F137S,核苷酸序列为SEQ ID NO.9所示)。The six halohydrin dehalogenase mutants L105N, mutant A134L, mutant P136N, mutant F137S, mutant Y178M, and mutant Y186N obtained through high-throughput screening were combined in pairs to construct double mutants of halohydrin dehalogenase Libraries, 15 kinds of possible double mutation libraries were constructed in pairs. The PCR reaction conditions and construction method were the same as in Example 2. The primary screening method of the double mutation library was the same as in Example 3. In the screening process of Example 3, each A total of about 400 single colonies were screened from the double mutation library, and about 6,000 single colonies were screened from the 15 possible double mutation libraries constructed. In the same time period, according to the color change speed and depth of the pH indicator, it was compared with the parental HheC control group. After comparison, it was found that the color of the reaction solution with higher enzyme activity than that of the parent was more yellow than that of the parent reaction solution, thus obtaining a double mutant with higher activity, which was sequenced to obtain E.coli BL21(DE3)- P136N/F137S (denoted as mutant P136N/F137S, nucleotide sequence shown in SEQ ID NO.9).
实施例5:含卤醇脱卤酶及其突变体的重组菌菌体的制备Example 5: Preparation of recombinant bacterial cells containing halohydrin dehalogenase and its mutants
分别将实施例3和实施例4筛选的突变体菌株接种到含有终浓度为50μg/mL卡那霉素的LB液体培养基中,在37℃条件下培养12h,获得种子液;在以体积浓度为2%的接种量将新鲜的种子液接种到新鲜的含有终浓度为50μg/mL卡那霉素的LB液体培养基中,在37℃条件下培养约1.5-2.5h,使得菌体浓度度OD600为0.4-0.8,再向培养液中加入终浓度为0.1mM的异丙基硫代-β-D-半乳糖苷(IPTG),在28℃诱导表达12h后,4℃、8000rpm离心10min,收集重组细胞湿菌体,用于催化1,3-DCP不对称脱卤反应所需要的湿菌体。The mutant strains screened in Example 3 and Example 4 were inoculated into LB liquid medium containing kanamycin at a final concentration of 50 μg/mL, and cultivated at 37°C for 12 hours to obtain seed liquid; Inoculate fresh seed liquid into fresh LB liquid medium containing kanamycin with a final concentration of 50 μg/mL at an inoculum size of 2%, and cultivate at 37°C for about 1.5-2.5h, so that the cell concentration OD 600 is 0.4-0.8, then add isopropylthio-β-D-galactoside (IPTG) at a final concentration of 0.1mM to the culture medium, induce expression at 28°C for 12h, then centrifuge at 4°C, 8000rpm for 10min , to collect the wet cells of the recombinant cells, which are used to catalyze the wet cells required for the asymmetric dehalogenation reaction of 1,3-DCP.
亲本湿菌体制备方法及条件同突变体湿菌体。The preparation method and conditions of the parental wet cells are the same as those of the mutant wet cells.
实施例6:卤醇脱卤酶不对称催化合成(S)-ECH的活性比较Example 6: Activity Comparison of Halohydrin Dehalogenase Asymmetric Catalytic Synthesis of (S)-ECH
卤醇脱卤酶酶活单位(U)定义:在37℃、pH 8.0条件下,1min内催化底物(1,3-DCP)生成1mmoL产物手性环氧氯丙烷所需要的菌体量定义为1U。Halohydrin dehalogenase enzyme activity unit (U) definition: under the conditions of 37°C and pH 8.0, the definition of the amount of bacteria required to catalyze the substrate (1,3-DCP) to generate 1mmoL product chiral epichlorohydrin within 1min 1U.
反应体系:终浓度20mM 1,3-DCP,终浓度20g/L的实施例5制备的湿菌体,0.2M、pH8.0磷酸盐缓冲液与乙酸乙酯以体积比6:4混合液为反应介质10mL构成反应体系。在37℃,600rpm反应30min,取500μL反应液加入1mL乙酸乙酯的进行萃取,12000rpm离心1min,取有机相用无水硫酸钠干燥后,气相检测手性环氧氯丙烷产物峰面积以及残留底物1,3-dcp峰面积(图2),通过产物和底物的标准曲线计算产物生产量和底物消耗量,根据酶活定义,进行酶活的计算。产物(S)-ECH标准曲线方程为:Y=15.103X-0.5779(R2=0.9993);(R)-ECH标准曲线方程为:Y=15.334X-0.9597(R2=0.9984);底物1,3-dcp标准曲线方程为:Y=14.869X+9.6286(R2=0.9919)。Reaction system: final concentration of 20mM 1,3-DCP, wet bacteria prepared in Example 5 with a final concentration of 20g/L, 0.2M, pH8.0 phosphate buffer and ethyl acetate at a volume ratio of 6:4 10 mL of the reaction medium constitutes the reaction system. React at 37°C and 600rpm for 30min, take 500μL of the reaction solution and add 1mL of ethyl acetate for extraction, centrifuge at 12000rpm for 1min, take the organic phase and dry it with anhydrous sodium sulfate, and detect the peak area of the chiral epichlorohydrin product and the residual bottom by gas phase Product 1,3-dcp peak area (Figure 2), product production and substrate consumption were calculated by the standard curve of product and substrate, and enzyme activity was calculated according to the definition of enzyme activity. The product (S)-ECH standard curve equation is: Y=15.103X-0.5779 (R 2 =0.9993); (R)-ECH standard curve equation is: Y=15.334X-0.9597 (R 2 =0.9984); substrate 1 , The equation of the 3-dcp standard curve is: Y=14.869X+9.6286 (R 2 =0.9919).
手性环氧氯丙烷的检测方法:采用Agilent GC-7890A系统,色谱柱类型:BGB-175毛细管柱,色谱条件:柱温90℃,进样室温度220℃,FID检测器220℃。The detection method of chiral epichlorohydrin: adopt Agilent GC-7890A system, column type: BGB-175 capillary column, chromatographic conditions: column temperature 90°C, injection chamber temperature 220°C, FID detector 220°C.
计算(S)-ECH的对映体过剩(e.e.)为[e.e.(%)=(S-R)/(S+R)×100],其中S为(S)-ECH的产率,R为(R)-ECH的产率。所有实验数据均来自三次重复实验。The enantiomeric excess (e.e.) of (S)-ECH was calculated as [e.e.(%)=(S-R)/(S+R)×100], where S was the yield of (S)-ECH and R was (R )-ECH yield. All experimental data are from triplicate experiments.
结果见表2,其酶活分别从23.56U/mL提高至100-600U/mL,(S)-ECH的e.e.值从70%提高至>99%。The results are shown in Table 2. The enzyme activities were increased from 23.56U/mL to 100-600U/mL, and the e.e. value of (S)-ECH was increased from 70% to >99%.
表2:卤醇脱卤酶突变体催化合成(S)-ECH的酶活及e.e.值比较Table 2: Enzyme activity and e.e. value comparison of halohydrin dehalogenase mutants catalyzing the synthesis of (S)-ECH
实施例7:卤醇脱卤酶不对称催化合成(S)-ECHExample 7: Asymmetric Catalytic Synthesis of (S)-ECH by Halohydrin Dehalogenase
由于卤醇脱卤酶在水相中催化反应易发生消旋和水解反应,本实施例采用乙酸乙酯和磷酸钠缓冲液(200mM、pH=8.0)体积比为6:4的两相体系作为反应介质,以1,3-二氯-2-丙醇为底物,以实施例5方法制备的湿菌体为催化剂进行(S)-ECH生物合成的不对称反应,具体如下:Since the halohydrin dehalogenase catalyzes the reaction in the aqueous phase, racemization and hydrolysis reactions are likely to occur. In this example, a two-phase system with a volume ratio of ethyl acetate and sodium phosphate buffer (200mM, pH=8.0) of 6:4 was used as the In the reaction medium, 1,3-dichloro-2-propanol is used as a substrate, and the wet bacteria prepared by the method in Example 5 is used as a catalyst to carry out an asymmetric reaction of (S)-ECH biosynthesis, as follows:
1)反应体系:反应介质10mL,加入终浓度为10mM的底物,终浓度10g/L催化剂组成反应体系,在37℃,600rpm的条件下反应30min,取500μL反应液加1mL乙酸乙酯稀释,12000rpm离心3min,收集上清,经无水硫酸钠干燥后气相分析(检测方法同实施例6),结果见表3。1) Reaction system: 10 mL of reaction medium, add substrate with a final concentration of 10 mM, and a catalyst with a final concentration of 10 g/L to form a reaction system. React for 30 min at 37°C and 600 rpm, take 500 μL of the reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 3.
表3:卤醇脱卤酶突变体催化合成(S)-ECH的产率比较Table 3: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants
2)反应体系:反应介质10mL,加入终浓度为20mM的底物,终浓度10g/L催化剂组成反应体系,在37℃,600rpm的条件下反应30min;取500μL反应液加1mL乙酸乙酯稀释,12000rpm离心3min,收集上清,经无水硫酸钠干燥后气相分析(检测方法同实施例6),结果见表4。2) Reaction system: 10 mL of reaction medium, adding substrate with a final concentration of 20 mM, and catalyst with a final concentration of 10 g/L to form a reaction system, reacted at 37°C and 600 rpm for 30 min; take 500 μL of reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 4.
表4:卤醇脱卤酶突变体催化合成(S)-ECH的产率比较Table 4: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants
3)反应体系:反应介质10mL,加入终浓度为20mM的底物,终浓度20g/L催化剂组成反应体系,在37℃,600rpm的条件下反应30min;取500μL反应液加1mL乙酸乙酯稀释,12000rpm离心3min,收集上清,经无水硫酸钠干燥后气相分析(检测方法同实施例6),结果见表5。3) Reaction system: 10 mL of reaction medium, adding substrate with a final concentration of 20 mM, and a catalyst with a final concentration of 20 g/L to form a reaction system, reacted at 37°C and 600 rpm for 30 min; take 500 μL of the reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 5.
表5:卤醇脱卤酶突变体催化合成(S)-ECH的产率比较Table 5: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants
4)反应体系:反应介质10mL,加入终浓度为30mM的底物,终浓度20g/L催化剂组成反应体系,在37℃,600rpm的条件下反应30min;取500μL反应液加1mL乙酸乙酯稀释,12000rpm离心3min,收集上清,经无水硫酸钠干燥后气相分析(检测方法同实施例6),结果见表6。4) Reaction system: 10 mL of reaction medium, adding substrate with a final concentration of 30 mM, and a catalyst with a final concentration of 20 g/L to form a reaction system, reacted at 37°C and 600 rpm for 30 min; take 500 μL of the reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 6.
表6:卤醇脱卤酶突变体催化合成(S)-ECH的产率比较Table 6: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants
5)反应体系:反应介质10mL,加入终浓度为40mM的底物,终浓度20g/L催化剂组成反应体系,在37℃,600rpm的条件下反应30min;取500μL反应液加1mL乙酸乙酯稀释,12000rpm离心3min,收集上清,经无水硫酸钠干燥后气相分析(检测方法同实施例6),结果见表7。5) Reaction system: 10 mL of reaction medium, adding substrate with a final concentration of 40 mM, and catalyst with a final concentration of 20 g/L to form a reaction system, reacted at 37°C and 600 rpm for 30 min; take 500 μL of reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 7.
表7:卤醇脱卤酶突变体催化合成(S)-ECH的产率比较Table 7: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants
6)反应体系:反应介质10mL,加入终浓度为80mM的底物,终浓度40g/L催化剂组成反应体系,在37℃,600rpm的条件下反应30min;取500μL反应液加1mL乙酸乙酯稀释,12000rpm离心3min,收集上清,经无水硫酸钠干燥后气相分析(检测方法同实施例6),结果见表8。6) Reaction system: 10 mL of reaction medium, adding substrate with a final concentration of 80 mM, and catalyst with a final concentration of 40 g/L to form a reaction system, reacted at 37°C and 600 rpm for 30 min; take 500 μL of reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 8.
表8:卤醇脱卤酶突变体催化合成(S)-ECH的产率比较Table 8: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants
最终,优选的底物浓度为20mM,催化剂用量为20g/L湿菌体,优选突变株为P136N,F137S,Y178M,P136N/F137S。Finally, the preferred substrate concentration is 20mM, the catalyst dosage is 20g/L wet bacteria, and the preferred mutants are P136N, F137S, Y178M, and P136N/F137S.
由于在水相中,卤醇脱卤酶催化合成(S)-ECH过程中,易发生产物自身降解和HHDH的外消旋问题,考虑到工业化生产应用,所以经过筛选得到的突变体,采用水-有机双相缓冲溶液作为反映介质,一方面解决产物在水相中发生自水解问题,另一方面通过改造设计的酶在水-有机双相缓冲溶液以消除外消旋化副反应。Since in the water phase, during the synthesis of (S)-ECH catalyzed by halohydrin dehalogenase, the product self-degradation and the racemization of HHDH are prone to occur. Considering the application of industrial production, the mutants obtained through screening were obtained by using water -The organic biphasic buffer solution is used as the reaction medium, on the one hand, it solves the problem of self-hydrolysis of the product in the aqueous phase, and on the other hand, it eliminates the side reaction of racemization by modifying the designed enzyme in the water-organic biphasic buffer solution.
本发明不受上述具体文字描述的限制。本发明可在权利要求书所概括的范围内作各种改变,这些改变均在本发明的范围之内。The present invention is not limited by the above specific written description. The present invention can be modified within the range outlined by the claims, and these changes are within the scope of the present invention.
序列表sequence listing
<110> 浙江工业大学<110> Zhejiang University of Technology
<120> 一种卤醇脱卤酶突变体、工程菌及其应用<120> A mutant of halohydrin dehalogenase, engineering bacteria and its application
<160> 9<160> 9
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 774<211> 774
<212> DNA<212>DNA
<213> 放射性农杆菌(Agrobacterium radiobacter)<213> Agrobacterium radiobacter
<400> 1<400> 1
atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60
cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120
gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180
gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240
gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300
gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360
aagaaacgta aatctggcca catcatcttc attacctctg caactccatt cggtccgtgg 420aagaaacgta aatctggcca catcatcttc attacctctg caactccat cggtccgtgg 420
aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480
tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540
tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600
gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660
ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720
gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774
<210> 2<210> 2
<211> 257<211> 257
<212> PRT<212> PRT
<213> 放射性农杆菌(Agrobacterium radiobacter)<213> Agrobacterium radiobacter
<400> 2<400> 2
Met Ala Ser Thr Ala Ile Val Thr Asn Val Lys His Phe Gly Gly MetMet Ala Ser Thr Ala Ile Val Thr Asn Val Lys His Phe Gly Gly Met
1 5 10 151 5 10 15
Gly Ser Ala Leu Arg Leu Ser Glu Ala Gly His Thr Val Ala Cys HisGly Ser Ala Leu Arg Leu Ser Glu Ala Gly His Thr Val Ala Cys His
20 25 30 20 25 30
Asp Glu Ser Phe Lys Gln Lys Asp Glu Leu Glu Ala Phe Ala Glu ThrAsp Glu Ser Phe Lys Gln Lys Asp Glu Leu Glu Ala Phe Ala Glu Thr
35 40 45 35 40 45
Tyr Pro Gln Leu Lys Pro Met Ser Glu Gln Glu Pro Ala Glu Leu IleTyr Pro Gln Leu Lys Pro Met Ser Glu Gln Glu Pro Ala Glu Leu Ile
50 55 60 50 55 60
Glu Ala Val Thr Ser Ala Tyr Gly Gln Val Asp Val Leu Val Ser AsnGlu Ala Val Thr Ser Ala Tyr Gly Gln Val Asp Val Leu Val Ser Asn
65 70 75 8065 70 75 80
Asp Ile Phe Ala Pro Glu Phe Gln Pro Ile Asp Lys Tyr Ala Val GluAsp Ile Phe Ala Pro Glu Phe Gln Pro Ile Asp Lys Tyr Ala Val Glu
85 90 95 85 90 95
Asp Tyr Arg Gly Ala Val Glu Ala Leu Gln Ile Arg Pro Phe Ala LeuAsp Tyr Arg Gly Ala Val Glu Ala Leu Gln Ile Arg Pro Phe Ala Leu
100 105 110 100 105 110
Val Asn Ala Val Ala Ser Gln Met Lys Lys Arg Lys Ser Gly His IleVal Asn Ala Val Ala Ser Gln Met Lys Lys Arg Lys Ser Gly His Ile
115 120 125 115 120 125
Ile Phe Ile Thr Ser Ala Thr Pro Phe Gly Pro Trp Lys Glu Leu SerIle Phe Ile Thr Ser Ala Thr Pro Phe Gly Pro Trp Lys Glu Leu Ser
130 135 140 130 135 140
Thr Tyr Thr Ser Ala Arg Ala Gly Ala Cys Thr Leu Ala Asn Ala LeuThr Tyr Thr Ser Ala Arg Ala Gly Ala Cys Thr Leu Ala Asn Ala Leu
145 150 155 160145 150 155 160
Ser Lys Glu Leu Gly Glu Tyr Asn Ile Pro Val Phe Ala Ile Gly SerSer Lys Glu Leu Gly Glu Tyr Asn Ile Pro Val Phe Ala Ile Gly Ser
165 170 175 165 170 175
Asn Tyr Leu His Ser Glu Asp Ser Pro Tyr Phe Tyr Pro Thr Glu ProAsn Tyr Leu His Ser Glu Asp Ser Pro Tyr Phe Tyr Pro Thr Glu Pro
180 185 190 180 185 190
Trp Lys Thr Asn Pro Glu His Val Ala His Val Lys Lys Val Thr AlaTrp Lys Thr Asn Pro Glu His Val Ala His Val Lys Lys Val Thr Ala
195 200 205 195 200 205
Leu Gln Arg Leu Gly Thr Gln Lys Glu Leu Gly Glu Leu Val Ala PheLeu Gln Arg Leu Gly Thr Gln Lys Glu Leu Gly Glu Leu Val Ala Phe
210 215 220 210 215 220
Leu Ala Ser Gly Ser Cys Asp Tyr Leu Thr Gly Gln Val Phe Trp LeuLeu Ala Ser Gly Ser Cys Asp Tyr Leu Thr Gly Gln Val Phe Trp Leu
225 230 235 240225 230 235 240
Ala Gly Gly Phe Pro Met Ile Glu Arg Pro Pro Gly Met Pro Glu LeuAla Gly Gly Phe Pro Met Ile Glu Arg Pro Pro Gly Met Pro Glu Leu
245 250 255 245 250 255
GluGlu
<210> 3<210> 3
<211> 774<211> 774
<212> DNA<212>DNA
<213> 放射性农杆菌(Agrobacterium radiobacter)<213> Agrobacterium radiobacter
<400> 3<400> 3
atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60
cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120
gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180
gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240
gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300
gctgtcgaag ctaatcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360gctgtcgaag ctaatcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360
aagaaacgta aatctggcca catcatcttc attacctctg caactccatt cggtccgtgg 420aagaaacgta aatctggcca catcatcttc attacctctg caactccat cggtccgtgg 420
aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480
tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540
tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600
gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660
ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720
gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774
<210> 4<210> 4
<211> 774<211> 774
<212> DNA<212>DNA
<213> 放射性农杆菌(Agrobacterium radiobacter)<213> Agrobacterium radiobacter
<400> 4<400> 4
atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60
cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120
gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180
gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240
gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300
gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360
aagaaacgta aatctggcca catcatcttc attacctctc tgactccatt cggtccgtgg 420aagaaacgta aatctggcca catcatcttc attacctctc tgactccatt cggtccgtgg 420
aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480
tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540
tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600
gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660
ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720
gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774
<210> 5<210> 5
<211> 774<211> 774
<212> DNA<212>DNA
<213> 放射性农杆菌(Agrobacterium radiobacter)<213> Agrobacterium radiobacter
<400> 5<400> 5
atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60
cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120
gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180
gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240
gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300
gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360
aagaaacgta aatctggcca catcatcttc attacctctg caactaattt cggtccgtgg 420aagaaacgta aatctggcca catcatcttc attacctctg caactaattt cggtccgtgg 420
aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480
tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540
tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600
gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660
ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720
gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774
<210> 6<210> 6
<211> 774<211> 774
<212> DNA<212>DNA
<213> 放射性农杆菌(Agrobacterium radiobacter)<213> Agrobacterium radiobacter
<400> 6<400> 6
atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60
cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120
gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180
gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240
gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300
gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360
aagaaacgta aatctggcca catcatcttc attacctctg caactccaag cggtccgtgg 420aagaaacgta aatctggcca catcatcttc attacctctg caactccaag cggtccgtgg 420
aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480
tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540
tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600
gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660
ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720
gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774
<210> 7<210> 7
<211> 774<211> 774
<212> DNA<212>DNA
<213> 放射性农杆菌(Agrobacterium radiobacter)<213> Agrobacterium radiobacter
<400> 7<400> 7
atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60
cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120
gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180
gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240
gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300
gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360
aagaaacgta aatctggcca catcatcttc attacctctg caactccatt cggtccgtgg 420aagaaacgta aatctggcca catcatcttc attacctctg caactccat cggtccgtgg 420
aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480
tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa catgctgcac 540tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa catgctgcac 540
tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600
gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660
ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720
gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774
<210> 8<210> 8
<211> 774<211> 774
<212> DNA<212>DNA
<213> 放射性农杆菌(Agrobacterium radiobacter)<213> Agrobacterium radiobacter
<400> 8<400> 8
atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60
cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120
gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180
gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240
gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300
gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360
aagaaacgta aatctggcca catcatcttc attacctctg caactccatt cggtccgtgg 420aagaaacgta aatctggcca catcatcttc attacctctg caactccat cggtccgtgg 420
aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480
tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540
tctgaagaca gcccgaattt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600tctgaagaca gcccgaattt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600
gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660
ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720
gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774
<210> 9<210> 9
<211> 774<211> 774
<212> DNA<212>DNA
<213> 放射性农杆菌(Agrobacterium radiobacter)<213> Agrobacterium radiobacter
<400> 9<400> 9
atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60
cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120
gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180
gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240
gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300
gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360
aagaaacgta aatctggcca catcatcttc attacctctg caactaatag cggtccgtgg 420aagaaacgta aatctggcca catcatcttc attacctctg caactaatag cggtccgtgg 420
aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480
tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540
tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600
gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660
ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720
gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774
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