CN106754782B - 一种氧化酶及其应用 - Google Patents

一种氧化酶及其应用 Download PDF

Info

Publication number
CN106754782B
CN106754782B CN201710006551.2A CN201710006551A CN106754782B CN 106754782 B CN106754782 B CN 106754782B CN 201710006551 A CN201710006551 A CN 201710006551A CN 106754782 B CN106754782 B CN 106754782B
Authority
CN
China
Prior art keywords
alpha
ester
acid
leu
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201710006551.2A
Other languages
English (en)
Other versions
CN106754782A (zh
Inventor
蔡宇杰
武旭攀
曹憬
白亚军
郑晓晖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhuohong Chaoyuan Biotechnology Zhengzhou Co ltd
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN201710006551.2A priority Critical patent/CN106754782B/zh
Publication of CN106754782A publication Critical patent/CN106754782A/zh
Application granted granted Critical
Publication of CN106754782B publication Critical patent/CN106754782B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明涉及一种来源于沙门氏菌(Salmonella enterica)的D‑乳酸氧化酶基因的获得及其克隆表达，属于生物工程领域。公开了其底物特异性，同时该D‑乳酸氧化酶可以氧化(R)‑α‑羟酸酯，可应用于光学纯(S)‑α‑羟酸酯的制备。

Description

一种氧化酶及其应用
技术领域
本发明克隆表达了一种D-乳酸氧化酶，并公开了其核苷酸序列和氨基酸序列及酶学性质和应用，属于工业微生物领域。
背景技术
D-乳酸氧化酶(D-lactate oxidase)是一种以FAD(FMN)为辅酶的α-羟酸氧化酶(习惯上均称之为D-乳酸氧化酶)。D-乳酸氧化酶可用于生物传感器中测定乳酸的含量，或氧化D-乳酸生产丙酮酸。也有被用于光学纯α-羟酸的制备(中国专利201210109290.4)
目前为止，已经在迟钝爱德华菌(Edwardsiella tarda)和运动发酵单胞菌(Zymomonas mobilis)等中发现了D-乳酸氧化酶。(Kalnenieks U,Galinina N, Bringer-Meyer S,et al.Membrane D-lactate oxidase in Zymomonas mobilis: evidence for abranched respiratory chain[J].FEMS microbiology letters,1998, 168(1):91-97)
本发明首次从沙门氏菌(Salmonella enterica)中克隆表达出一种新型的D- 乳酸氧化酶，该酶不仅可以氧化(R)-α-羟酸，而且可以氧化(R)-α-羟酸酯，该反应与NAD(NADP)为辅酶的乳酸脱氢酶参与的反应相比逆反应极微弱，可应用于光学纯(S)-α-羟酸酯和(S)-α-羟酸的制备。
发明内容
本发明从沙门氏菌(Salmonella enterica)中克隆得到了一种以FAD为辅酶的D-乳酸氧化酶的基因，利用大肠杆菌工程菌异源表达，公开了其相关的酶学特性，并进行了应用研究。
本发明的技术方案如下：
1、菌株
本发明D-乳酸氧化酶基因的来源菌株为：Salmonella enterica ATCC 43845，购自美国ATCC菌种库。
2、D-乳酸氧化酶基因的克隆
提取Salmonella enterica ATCC 43845菌体基因组总DNA。设计特异性引物，应用PCR方法，扩增出D-乳酸氧化酶基因全长编码框序列。并构建重组质粒。
3、D-乳酸氧化酶表达与纯化
将重组质粒导入E.coli BL21(DE3)中，诱导表达。菌体破碎后得到粗酶液，纯化后冷冻干燥备用。
4、D-乳酸氧化酶的酶学性质分析
以D-乳酸为底物研究pH对本发明所述D-乳酸氧化酶酶活的影响。
以D-乳酸为底物研究温度对本发明所述D-乳酸氧化酶酶活的影响。
D-乳酸氧化酶的底物特异性分析：所用的底物有D-乳酸、乙醇酸、D-苯乳酸、D-对羟基苯乳酸、D-酒石酸、D-苹果酸、D-扁桃酸、D-丹参素。
酶活测定方法为：根据Characterization of a Lactate Oxidase from aStrain of Gram Negative Bacterium from Soil，Applied Biochemistry andBiotechnology,56, 1996,278-288。所述方法进行。
5、D-乳酸氧化酶拆分混旋的α-羟酸酯
拆分α-羟酸酯(alpha-hydroxy esters)的方法为：取纯化好的酶0.1克于50 mL三角瓶中，加入溶有α-羟酸酯5mM的pH 7的磷酸盐缓冲液中，于30℃， 150rpm水浴摇床中转化16h，转化后液相色谱分析上清液。(R)-α-羟酸酯中的α-羟基被脱氢氧化成对应的α-酮酸酯，(S)-α-羟酸酯不被氧化。
产物(S)-α-羟酸酯的光学纯度通过对映体过量值(％e.e)来评价：
对映体过量值％e.e＝[(SS-SR)/(SS+SR)]×100％
(S)-α-羟酸酯得率(％)＝(SS/S0)×100％
式中SR为反应后(R)-对映体的峰面积，SS为反应后(S)-对映体的液相色谱峰面积，S0为反应前(R)-和(S)-对映体的液相色谱峰面积之和。
产物测定液相色谱条件为：Chiralcel OD-H手性柱(4.6×250mm)，流动相体积比为正己烷：异丙醇：三氟乙酸＝80:20:0.1，流速为0.5mL/min，柱温 25℃，检测波长210nm，进样量20μL。
所述的α-羟酸酯为下列之一：丹参素冰片酯、丹参素异丙酯、苯乳酸冰片酯、苯乳酸异丙酯、对羟基苯乳酸冰片酯、对羟基苯乳酸异丙酯、扁桃酸冰片酯、扁桃酸异丙酯、丹参素细辛醇酯、乳酸冰片酯、苯乳酸细辛醇酯、对羟基苯乳酸细辛醇酯。
所述的α-羟酸酯，根据中国专利200610042787.3、201410180490.8、201410175950.8和20140699506.6公布的方法合成。
本发表明的有益之处：从Salmonella enterica ATCC 43845中克隆表达出一种新型的D-乳酸氧化酶，该酶可以氧化(R)-α-羟酸和(R)-α-羟酸酯，可用于规模化制备手性纯(S)-α-羟酸酯，具有重要的工业应用价值。
具体实施方式
实施例1
本实施例为本发明所述D-乳酸氧化酶基因的克隆与大肠杆菌工程菌构建。
1、Salmonella enterica ATCC 43845DNA的提取
将Salmonella enterica ATCC 43845菌株在LB培养基中培养12h，12,000 rmp/min离心10min得到菌体，应用细菌基因组DNA抽提试剂盒(TaKaRa公司)按照其操作提取菌体基因组总DNA，放冰箱备用。
2、大肠杆菌感受态制备
(1)接种E.coli DH5α和BL21(DE3)分别于含有20mL LB培养基的250mL 摇瓶中，37℃、200rpm/min培养过夜。
(2)按1％接种量接种于50mL LB培养基中，37℃培养至OD600约0.6(约 2～3h)。
(3)将菌液转移到50mL预冷的离心管中，冰上放置30min，8000rpm/min、 4℃离心5min。
(4)弃上清，加入5mL预冷的0.1mol/L CaCl2溶液，使菌体悬浮，冰上放置20min，8000rpm/min、4℃离心5min。重复2次。
(5)弃上清，加入1.5mL预冷的0.1mol/L CaCl2溶液(含15％甘油)，轻轻悬浮菌体，然后按每个离心管(1.5mL)加入100μL菌液分装，-70℃冰箱保藏备用。
3、D-乳酸氧化酶基因的克隆
(1)引物设计
设计引物序列为：
引物1：5'GCCGGGATCCATGTCTTCCATAACAATGACTGAT 3'
引物2：5'GCCGTCTAGAGTTTCATCGGGCGTATTT 3'
用以上合成的两条引物，以Salmonella enterica ATCC 43845的基因组 DNA为模板进行PCR扩增。
本步骤中扩增体系为：
扩增程序为：
98℃，10min
98℃，10sec；55℃，15sec；72℃，2min反应30个循环
72℃，10min
PCR产物送华大基因测序后得到该酶的基因序列，如SEQ ID NO:1所示。根据该基因序列得到的氨基酸序列如SEQ ID NO:2所示。
(3)双酶切和连接
将pColdⅡ质粒和PCR产物进行双酶切，酶切体系为：10×cut buffer 3μl, DNA 4μl，酶BamHI和XbaI各0.5μl，无菌水2μl共30μl。37℃水浴下双酶切1h。将DNA片段克隆到pColdⅡ载体上，并转化到E.coli DH5α感受态细胞中。连接体系：10×DNA ligase buffer2.5μl，DNA片段8μl，载体DNA 2μl， T4DNA ligase 1μl，无菌水11.5μl共25μl。16℃水浴下连接12h-16h。
(4)转化
步骤：
1在连接体系中加入100μl DH5α感受态细菌，轻混匀，冰浴30min。
2放入预热的42℃水浴中，放置90s进行热休克处理。
3立即冰浴2min。
4加入1ml不含抗生素的LB培养液，37℃培养1h使菌体复苏。
5将菌体均匀涂布在含抗生素的LB平板上。
6培养24h长势良好。挑单菌落进行菌落PCR，核酸电泳验证，提取重组质粒。将重组质粒导入BL21大肠杆菌感受态中，保存备用。
实施例2
本实施例为本发明所述D-乳酸氧化酶的诱导表达及分离纯化。
1、加500μl重组菌液到50ml LB培养液中。37℃培养2.5h，15℃下静置0.5h。再加20μl 0.5M的IPTG，15℃下冷诱导培养24h。将发酵液进行离心(8000rmp/min，10min)得到菌体，用磷酸氢二钠-磷酸二氢钠缓冲溶液 (20mmol/L，pH 7.0)复溶菌体，超声破碎仪破碎，离心(8000rmp/min，10 min)收集上清得到粗酶液。
2、将步骤1得到的粗酶液采用AKTA avant 150蛋白纯化系统操作进行镍柱纯化，洗脱方法为：将A1、A2、B1、B2四根管路都放进水里，设置system flow 20ml/min流速，进行排气。然后设置system flow 1ml/min、flow path (column position 3)、delta pressure0.3、pre-pressure 0.5、Gradient 0、inset A1，待水滴均匀流出后装柱子，平衡十分钟之后把A1放进结合液中，B1放进洗脱液中，再进行排气一次，平衡二十分钟，然后上样粗酶液，用500mM的高浓度咪唑缓冲液(B1所处溶液)梯度洗脱目的蛋白，将吸附在离子柱上的蛋白洗脱下来得到纯化的酶。纯化后的酶经冷冻干燥备用。
实施例3
本实施例为本发明所述D-乳酸氧化酶的最适温度。以D-乳酸为底物，将底物与pH为5.0的磷酸缓冲液在30-60℃不同的温度条件下水浴15min，测定 D-乳酸氧化酶的酶活，确定酶的最适反应温度为45℃。
实施例4
本实施例为本发明所述D-乳酸氧化酶的最适pH值。以D-乳酸为底物，将底物在pH3-9，45℃水浴15min测定酶的酶活，结果发现在pH 5.0条件下D- 乳酸氧化酶酶活最高。
实施例5
本实施例为本发明所述D-乳酸氧化酶与不同底物的反应特性，列于表1 中。
表1 D-乳酸氧化酶对不同底物的活性
实施例6
根据发明内容中的方法拆分各种外消旋α-羟酸酯，结果如下表所示：
表2 拆分各种外消旋α-羟酸酯的效果
由上表可以看出，当在反应时间充分时，可以得到各类高光学纯的(S)-α- 羟酸酯，该酶的光学专一性非常好。
SEQUENCE LISTING
<110> 江南大学
<120> 一种氧化酶及其应用
<130> No
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1731
<212> DNA
<213> Salmonella enterica ATCC 43845
<400> 1
atgtcttcca taacaatgac tgataataaa acgttcctga atgagctggc ccgcctggta 60
ggccattctc acctgctgac agaccccgcc aaaaccgccc gctatcgcaa gggtttccgt 120
tccggtcagg gcgatgcgct ggccgttgtt ttcccaagtt cgttactgga actgtggcgt 180
gtgctcaacg cctgcgttaa cgctgacaaa attattctga tgcaggccgc caacaccggt 240
ctgacggaag gctccacgcc gaacggcaac gactacgatc gcgatattgt gattatcagc 300
acccagcggc tggataagct gcacttactg gataacggcc aacaggttct ggcctggccc 360
ggtacgacgc tctatgcgct ggaaaaagcg cttaaaccgc tgggccgtga accgcactcg 420
gtgattggtt cgtcgtgtat cggcgcgtcg gtcatcggcg gcatctgtaa taactccggc 480
ggctcgctgg tgcagcgcgg cccggcctat accgagatgt cgctctttgc ccgtattgac 540
gagcaaggca aactgcaact ggtcaatcat ctgggtatcg atcttgggca tacgccggag 600
cagatcctga gcaagctgga tgatgaacgg attaaagatg aggatgtgcg tcacgatggt 660
cgtcatgcgc acgatcacga ctacgtgacg cgggtacgag atatcaacgc cgatacgccc 720
gcccgctata acgccgaccc ggaccgtttg tttgaatctt ccggctgcgc cggaaaactg 780
gcggttttta ccgttcgcct tgataccttt gcagcggaaa aaaatcagca ggttttttat 840
atcggcacca atcagcccgc ggtactgact gaaatccgtc ggcatattct ggcgaacttc 900
gacaatctgc cagtggcggg agaatatatg catcgcgata tctacgatat tgcggagcag 960
tacggtaaag atacctttct gatgatcgat aaactcggca ccgacaagat gccattcttt 1020
ttcacgctaa aaggccgaac cgatgcgatg ctggaaaaag taaaattttt ccgaccgcat 1080
ttcaccgacc gcgcgatgca gaaattcggc cacctgtttc ccagccatct gccgccgcga 1140
atgaaaaact ggcgcgataa atatgagcat catctgctgt taaaaatggc gggagacggc 1200
gtcgcagaag cgcaacgctg gctgaatgaa ttttttaaat ccgctgaggg cggtttcttc 1260
acctgtacgc cggaagaagg cagcaaagcc ttcctgcacc gcttcgccgc tgctggcgcg 1320
gcgattcgtt atcaggccgt acacgctgat gaagtggaag atattctggc gctggatatc 1380
gctctgcgcc gcaacgatac cgactggttc gagcatctgc cgccggaaat cgacagccaa 1440
ctggtacata aactctatta cggccacttt atgtgccacg tttttcatca ggactatatc 1500
gtgaagaaag gcgtcgacgt ccatgcgctg aaagcgcaaa tgttggagct gttgcaggcg 1560
cgcggggcac aatacccggc ggagcacaac gttggacatc tgtataaagc gccggaaacg 1620
ctgacgcgtt tttatcggca aaatgaccca accaacagta tgaaccccgg tatcggcaaa 1680
acgagtaagc ggaaattctg gcaggaaaat acgcccgatg aaacgcatta a 1731
<210> 2
<211> 576
<212> PRT
<213> Salmonella enterica ATCC 43845
<400> 2
Met Ser Ser Ile Thr Met Thr Asp Asn Lys Thr Phe Leu Asn Glu Leu
1 5 10 15
Ala Arg Leu Val Gly His Ser His Leu Leu Thr Asp Pro Ala Lys Thr
20 25 30
Ala Arg Tyr Arg Lys Gly Phe Arg Ser Gly Gln Gly Asp Ala Leu Ala
35 40 45
Val Val Phe Pro Ser Ser Leu Leu Glu Leu Trp Arg Val Leu Asn Ala
50 55 60
Cys Val Asn Ala Asp Lys Ile Ile Leu Met Gln Ala Ala Asn Thr Gly
65 70 75 80
Leu Thr Glu Gly Ser Thr Pro Asn Gly Asn Asp Tyr Asp Arg Asp Ile
85 90 95
Val Ile Ile Ser Thr Gln Arg Leu Asp Lys Leu His Leu Leu Asp Asn
100 105 110
Gly Gln Gln Val Leu Ala Trp Pro Gly Thr Thr Leu Tyr Ala Leu Glu
115 120 125
Lys Ala Leu Lys Pro Leu Gly Arg Glu Pro His Ser Val Ile Gly Ser
130 135 140
Ser Cys Ile Gly Ala Ser Val Ile Gly Gly Ile Cys Asn Asn Ser Gly
145 150 155 160
Gly Ser Leu Val Gln Arg Gly Pro Ala Tyr Thr Glu Met Ser Leu Phe
165 170 175
Ala Arg Ile Asp Glu Gln Gly Lys Leu Gln Leu Val Asn His Leu Gly
180 185 190
Ile Asp Leu Gly His Thr Pro Glu Gln Ile Leu Ser Lys Leu Asp Asp
195 200 205
Glu Arg Ile Lys Asp Glu Asp Val Arg His Asp Gly Arg His Ala His
210 215 220
Asp His Asp Tyr Val Thr Arg Val Arg Asp Ile Asn Ala Asp Thr Pro
225 230 235 240
Ala Arg Tyr Asn Ala Asp Pro Asp Arg Leu Phe Glu Ser Ser Gly Cys
245 250 255
Ala Gly Lys Leu Ala Val Phe Thr Val Arg Leu Asp Thr Phe Ala Ala
260 265 270
Glu Lys Asn Gln Gln Val Phe Tyr Ile Gly Thr Asn Gln Pro Ala Val
275 280 285
Leu Thr Glu Ile Arg Arg His Ile Leu Ala Asn Phe Asp Asn Leu Pro
290 295 300
Val Ala Gly Glu Tyr Met His Arg Asp Ile Tyr Asp Ile Ala Glu Gln
305 310 315 320
Tyr Gly Lys Asp Thr Phe Leu Met Ile Asp Lys Leu Gly Thr Asp Lys
325 330 335
Met Pro Phe Phe Phe Thr Leu Lys Gly Arg Thr Asp Ala Met Leu Glu
340 345 350
Lys Val Lys Phe Phe Arg Pro His Phe Thr Asp Arg Ala Met Gln Lys
355 360 365
Phe Gly His Leu Phe Pro Ser His Leu Pro Pro Arg Met Lys Asn Trp
370 375 380
Arg Asp Lys Tyr Glu His His Leu Leu Leu Lys Met Ala Gly Asp Gly
385 390 395 400
Val Ala Glu Ala Gln Arg Trp Leu Asn Glu Phe Phe Lys Ser Ala Glu
405 410 415
Gly Gly Phe Phe Thr Cys Thr Pro Glu Glu Gly Ser Lys Ala Phe Leu
420 425 430
His Arg Phe Ala Ala Ala Gly Ala Ala Ile Arg Tyr Gln Ala Val His
435 440 445
Ala Asp Glu Val Glu Asp Ile Leu Ala Leu Asp Ile Ala Leu Arg Arg
450 455 460
Asn Asp Thr Asp Trp Phe Glu His Leu Pro Pro Glu Ile Asp Ser Gln
465 470 475 480
Leu Val His Lys Leu Tyr Tyr Gly His Phe Met Cys His Val Phe His
485 490 495
Gln Asp Tyr Ile Val Lys Lys Gly Val Asp Val His Ala Leu Lys Ala
500 505 510
Gln Met Leu Glu Leu Leu Gln Ala Arg Gly Ala Gln Tyr Pro Ala Glu
515 520 525
His Asn Val Gly His Leu Tyr Lys Ala Pro Glu Thr Leu Thr Arg Phe
530 535 540
Tyr Arg Gln Asn Asp Pro Thr Asn Ser Met Asn Pro Gly Ile Gly Lys
545 550 555 560
Thr Ser Lys Arg Lys Phe Trp Gln Glu Asn Thr Pro Asp Glu Thr His
565 570 575

Claims (2)

1.一种拆分α-羟酸酯(alpha-hydroxy esters)的方法，其特征在于，所述方法为：取纯化好的酶0.1克于50mL三角瓶中，加入溶有α-羟酸酯5mM的pH 7的磷酸盐缓冲液中，于30℃，150rpm水浴摇床中转化16h，转化后液相色谱分析上清液；所述酶为来源于沙门氏菌(Salmonella enterica)的D-乳酸氧化酶，其氨基酸序列为SEQ ID NO:2所示；所述的α-羟酸酯为下列之一：丹参素冰片酯、丹参素异丙酯、苯乳酸冰片酯、苯乳酸异丙酯、对羟基苯乳酸冰片酯、对羟基苯乳酸异丙酯、乳酸冰片酯、扁桃酸冰片酯、扁桃酸异丙酯、丹参素细辛醇酯、苯乳酸细辛醇酯、对羟基苯乳酸细辛醇酯。
2.根据权利要求1所述的D-乳酸氧化酶，其核苷酸序列为SEQ ID NO:1所示。
CN201710006551.2A 2017-01-05 2017-01-05 一种氧化酶及其应用 Active CN106754782B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710006551.2A CN106754782B (zh) 2017-01-05 2017-01-05 一种氧化酶及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710006551.2A CN106754782B (zh) 2017-01-05 2017-01-05 一种氧化酶及其应用

Publications (2)

Publication Number Publication Date
CN106754782A CN106754782A (zh) 2017-05-31
CN106754782B true CN106754782B (zh) 2019-11-15

Family

ID=58949594

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710006551.2A Active CN106754782B (zh) 2017-01-05 2017-01-05 一种氧化酶及其应用

Country Status (1)

Country Link
CN (1) CN106754782B (zh)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102660470B (zh) * 2012-04-13 2013-07-31 浙江工业大学 中华根瘤菌及其生物拆分外消旋α-羟酸生产手性α-羟酸

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WP_023205328.1;无;《NCBI》;20160108;第1页 *

Also Published As

Publication number Publication date
CN106754782A (zh) 2017-05-31

Similar Documents

Publication Publication Date Title
CN106754778B (zh) 一种氧化酶及其应用
CN106591250B (zh) 一种氧化酶及其应用
CN106754801B (zh) 一种氧化酶及其应用
CN106754785B (zh) 一种氧化酶及其应用
CN106754782B (zh) 一种氧化酶及其应用
CN106701702B (zh) 一种氧化酶及其应用
CN106754793B (zh) 一种氧化酶及其应用
CN106701705B (zh) 一种氧化酶及其应用
CN106754791B (zh) 一种氧化酶及其应用
CN106754780B (zh) 一种氧化酶及其应用
CN106754781B (zh) 一种氧化酶及其应用
CN106754792B (zh) 一种氧化酶及其应用
CN106754788B (zh) 一种氧化酶及其应用
CN106591252B (zh) 一种氧化酶及其应用
CN106754779B (zh) 一种氧化酶及其应用
CN106754784B (zh) 一种氧化酶及其应用
CN106754783B (zh) 一种氧化酶及其应用
CN106754789B (zh) 一种氧化酶及其应用
CN106754798B (zh) 一种氧化酶及其应用
CN106754797B (zh) 一种氧化酶及其应用
CN106754800B (zh) 一种氧化酶及其应用
CN106754786B (zh) 一种氧化酶及其应用
CN106754795B (zh) 一种氧化酶及其应用
CN106701700A (zh) 一种氧化酶及其应用
CN106754790B (zh) 一种氧化酶及其应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20230217

Address after: Floor 20, Unit 2, Building 1, Jinlan West Jingyuan, No. 56, Shinan Road, Science Avenue, High-tech Industrial Development Zone, Zhengzhou City, Henan Province, 450000

Patentee after: Zhuohong Chaoyuan Biotechnology (Zhengzhou) Co.,Ltd.

Address before: No. 1800 road 214122 Jiangsu Lihu Binhu District City of Wuxi Province

Patentee before: Jiangnan University