CN115305243B - 一种Baeyer-Villiger单加氧酶突变体及其应用 - Google Patents
一种Baeyer-Villiger单加氧酶突变体及其应用 Download PDFInfo
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- CN115305243B CN115305243B CN202210782944.3A CN202210782944A CN115305243B CN 115305243 B CN115305243 B CN 115305243B CN 202210782944 A CN202210782944 A CN 202210782944A CN 115305243 B CN115305243 B CN 115305243B
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- baeyer
- villiger monooxygenase
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Abstract
本发明公开了一种Baeyer‑Villiger单加氧酶突变体及其应用,本发明的Baeyer‑Villiger单加氧酶突变体不对称氧化潜手性硫醚生产手性亚砜有较好的催化效率和转化效率,因此,本发明的Baeyer‑Villiger单加氧酶突变体在生产如(S)‑苄基苯基亚砜、(S)‑奥美拉唑等的手性亚砜中具有极高的应用前景。
Description
技术领域
本发明涉及一种Baeyer-Villiger单加氧酶突变体及其应用,属于酶工程技术领域。
背景技术
手性过渡金属配合物、有机催化剂或Baeyer-Villiger单加氧酶(BVMOs)介导的前手性硫醚催化不对称亚砜化是有机化学中的一个重要转变。BVMOs为合成亚砜提供了一种绿色、实用的方法,在硫醚的立体选择性催化氧化合成亚砜中得到了很好的应用。亚砜是一种很有价值的有机含硫化合物,广泛用于手性辅助试剂和手性配体,也广泛应用于医药领域。奥美拉唑硫醚是合成手性抗胃溃疡药物埃索美拉唑的关键前体,而埃索美拉唑((S)-奥美拉唑)是重要的质子泵抑制剂(PPI,用于胃食管反流和消化性溃疡的治疗)。
已报道利用天然酶全细胞生物氧化奥美拉唑硫醚形成埃索美拉唑。当奥美拉唑硫醚累积量分别达到0.05 g/L和0.115 g/L时,利用全细胞的Aspergillus carbonarius和Lysinibacillussp. B71对奥美拉唑具有较高的对映选择性(≥95%ee,S)。然而,相关酶的遗传信息和生化特性既未披露也未表征。最近,通过基因挖掘鉴定出了两种重组的BVMOs,它们能够对奥美拉唑硫醚进行不对称氧化,并具有良好的对映选择性(≥99%,ee,R),在3或5 g/l底物负载的情况下,6 h内转化率分别达到89%和71%。到目前为止,只有少数几种天然酶被证实能对大体积的吡唑硫醚进行不对称亚砜化。因此,利用蛋白质工程进化酶的活性和对映选择性对非天然底物也被探索。例如,Codexis专利中 (Acinetobacter sp. NCIMB9871) 30多个突变位点的工程化CHMO可以有效氧化奥美拉唑硫醚形成(S)-奥美拉唑,通过多次添加工程化CHMO, 50 g/l奥美拉唑硫醚在36 h内完全转化。此外,另一个工程AcCHMO来自Acinetobacter calcoaceticus(AcCHMO,与CHMONCIMB9871具有70%的同源性)具有15个以上突变位点,选择性氧化奥美拉唑硫醚生成所需的(S)-奥美拉唑。奥美拉唑硫醚加载为15mM底物时,在6 h内完全转化。利用蛋白质工程进化酶的活性和对映选择性对非天然底物,虽然通常需要大量的突变文库,但上述工程酶的成功案例激发了人们探索其他工程酶的可能性,这些酶可以转化大体积的硫醚。为了减少筛选工作,结构导向的理性或半理性设计策略是很有希望的。目前只有极少数野生酶或某些酶的突变体报道能够催化大体积的奥美拉唑硫醚生成(S)-奥美拉唑,这大大阻碍了利用酶不对称氧化法生产手性(S)-奥美拉唑的工业化进程。但是,与使用金属催化剂和过酸或过氧化氢对奥美拉唑硫醚进行化学氧化不同,这种酶催化过程使用氧和葡萄糖作为可持续的共底物,提供水作为清洁的副产物,使这种方法成为一种有前途的绿色化学方法来生产埃索美拉唑。
因此,急需找到不对称氧化潜手性美拉唑硫醚生产手性(S)-奥美拉唑的转化效率高的Baeyer-Villiger单加氧酶以早日实现手性(S)-奥美拉唑的大规模工业化生产以及在医药领域的大规模应用。
发明内容
为解决上述技术问题,本发明提供一种不对称氧化潜手性硫醚生产手性亚砜的转化效率高的Baeyer-Villiger单加氧酶(Baeyer-Villiger Monooxygenase,简称BVMO,EC1.1.1.1)。
本发明的第一个目的是提供一种Baeyer-Villiger单加氧酶突变体,所述Baeyer-Villiger单加氧酶突变体是将氨基酸序列如SEQ ID NO.2所示的Baeyer-Villiger单加氧酶亲本的第442位苯丙氨酸突变为丙氨酸得到,或是将氨基酸序列如SEQ ID NO.2所示的Baeyer-Villiger单加氧酶亲本的第337位精氨酸突变为脯氨酸,并将第442位苯丙氨酸突变为丙氨酸得到。
本发明的第二个目的是提供一种编码所述Baeyer-Villiger单加氧酶突变体的基因。
本发明的第三个目的是提供一种携带所述基因的重组质粒。
进一步地,所述重组质粒的载体为pET-28a(+)质粒。
本发明的第四个提供一种携带所述基因或所述重组质粒的重组细胞。
进一步地,所述重组细胞的宿主细胞为大肠杆菌E.coliBL21(DE3)。
本发明的第五个目的是提供了所述Baeyer-Villiger单加氧酶突变体的制备方法,所述方法为将上述重组细胞接种至发酵培养基中进行发酵,获得发酵液;将发酵液进行离心,收集菌体;将菌体进行破碎后离心,获得细胞破碎上清液;将细胞破碎上清液进行提取,获得所述Baeyer-Villiger单加氧酶突变体。
本发明的第六个目的是提供了一种生产手性亚砜的方法,所述方法为将所述Baeyer-Villiger单加氧酶突变体添加至含有潜手性硫醚的反应体系中进行反应,得到反应液;将反应液进行提取,获得手性亚砜。
进一步地,所述反应体系中还含有辅酶和辅酶循环系统;所述辅酶循环系统含有D-葡萄糖和葡萄糖脱氢酶,或者,含有亚磷酸盐和亚磷酸盐脱氢酶,或者,含有甲酸和甲酸脱氢酶,或者,含有乳酸和乳酸脱氢酶,或者,含有甘油和甘油脱氢酶。
进一步地,所述辅酶为NADP+、NADPH、NAD+和/或NADH。
进一步地,所述Baeyer-Villiger单加氧酶突变体在反应体系中的添加量为1~10kU/L。
进一步地,所述反应体系中,潜手性硫醚的浓度为1~10 mmol/L。
进一步地,所述反应体系中,辅酶的浓度为0.1~1 mmol/L。
进一步地,所述反应体系中,葡萄糖脱氢酶的浓度为1~10 kU/L。
进一步地,所述反应体系中,D-葡萄糖的浓度为2~100 mmol/L。
进一步地,所述反应体系为含有潜手性硫醚、辅酶以及辅酶循环系统的缓冲液。
进一步地,所述缓冲液为Tris-HCl缓冲液。
进一步地,所述Tris-HCl缓冲液的浓度为0.05~0.1 mol/L。
进一步地,所述反应的温度为30~35℃、pH为8~9。
进一步地,所述潜手性硫醚为潜手性苯甲硫醚、潜手性2-甲硫基萘、潜手性苄基苯基硫醚或潜手性奥美拉唑硫醚。
进一步地,潜手性苯甲硫醚时,所述手性亚砜为苯甲亚砜;潜手性2-甲硫基萘时,所述手性亚砜为2-(甲基亚磺酰基)萘;潜手性苄基苯基硫醚时,所述手性亚砜为苄基苯基亚砜;潜手性奥美拉唑硫醚时,所述手性奥美拉唑。
本发明的有益效果是:
(1)本发明的Baeyer-Villiger单加氧酶突变体不对称氧化潜手性硫醚生产手性亚砜的转化效率高,其中,突变体M1不对称氧化潜手性奥美拉唑硫醚生产手性奥美拉唑,而野生型同条件下不能够转化奥美拉唑硫醚。
(2)本发明的Baeyer-Villiger单加氧酶突变体不对称氧化潜手性硫醚生产手性亚砜的催化效率高,其中,突变体M2不对称氧化潜手性奥美拉唑硫醚生产手性(S)-奥美拉唑的催化效率为2.5 u/mg,是M1的105倍;突变体M2不对称氧化潜手性奥美拉唑硫醚生产手性(S)-奥美拉唑的催化效率为0.55 min–1·mM–1。
(3)野生型Baeyer-Villiger单加氧酶可不对称氧化潜手性2-甲硫基萘生成(S)-2-(甲基亚磺酰基)萘,且ee值可达91.1%(S),而本发明的Baeyer-Villiger单加氧酶突变体不对称氧化潜手性2-甲硫基萘生成手性2-(甲基亚磺酰基)萘时具有反转的立体选择性,其中,突变体M1可不对称氧化潜手性2-甲硫基萘生成(R)- 2-(甲基亚磺酰基)萘,且ee值可达99.6%(R)。
(4)本发明的Baeyer-Villiger单加氧酶突变不对称氧化潜手性硫醚生产手性亚砜的催化效率和对映选择性高,因此,本发明的Baeyer-Villiger单加氧酶突变体在生产如(S)-奥美拉唑、 (S)-苄基苯基亚砜等的手性亚砜中具有极高的应用前景。
附图说明
图1为重组质粒的的PCR扩增电泳图谱;其中,M:Marker,泳道1~3:重组质粒pET28a-RaBVMO-1、重组质粒pET28a-RaBVMO-2、重组质粒pET28a-RaBVMO-3的全质粒PCR产物。
图2为重组大肠杆菌摇瓶诱导发酵获得的表达产物的SDS-PAGE电泳分析结果;其中,M:标准蛋白Maker,泳道1~3:重组大肠杆菌E. coli BL21/pET28a-RaBVMO-1、重组大肠杆菌E. coli BL21/pET28a-RaBVMO-2、重组大肠杆菌E. coli BL21/pET28a-RaBVMO-3摇瓶诱导发酵获得的野生型及突变体M1和M2的纯酶。
图3为突变体M2不对称氧化潜手性奥美拉唑硫醚所得产物的手性色谱图。
实施方式
下面结合具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
下述实施例中涉及的大肠杆菌E.coliBL21(DE3)购自北纳生物;下述实施例中涉及的pET-28a(+)质粒、NADPH购自Novagen公司;下述实施例中涉及的葡萄糖脱氢酶(GDH)、乳酸脱氢酶(LDH)购自诺唯赞公司;下述实施例中涉及的D-葡萄糖购自捷瑞公司;下述实施例中涉及的潜手性奥美拉唑硫醚购自阿拉丁公司(上述菌株大肠杆菌E.coliBL21(DE3)可以购买得到,不需要进行用于专利程序的保藏)。
下述实施例中涉及的培养基如下:
LB液体培养基:酵母粉5.0 g×L-1、胰蛋白胨10.0 g×L-1、NaCl 10.0 g×L-1、卡那霉素100 mg×L-1。
LB固体培养基:酵母粉5.0 g×L-1、胰蛋白胨10.0 g×L-1、NaCl 10.0 g×L-1、琼脂粉 15 g/L、卡那霉素50 mg×L-1。
下述实施例中涉及的检测方法如下:
Baeyer-Villiger单加氧酶酶突变体活的检测方法如下:
将含有1 mM NADPH、1.0 mM底物潜手性奥美拉唑硫醚的Tris-HCl冲液(PBS,100mM,pH 7.0)于30℃保温2 min后,取100 μL纯酶液加入Tris-HCl冲液中于30℃进行反应,反应过程中,使用高效液相色谱,并以此为依据计算酶活;
酶活的计算公式如下:
酶活力(u/mg)=(C1×V1)×103/(T×C2×V2);
式中,C1为生成产物的浓度,单位为M/L;V1为反应液的体积,单位为mL;C2为酶的蛋白浓度,单位为mg/ml;V2为反应体系里添加的酶的体积,单位为µL;T为反应时间,单位为min;
酶活的定义:在该条件下每分钟催化氧化l µmol 底物产生l µmol产物所需酶量为一个酶活力单位(1 u)。
Baeyer-Villiger单加氧酶不对称氧化潜手性奥美拉唑硫醚生成手性(S)-奥美拉唑的转化效率和立体选择性的检测方法如下:
将含有1 mM NADPH、1.0 mM底物潜手性奥美拉唑硫醚的Tris-HCl冲液(100 mM,pH9.0)于30℃保温2 min后,取100 μL纯酶液加入Tris-HCl冲液中于30℃反应30 min;反应结束后,加入500 µL乙酸乙酯,震荡1~2 min,12000 rpm离心2~5 min,取上清到离心管中,待有机相自然挥发完全,加入500 μL色谱纯甲醇,进行手性液相色谱分析转化效率和ee值;
色谱条件具体如下:Daicel Chiralcel AD-3 (5µm,250 mm×4.6 mm)液相色谱柱,流动相为正已烷:异丙醇:乙酸(50:50:0.01,v/v/v),流速0.5 mL/min,柱温30℃,紫外检测波长302 nm,进样量10 μL,(S)-奥美拉唑和(R)-奥美拉唑保留时间分别为11.12 min和13.71 min;
转化效率的计算方法如下:
ee值的计算方法如下:
As:反应液中(S)-奥美拉唑的摩尔浓度;AR:反应液中(R)-奥美拉唑的摩尔浓度;Asub:反应液中未反应完的奥美拉唑硫醚的摩尔浓度。
实施例1 Baeyer-Villiger单加氧酶突变体的制备、表达及纯化
化学合成编码氨基酸序列如SEQ ID NO.2所示的Baeyer-Villiger单加氧酶的基因(基因的核苷酸序列如SEQ ID NO.1所示);将获得的基因与pET-28a(+)质粒经双酶切(Nde Ⅰ和Xho Ⅰ)后进行连接,连接产物转化大肠杆菌E.coliBL21(DE3),转化产物涂布于LB固体培养基,于37℃培养8~10 h,在LB固体培养基上挑取5个转化子,接入LB液体培养基培养,于37℃培养10 h后提取质粒,将提取得到的质粒进行酶切验证以及测序验证,验证正确即获得含有编码野生型Baeyer-Villiger单加氧酶的基因的重组质粒pET28a-RaBVMO以及含有编码野生型Baeyer-Villiger单加氧酶的基因的重组菌E. coli BL21/pET28a-RaBVMO。
利用全质粒PCR技术,以获得的重组质粒pET28a-RaBVMO为模板进行定点突变,获得含有编码Baeyer-Villiger单加氧酶F442A(第442位苯丙氨酸突变为丙氨酸),R337P/F442A (第337位精氨酸突变为脯氨酸,第442位苯丙氨酸突变为丙氨酸)的基因的重组质粒,将上述Baeyer-Villiger单加氧酶突变体分别命名为M1和M2;
其中,突变R337P、F442A所用引物如下:
R337P-F:TTCGGTGCGAAAcctCCACCA(SEQ ID No.3);
R337P-R:ACCGCTTGGTGGaggTTTCGC(SEQ ID No.4);
F442A-F:CCACAGAGCCCAgctACCAAT(SEQ ID No.5);
F442A-R:TGGGATATTGGTagcTGGGCT(SEQ ID No.6);
PCR反应体系(50 μL)为:KOD酶(2.5 U/mL) l.0 μL,模板(5~50 ng) l.0 μL,dNTP4.0 μL,10×reaction buffer 5.0 μL,上下游引物各1.0 μL,ddH2O补足至50 μL;
PCR产物扩增条件均为:(1)94℃变性3 min,(2)95℃变性30 sec,(3)50℃退火30sec,(4)72 ℃延伸3 min 40 sec,重复步骤(2)~(4)进行20~25个循环,最后72℃延伸10min,16℃保存PCR扩增产物。
PCR扩增产物用1%琼脂糖凝胶电泳进行检测,检测结束后,向10 µL扩增产物中加入0.5 µL甲基化模板消化酶(DpnI),枪头吹吸进行混匀,于37°C条件下反应1 h,将DpnI处理后的扩增产物转化大肠杆菌E.coliBL21(DE3),转化产物涂布于LB固体培养基,于37℃培养8~10 h,在LB固体培养基上挑取5个转化子,接入LB液体培养基培养,于37℃培养10 h后提取质粒,将提取得到的质粒进行酶切验证(验证结果可见图1~3)以及测序验证,验证正确即获得分别含有编码Baeyer-Villiger单加氧酶突变体M1和M2的基因的重组质粒pET28a-RaBVMO-2~重组质粒pET28a-RaBVMO-3以及分别含有编码Baeyer-Villiger单加氧酶突变体M1和M2的基因的重组菌E. coli BL21/pET28a-RaBVMO-2~重组菌E. coli BL21/pET28a-RaBVMO-3。
将获得的重组菌E. coli BL21/pET28a-RaBVMO-2以及重组菌E. coli BL21/pET28a-RaBVMO-3分别涂布于LB固体培养基,于37℃培养8~10 h,获得单菌落;挑取单菌落接入LB液体培养基,于37℃培养12~14 h,获得种子液;将种子液按照2%(v/v)的接种量接入LB液体培养基,于37℃、200 rpm培养至OD600达到0.8后,在发酵液中加入终浓度为0.2 mM的IPTG,于25℃继续诱导培养8 h,得到发酵液;将发酵液于4℃、8000 rpm离心10 min后,收集细胞;将收集得到的细胞悬浮于Tris-HCl缓冲液(100 mmol·L-1,pH 9.0)中进行超声破碎,收集分别含有野生型Baeyer-Villiger单加氧酶、Baeyer-Villiger单加氧酶突变体M1和M2的细胞破碎上清液。
将获得的细胞破碎上清液使用亲和柱HisTrap FF crude(镍柱)进行纯化,纯化过程如下:先使用缓冲液A(25 mmol·L-1 Tris-HCl,500 mmol·L-1 NaCl,20 mmol·L-1 咪唑,pH 7.4)平衡镍柱,并实施例1获得的细胞破碎上清液过镍柱,继续使用缓冲液A洗脱未与镍柱结合的蛋白,待穿透峰流尽后,从缓冲液A到缓冲液B(25 mmol·L-1 Tris-HCl,500mmol·L-1 NaCl,500 mmol·L-1 咪唑,pH 7.4)进行梯度洗脱,将结合到镍柱上的重组蛋白洗脱下来,获得野生型Baeyer-Villiger单加氧酶、Baeyer-Villiger单加氧酶突变体M1和M2的纯酶液。
将获得的Baeyer-Villiger单加氧酶突变体M1和M2的纯酶液进行SDS-PAGE分析,Baeyer-Villiger单加氧酶突变体M1和M2的纯酶液均在61 kDa左右显示单条带,且杂蛋白较少,说明镍柱纯化效果较好。
实施例2 Baeyer-Villiger单加氧酶突变体的动力学参数以及不对称氧化潜手性硫醚生成手性亚砜的立体选择性
选择实施例1获得的野生型Baeyer-Villiger单加氧酶以及Baeyer-Villiger单加氧酶突变体M2,分别以浓度为0.1~1mM的潜手性奥美拉唑硫醚为底物,测定实施例1获得Baeyer-Villiger单加氧酶突变体M2的氧化活力,采用Graph Pad Prism 7.0软件中的非线性回归方法对数据进行拟合,分别得到米氏(Michaelis-Menten)方程的K m值,再计算得到K cat和K cat/K m值,计算结果见表1;
其中,K cat值的计算公式为:K cat=V max·M/60;其中,M为酶的分子质量,单位为kDa。
由表2可知,Baeyer-Villiger单加氧酶突变体M2不对称氧化潜手性奥美拉唑硫醚生成(S)-奥美拉唑的催化效率较野生型Baeyer-Villiger单加氧酶有了明显的提升,是Baeyer-Villiger单加氧酶突变体M2的105倍;Baeyer-Villiger单加氧酶突变体M2不对称氧化潜手性苄基苯基硫醚生成(S)-苄基苯基亚砜的催化效率比野生型有6倍的提升;Baeyer-Villiger单加氧酶突变体M2不对称氧化潜手性2-甲硫基萘生成(R)- 2-(甲基亚磺酰基)萘的催化效率则较野生型有了一定的下降。
检测实施例1获得的Baeyer-Villiger单加氧酶突变体M1和M2不对称氧化潜手性硫醚生成手性亚砜的立体选择性,检测结果见表2。
由表2可知,Baeyer-Villiger单加氧酶突变体M1和M2不对称氧化潜手性硫醚生成手性(S)-奥美拉唑的立体选择性活性均逐渐提高;并且,由表2可知,Baeyer-Villiger单加氧酶突变体M2可不对称氧化潜手性奥美拉唑硫醚生成(S)-奥美拉唑,且ee值可达99.1%(S);与野生型Baeyer-Villiger单加氧酶相比,Baeyer-Villiger单加氧酶突变体M1不对称氧化潜手性2-甲硫基萘时具有反转的立体选择性,且ee值可达99.3%(R)。
表1 野生型Baeyer-Villiger单加氧酶以及Baeyer-Villiger单加氧酶突变体M1和M2的动力学参数
ND:没有测定。
表2 野生型Baeyer-Villiger单加氧酶以及Baeyer-Villiger单加氧酶突变体M1和M2不对称氧化潜手性硫醚生成手性亚砜的立体选择性
实施例3 Baeyer-Villiger单加氧酶突变体不对称氧化潜手性奥美拉唑硫醚生成(S)-奥美拉唑的转化效率
选择实施例1获得的野生型Baeyer-Villiger单加氧酶以及Baeyer-Villiger单加氧酶突变体M2,将实施例1获得的野生型Baeyer-Villiger单加氧酶以及Baeyer-Villiger单加氧酶突变体M2分别以4 g/L的添加量添加至分别含有1 mM、3 mM、5 mM潜手性奥美拉唑硫醚的100 mM Tris-HCl缓冲液(pH 9.0)中,在30℃、pH 9.0、200 rpm条件下反应12 h,得到反应液;除潜手性奥美拉唑硫醚外,Tris-HCl缓冲液中还含有浓度为0.02 mM的辅酶NADP+、浓度为1.5 mM的葡萄糖、浓度为1.5 mM的葡萄糖脱氢酶GDH和浓度为5%(v/v)的甲醇。
分别检测反应不同时间时野生型Baeyer-Villiger单加氧酶以及Baeyer-Villiger单加氧酶突变体M2不对称氧化潜手性奥美拉唑硫醚生成(S)-奥美拉唑的转化率,检测结果见表3。
由表3可知,1 mM底物浓度下,Baeyer-Villiger单加氧酶突变体M2在反应3 h时达到>99.9%的转化率,可见,Baeyer-Villiger单加氧酶突变体M2不对称氧化潜手性奥美拉唑硫醚生成(S)-奥美拉唑的转化效率较野生型Baeyer-Villiger单加氧酶有了显著的提升。
表3 野生型Baeyer-Villiger单加氧酶不对称氧化潜手性奥美拉唑硫醚生成(S)-奥美拉唑的转化率
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
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Claims (10)
1.一种Baeyer-Villiger单加氧酶突变体,其特征在于,所述Baeyer-Villiger单加氧酶突变体是将氨基酸序列如SEQ ID NO.2所示的Baeyer-Villiger单加氧酶亲本的第442位苯丙氨酸突变为丙氨酸得到,或是将氨基酸序列如SEQ ID NO.2所示的Baeyer-Villiger单加氧酶亲本的第337位精氨酸突变为脯氨酸,并将第442位苯丙氨酸突变为丙氨酸得到。
2.一种编码权利要求1所述Baeyer-Villiger单加氧酶突变体的基因。
3.一种携带权利要求2所述基因的重组质粒。
4.一种携带权利要求2所述基因或权利要求3所述重组质粒的重组细胞。
5.一种利用权利要求4所述重组细胞制备权利要求1所述Baeyer-Villiger单加氧酶突变体的方法,其特征在于,所述方法为将所述重组细胞接种至发酵培养基中进行发酵,获得发酵液;将发酵液进行离心,收集菌体;将菌体进行破碎后离心,获得细胞破碎上清液;将细胞破碎上清液进行提取,获得所述Baeyer-Villiger单加氧酶突变体。
6.一种利用权利要求1所述Baeyer-Villiger单加氧酶突变体生产手性亚砜的方法,其特征在于,所述方法为将所述Baeyer-Villiger单加氧酶突变体添加至含有潜手性硫醚的反应体系中进行反应,得到反应液;将反应液进行提取,获得手性亚砜。
7.根据权利要求6所述的方法,其特征在于,所述反应体系中还含有辅酶和辅酶循环系统;所述辅酶循环系统含有D-葡萄糖和葡萄糖脱氢酶,或者,含有亚磷酸盐和亚磷酸盐脱氢酶,或者,含有甲酸和甲酸脱氢酶,或者,含有乳酸和乳酸脱氢酶,或者,含有甘油和甘油脱氢酶。
8.根据权利要求6所述的方法,其特征在于,所述潜手性硫醚为潜手性苯甲硫醚、潜手性2-甲硫基萘、潜手性苄基苯基硫醚或潜手性奥美拉唑硫醚。
9.根据权利要求6所述的方法,其特征在于,所述Baeyer-Villiger单加氧酶突变体在反应体系中的添加量为1~10 kU/L。
10.根据权利要求6所述的方法,其特征在于,所述反应体系中,潜手性硫醚的浓度为1~10 mmol/L。
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