CN106591252B - 一种氧化酶及其应用 - Google Patents

一种氧化酶及其应用 Download PDF

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CN106591252B
CN106591252B CN201710006629.0A CN201710006629A CN106591252B CN 106591252 B CN106591252 B CN 106591252B CN 201710006629 A CN201710006629 A CN 201710006629A CN 106591252 B CN106591252 B CN 106591252B
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蔡宇杰
王亚红
曹憬
白亚军
郑晓晖
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Zhuohong Chaoyuan Biotechnology Zhengzhou Co ltd
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Abstract

本发明涉及一种来源于肺炎克雷伯菌肺炎亚种(Klebsiella pneumoniae subsp.pneumoniae)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)
本发明首次从肺炎克雷伯氏菌(Klebsiella pneumoniae subsp.pneumoniae) 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
本发明从肺炎克雷伯菌肺炎亚种(Klebsiella pneumoniae subsp.pneumoniae)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：Klebsiella pneumoniae subsp.pneumoniae ATCC 700721，购自美国ATCC菌种库。
2、D-乳酸氧化酶基因的克隆
提取Klebsiella pneumoniae subsp.pneumoniae ATCC 700721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。(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Klebsiella pneumoniae subsp.pneumoniae ATCC700721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、Klebsiella pneumoniae subsp.pneumoniae ATCC 700721 DNA的提取
将Klebsiella pneumoniae subsp.pneumoniae ATCC 700721菌株在LB培养基中培养12h，12,000rmp/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'GCCGGGATCCATGTCATCTGCACCCACTGACACCC 3'
引物2：5'GCCGTCTAGACGGATCCGCCGGAGAGGCT 3'
(2)PCR扩增
用以上合成的两条引物，以Klebsiella pneumoniae subsp.pneumoniae ATCC700721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(20 mmol/L，pH 7.0)复溶菌体，超声破碎仪破碎，离心(8000rmp/min，10min) 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8.0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40℃。
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pH3-9，40℃水浴15min测定酶的酶活，结果发现在pH 8.0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
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ggcaccaccc tctattcgct ggaaaaagcg ctcaaaccgc tgggacgcga accgcactcg 420
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gcggttttcg ccgtgcgtct cgatactttc ccggcggaaa agcgccagca ggtgttttac 840
attggcacca accagccgca ggtattgacc gagatccgcc gccatatcct cgccgagttc 900
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tatggcaaag acaccttcct gatgatcgac aagctcggca ccgacaaaat gccgttcttc 1020
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atgaaaacct ggcgtgataa atatgaacat catctgctgc tgaagatggc tggcgacggc 1200
atcgaagaag cgcagcgttg gttgacggag tacttccagc aggccgaggg ggatttcttc 1260
gcctgtacgc cggaggaagg cagcaaggcg ttcctccatc gctttgccgc cgccggcgcg 1320
gcgatccgct atcaggcggt gcatgccgac gaagtcgaag atattctggc gctggatatc 1380
gccctgcgcc gtaacgatac cgagtggttc gaacatctgc cgccggagat cgacacccag 1440
ctggtgcata agctctacta tggccacttt atgtgccatg tgttccatca agattacatc 1500
gtcaggaaag gggtcgacgc acacgcgctg aaggaaaaaa tgctggagtt actgaaagcg 1560
cgcggcgccc agtatccggc ggagcataac gtgggtcatc tgtatgaggc cccggagagc 1620
ctgcagcagt tctatcgcca gaacgatccc accaacagca tgaacccggg gattggtaaa 1680
accagcaagc agaaatactg gggtgaagcg gccccgacgc cagcctctcc ggcggatccg 1740
caataa 1746
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<213> Klebsiella pneumoniae subsp. pneumoniae ATCC 700721
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Met Ser Ser Ala Pro Thr Asp Thr His Lys Thr Phe Leu Ala Asp Leu
1 5 10 15
Ala Arg Leu Val Gly Pro Ser His Leu Leu Thr Asp Pro Ala Lys Thr
20 25 30
Gln Arg Tyr Arg Lys Gly Phe Arg Ser Gly Gln Gly Glu Ala Leu Ala
35 40 45
Val Val Phe Pro Gly Thr Leu Leu Glu Leu Trp Arg Val Leu Asn Ala
50 55 60
Cys Val Asp 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 Glu Ile
85 90 95
Val Ile Ile Ser Thr Leu Arg Leu Asp Lys Leu His Leu Leu Asp Lys
100 105 110
Gly Glu Gln Val Leu Ala Trp Pro Gly Thr Thr Leu Tyr Ser 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 Gln Ile Asp Ala Asp Gly Lys Leu Lys Leu Val Asn His Leu Gly
180 185 190
Ile Asp Leu Gly Ser Thr Pro Glu Gln Ile Leu Ser Arg Leu Asp Asp
195 200 205
Glu Arg Ile Ser Asp Ser Asp Val Leu His Asp Gly Arg His Ala His
210 215 220
Asp His Asp Tyr Val Thr Arg Val Arg Asp Val Asp 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 Ala Val Arg Leu Asp Thr Phe Pro Ala
260 265 270
Glu Lys Arg Gln Gln Val Phe Tyr Ile Gly Thr Asn Gln Pro Gln Val
275 280 285
Leu Thr Glu Ile Arg Arg His Ile Leu Ala Glu Phe Gln His Leu Pro
290 295 300
Val Ala Gly Glu Tyr Met His Arg Asp Ile Tyr Asp Ile Ala Glu Lys
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 Met Lys Gly Arg Thr Asp Ala Met Leu Glu
340 345 350
Lys Val Ser Leu Phe Lys Pro His Phe Thr Asp Arg Phe Met Gln Lys
355 360 365
Leu Gly His Val Phe Pro Ala His Leu Pro Glu Arg Met Lys Thr Trp
370 375 380
Arg Asp Lys Tyr Glu His His Leu Leu Leu Lys Met Ala Gly Asp Gly
385 390 395 400
Ile Glu Glu Ala Gln Arg Trp Leu Thr Glu Tyr Phe Gln Gln Ala Glu
405 410 415
Gly Asp Phe Phe Ala 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 Glu Trp Phe Glu His Leu Pro Pro Glu Ile Asp Thr 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 Arg Lys Gly Val Asp Ala His Ala Leu Lys Glu
500 505 510
Lys Met Leu Glu Leu Leu Lys Ala Arg Gly Ala Gln Tyr Pro Ala Glu
515 520 525
His Asn Val Gly His Leu Tyr Glu Ala Pro Glu Ser Leu Gln Gln 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 Gln Lys Tyr Trp Gly Glu Ala Ala Pro Thr Pro Ala Ser
565 570 575
Pro Ala Asp Pro Gln
580

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；所述酶为来源于肺炎克雷伯菌肺炎亚种(Klebsiella pneumoniae subsp.pneumoniae)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所述的方法，其特征在于，所述D-乳酸氧化酶的核苷酸序列为SEQ IDNO:1所示。
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