CN114150024B

CN114150024B - 一种双功能酶生物催化剂及其制备方法和应用

Info

Publication number: CN114150024B
Application number: CN202111470133.1A
Authority: CN
Inventors: 贾振华; 李冉; 宋聪; 宋水山; 张翔
Original assignee: Institute of Biology of Hebei Academy of Sciences
Current assignee: Institute of Biology of Hebei Academy of Sciences
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2023-06-20
Anticipated expiration: 2041-12-03
Also published as: CN114150024A

Abstract

本发明公开了一种双功能酶生物催化剂及其制备方法和应用。该方法包括如下步骤：以4‑羟基‑2‑丁酮为底物，经醇脱氢酶或醇脱氢酶酶的突变体催化反应生成R‑1,3‑丁二醇。本发明提供了一种以4‑羟基‑2‑丁酮为原料，通过单酶催化合成R‑1,3‑丁二醇的合成方法。

Description

一种双功能酶生物催化剂及其制备方法和应用

技术领域

本发明属于生物技术领域，涉及一种R-1,3-丁二醇的合成方法，特别涉及一种以利用生物酶催化合成R-1,3-丁二醇的方法。

背景技术

(R)-1,3-丁二醇是一种重要的手性合成子，被广泛地用于碳青霉烯类抗生素母核氮杂环丁酮、香料、信息激素和杀虫剂等物质的合成。在这些化合物中，碳青霉烯类药物、青霉烯类药物和青霉素都同属于β-内酰胺类抗生素，而青霉素的滥用导致许多抗药性菌株产生，但碳青霉烯类药物能够有效地缓解这些抗药性菌株引起的症状，碳青霉烯类药物在国内被广泛的应用，因此，开发绿色、高效的(R)-1,3-丁二醇合成技术成为国内外重要研究的目的。

目前，国内外关于生物催化方法合成(R)-1,3-丁二醇的相关报道很少。1993年，Eguchi等利用固定化SP382脂肪酶重复催化双酰基化反应拆分(R,S)-1,3-丁二醇工艺制备(R)-1,3-丁二醇，其光学纯度达98％以上，但其收率较低(Tamotsu Eguchi,KenichiMochida.Lipase-catalyzed diacylation of 1,3-butanediol[J].Biotechnol.Lett.,1993,15(9):955-960.)。Akinobu Matsuyama报道了利用近平滑假丝酵母Candiaparapsilosis IFO 1396拆分外消旋体制备(R)-1,3-丁二醇，其光学纯度达到94％，得率达到48％，但这两种反应的立体选择性较低，很难满足药物的合成需求(Matsuyama A，Kobayashi Y，Ohnishi H.Microbial Production of Optically Active 1,3-Butanediolfrom 4-Hydroxy-2-butanone.Journal of the Agricultural Chemical Society,1993,57(4):685-686.)。

发明内容

本发明所要解决的技术问题是合成手性R-1,3-丁二醇。

为解决上述技术问题，本发明首先提供了一种R-1,3-丁二醇的合成方法，包括以下步骤：

以4-羟基-2-丁酮为底物，利用醇脱氢酶或所述醇脱氢酶的突变体进行催化反应，反应产物为R-1,3-丁二醇。

优选的，所述醇脱氢酶来源于：弓形脱硫菌(Desulfallas arcticus)、马钱子脱硫弧菌(Desulfonatronovibrio magnus)、消化球菌(Peptococcaceae bacterium BRH_c4b)、嗜热厌氧菌(Thermoanaerobacteraceae bacterium)、磺基磺酸盐脱硫弧菌(Desulfovibrio sulfodismutans)、斯氏假丝酵母(Candidatus Stahlbacteria)、硫氧化还原脱硫弧菌(Desulfolutivibrio sulfoxidireducens)、脱硫杆菌(Desulfobacteralesbacterium)、假丝酵母亚细菌(Candidatus Abyssubacteria bacterium SURF_5)、硝化螺旋菌(Nitrospirales bacterium)、扁平菌科细菌(Planctomycetaceae bacterium)或厌氧绳菌(Anaerolineales bacterium)。

优选的，来源于弓形脱硫菌的醇脱氢酶的氨基酸序列如SEQ ID No.2所示；

来源于马钱子脱硫弧菌的醇脱氢酶的氨基酸序列如SEQ ID No.4所示；

来源于消化球菌的醇脱氢酶的氨基酸序列如SEQ ID No.6所示；

来源于嗜热厌氧菌的醇脱氢酶的氨基酸序列如SEQ ID No.8所示；

来源于消化球菌的醇脱氢酶的氨基酸序列如SEQ ID No.10所示；

来源于磺基磺酸盐脱硫弧菌的醇脱氢酶的氨基酸序如SEQ ID No.12所示；

来源于斯氏假丝酵母的醇脱氢酶的氨基酸序列如SEQ ID No.14所示；

来源于硫氧化还原脱硫弧菌的醇脱氢酶的氨基酸序列如SEQ ID No.16所示；

来源于脱硫杆菌的醇脱氢酶的氨基酸序列如SEQ ID No.18所示；

来源于假丝酵母亚细菌的醇脱氢酶的氨基酸序列如SEQ ID No.20所示；

来源于硝化螺旋菌的醇脱氢酶的氨基酸序列如SEQ ID No.22所示；

来源于硝化螺旋菌的醇脱氢酶的氨基酸序列如SEQ ID No.24所示；

来源于硝化螺旋菌的醇脱氢酶的氨基酸序列如SEQ ID No.26所示；

来源于扁平菌科细菌的醇脱氢酶的氨基酸序列如SEQ ID No.28所示；

来源于厌氧绳菌的醇脱氢酶的氨基酸序列如SEQ ID No.30所示。

优选的，在所述醇脱氢酶的N端和/或C端连接标签。

优选的，所述醇脱氢酶的突变体包括：对SEQ ID No.2所示的氨基酸序列进行以下点突变：R80V和/或Y147N；

对SEQ ID No.4所示的氨基酸序列进行如下点突变中的至少一种：A84G、I85T、K110T、N198D。

优选的，所述醇脱氢酶或所述醇脱氢酶的突变体以粗酶液、粗酶液冻干粉、纯酶或全细胞的形式加入。

优选的，所述催化反应的温度为20～35℃，所述催化反应的时间为24～48h。

优选的，当所述醇脱氢酶是以粗酶液、粗酶液冻干粉或纯酶的形式加入时，所述醇脱氢酶在反应体系中的浓度为0.1g/L～10g/L；

当所述醇脱氢酶以全细胞的形式加入时，所述全细胞的湿重为100g/L。

优选的，所述催化反应在浓度为50～100mM，pH值为6.5～8.5的磷酸盐缓冲液中进行。

优选的，当所述醇脱氢酶是以粗酶液、粗酶液冻干粉或纯酶的形式加入时，所述催化反应的反应体系中除了含有4-羟基-2-丁酮、所述醇脱氢酶和辅酶NAD⁺外，还含有异丙醇。

本发明所提供的合成R-1,3-丁二醇的方法中，存在辅因子再生系统。所述辅因子再生系统为醇脱氢酶催化异丙醇氧化促进辅因子再生。在本发明合成R-1,3-丁二醇的方法中，醇脱氢酶催化4-羟基-2-丁酮还原成R-1,3-丁二醇，NAD(P)H被重新氧化成NAD(P)⁺，同时，醇脱氢酶催化异丙醇氧化成丙酮，NAD(P)+被还原成NAD(P)H，生成的NAD(P)H重新参与到4-羟基-2-丁酮还原成R-1,3-丁二醇的还原。

附图说明

图1为醇脱氢酶制备催化4-羟基-2-丁酮合成R-1,3-丁二醇的反应原理图；

图2为4-羟基-2-丁酮与R-1,3-丁二醇标准品气相检测峰图；

图3为反应液气相检测峰图。

具体实施方式

一种利用生物酶催化合成手性R-1,3-丁二醇的方法，可包括如下步骤(反应原理见图1)：以4-羟基-2-丁酮为底物，经醇脱氢酶及其辅酶催化反应生成R-1,3-丁二醇；

进一步地，所述醇脱氢酶均可来源于如下任一微生物：弓形脱硫菌(Desulfallasarcticus)、马钱子脱硫弧菌(Desulfonatronovibrio magnus)、消化球菌(Peptococcaceaebacterium BRH_c4b)、嗜热厌氧菌(Thermoanaerobacteraceae bacterium)、磺基磺酸盐脱硫弧菌(Desulfovibrio sulfodismutans)、斯氏假丝酵母(Candidatus Stahlbacteria)、硫氧化还原脱硫弧菌(Desulfolutivibrio sulfoxidireducens)、脱硫杆菌(Desulfobacterales bacterium)、假丝酵母亚细菌(Candidatus Abyssubacteriabacterium SURF_5)、硝化螺旋菌(Nitrospirales bacterium)、扁平菌科细菌(Planctomycetaceae bacterium)或厌氧绳菌(Anaerolineales bacterium)；

更进一步地，所述醇脱氢酶具体可为如下(a1)-(a15)中任一：

(a1)来源于弓形脱硫菌的醇脱氢酶，氨基酸序列为SEQ ID No.2；

(a2)来源于马钱子脱硫弧菌的醇脱氢酶，氨基酸序列为SEQ ID No.4；

(a3)来源于消化球菌的醇脱氢酶，氨基酸序列为SEQ ID No.6；

(a4)来源于嗜热厌氧菌的醇脱氢酶，氨基酸序列为SEQ ID No.8；

(a5)来源于消化球菌的醇脱氢酶，氨基酸序列为SEQ ID No.10；

(a6)来源于磺基磺酸盐脱硫弧菌的醇脱氢酶，氨基酸序SEQ ID No.12；

(a7)来源于斯氏假丝酵母的醇脱氢酶，氨基酸序列为SEQ ID No.14；

(a8)来源于硫氧化还原脱硫弧菌的醇脱氢酶，氨基酸序列为SEQ ID No.16；

(a9)来源于脱硫杆菌的醇脱氢酶，氨基酸序列为SEQ ID No.18；

(a10)来源于假丝酵母亚细菌的醇脱氢酶，氨基酸序列为SEQ ID No.20；

(a11)来源于硝化螺旋菌的醇脱氢酶，氨基酸序列为SEQ ID No.22；

(a12)来源于硝化螺旋菌的醇脱氢酶，氨基酸序列为SEQ ID No.24；

(a13)来源于硝化螺旋菌的醇脱氢酶，氨基酸序列为SEQ ID No.26；

(a14)来源于扁平菌科细菌的醇脱氢酶，氨基酸序列为SEQ ID No.28；

(a15)来源于厌氧绳菌的醇脱氢酶，氨基酸序列为SEQ ID No.30；

(a16)在(1)-(15)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白；

进一步地，所述醇脱氢酶的突变体具体可为如下(b1)-(b3)：

(b1)与SEQ ID No.2所示来源于弓形脱硫菌的醇脱氢酶相比，存在或仅存在如下突变中的至少一种:R80V、Y147K；

(b2)与SEQ ID No.4所示来源于马钱子脱硫弧菌的醇脱氢酶相比，存在或仅存在如下突变中的至少一种:A84G、I85T、K110T、N198D；

(b3)在(b1)-(b2)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。

所述方法中，所述醇脱氢酶是以粗酶液、粗酶液冻干粉、纯酶或全细胞的形式发生催化作用的；

进一步，所述粗酶液、粗酶液冻干粉和纯酶均按照包括如下步骤的方法制备得到:在宿主细胞中表达所述醇脱氢酶，得到重组细胞；裂解所述重组细胞获得所述粗酶液、粗酶液冻干粉或纯酶；

进一步，所述全细胞均按照包括如下步骤的方法制备得到:在宿主细胞中表达所述醇脱氢酶，得到的重组细胞即为所述全细胞；

再进一步，所述重组细胞是按照包括如下步骤的方法制备获得的：向所述宿主细胞导入能够表达所述醇脱氢酶的核酸分子，经诱导培养后获得表达所述醇脱氢酶的所述重组细胞；

更进一步，所述“能够表达所述醇脱氢酶的核酸分子”是通过重组载体的形式导入到所述宿主细胞中的；所述重组载体为携带有所述醇脱氢酶的编码基因的细菌质粒(如在细菌中表达的基于T7启动子的表达载体，具体如pET-28a等)、噬菌体、酵母质粒或逆转录病毒包装质粒；和/或所述宿主细胞为原核细胞或低等真核细胞；

在本发明的一个实施例中，所述重组载体具体为将所述酶A或所述酶B的编码基因替换pET-28a载体的酶切位点NdeI和XhoI之间的小片段后得到的重组质粒。

进一步地，所述宿主细胞可为原核细胞或低等真核细胞。

更进一步地，所述原核细胞具体可为细菌。所述低等真核细胞具体可为酵母细胞。

在本发明的一个实施例中，所述宿主细胞具体为大肠杆菌，更加具体的为E.coliBL21(DE3)。相应的，所述诱导培养为向培养体系中加IPTG至终浓度0.1-0.5mM(具体如0.1mM)，20-37℃诱导培养12-24h(具体如16h)。

所述来源于弓形脱硫菌的醇脱氢酶的编码基因的序列为SEQ ID No.1或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于马钱子脱硫弧菌的醇脱氢酶的编码基因的序列为SEQ ID No.3或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于消化球菌的醇脱氢酶的编码基因的序列为SEQ ID No.5或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于嗜热厌氧菌的醇脱氢酶的编码基因的序列为SEQ ID No.7或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于消化球菌的醇脱氢酶的编码基因的序列为SEQ ID No.9或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于磺基磺酸盐脱硫弧菌的醇脱氢酶的编码基因的序列为SEQ ID No.11或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于斯氏假丝酵母的醇脱氢酶的编码基因的序列为SEQ ID No.13或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于硫氧化还原脱硫弧菌的醇脱氢酶的编码基因的序列为SEQ ID No.15或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于脱硫杆菌的醇脱氢酶的编码基因的序列为SEQ ID No.17或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于假丝酵母亚细菌的醇脱氢酶的编码基因的序列为SEQ ID No.19或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于硝化螺旋菌的醇脱氢酶的编码基因的序列为SEQ ID No.21或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于硝化螺旋菌的醇脱氢酶的编码基因的序列为SEQ ID No.23或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于硝化螺旋菌的醇脱氢酶的编码基因的序列为SEQ ID No.25或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于扁平菌科细菌的醇脱氢酶的编码基因的序列为SEQ ID No.27或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于厌氧绳菌的醇脱氢酶的编码基因的序列为SEQ ID No.29或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于短小乳杆菌的醇脱氢酶的突变体的编码基因的序列为如下(c1)-(c3)中任一：(c1)与SEQ ID No.1相比，存在或仅存在如下突变中的至少一种:G239T、T439A；(c2)在(c1)所限定序列的5’端和/或3’端连接标签编码序列后得到的融合序列；(c3)与(c1)或(c2)所限定的序列相比保有功能且编码相同蛋白的随机或/和定点诱变序列；

所述来源于高温厌氧杆菌的醇脱氢酶的突变体的编码基因的序列为如下(d1)-(d3)中任一：(d1)与SEQ ID No.3相比，存在或仅存在如下突变中的至少一种：C251G、T253C、A329C、G593A；(d2)在(d1)所限定序列的5’端和/或3’端连接标签编码序列后得到的融合序列；(d3)与(d1)或(d2)所限定的序列相比保有功能且编码相同蛋白的随机或/和定点诱变序列；

在催化反应中，所述催化反应的温度均可为20～35℃，如25～30℃，具体如25℃。所述催化反应的时间均可为24～48h，如24h。

当所述醇脱氢酶是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，所述催化反应可在浓度为50～100mM，pH值为6.5～8.0的磷酸盐缓冲液中进行，具体如:浓度为50mM，pH值为7.0的磷酸盐缓冲液；当所述醇脱氢酶以全细胞的形式发生催化作用时，催化反应可在浓度为50～100mM，pH值为7.5～8.5的磷酸盐缓冲液，具体如:浓度为50mM，pH值为7.5的磷酸盐缓冲液。

当所述醇脱氢酶是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，所述醇脱氢酶在各自反应体系中的浓度均可为0.1g/L～10g/L，如10g/L。当所述醇脱氢酶以全细胞的形式发生催化作用时，所述反应体系中所述全细胞的浓度为100g/L(每升反应体系中含有所述全细胞的湿重为100g)。

在本发明中，所述醇脱氢酶的辅酶具体可为氧化型辅酶I(即NAD+)或氧化型辅酶II(即NADP+)。

在本发明中，当所述醇脱氢酶是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，所述醇脱氢酶的辅酶在其各自的反应体系中的浓度均可为0.1～5mM(具体如5mM)。

当所述醇脱氢酶是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，所述催化反应的反应体系中除了含有4-羟基-2-丁酮和所述醇脱氢酶及其辅酶外，还含有异丙醇。

具体的，所述催化反应的反应体系组成如下:浓度为50mM，pH值为7.0的磷酸盐缓冲、终浓度为500mM的4-羟基-2-丁酮、终浓度为5mM的氧化型辅酶I(即NAD+)或氧化型辅酶II(即NADP+)、终浓度为10％的异丙醇(v/v)、终浓度为10g/L的所述醇脱氢酶的粗酶液、粗酶液冻干粉或纯酶。

当所述醇脱氢酶以全细胞的形式发生催化作用时，所述反应体系中还含有异丙醇。

具体的，所述反应体系的组成可为如下：浓度为50mM、pH值为7.5的磷酸盐缓冲液；终浓度为500mM的4-羟基-2-丁酮、终浓度为10％的异丙醇(v/v)、终浓度为100g/L的所述全细胞(即每升反应体系中含有所述全细胞的湿重为100g)。

本发明还提供了一种酶及其相关产品。

本发明所提供的酶为前文所述醇脱氢酶。当然，也可以包括所述脱氢酶各自的辅酶。

所述相关产品可为能够表达所述酶系统中各酶的核酸分子，或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。

所述酶或所述相关产品在合成R-1,3-丁二醇中的应用也属于本发明的保护范围。

为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例对本发明进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。

下述实施例中，如无特殊说明，均为常规方法。

下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。

实施例1醇脱氢酶或其突变体工程菌的制备

将醇脱氢酶的编码基因分别进行全基因合成(根据需要以大肠杆菌为宿主进行密码子优化)，将合成的基因连接于pET28a(+)表达载体上，并经测序验证正确后得到重组载体。利用定点突变方法得到其相关基因突变体。

将上述测序验证正确的重组表达载体转化至大肠杆菌BL21(DE3)的微生物宿主中，得到本发明的基因工程菌株。其中所述转化方法为本领域常规转化方法，较佳的为电转法或化学转化法。

本实施例中所涉及的醇脱氢酶或其突变体详见表1。

表1醇脱氢酶及其突变体

编号	菌种	基因序列	蛋白序列
				a1	弓形脱硫菌(Desulfallas arcticus)	SEQID No.1	SEQID No.2
a2	马钱子脱硫弧菌(Desulfonatronovibrio magnus)	SEQID No.3	SEQID No.4
				a3	消化球菌(Peptococcaceae bacterium BRH_c4b)	SEQID No.5	SEQID No.6
a4	嗜热厌氧菌(Thermoanaerobacteraceae bacterium)	SEQID No.7	SEQID No.8
				a5	消化球菌(Peptococcaceae bacterium BRH_c4b)	SEQID No.9	SEQID No.10
a6	磺基磺酸盐脱硫弧菌(Desulfovibrio sulfodismutans)	SEQID No.11	SEQID No.12
				a7	斯氏假丝酵母(Candidatus Stahlbacteria)	SEQID No.13	SEQID No.14
a8	硫氧化还原脱硫弧菌(Desulfolutivibrio sulfoxidireducens)	SEQID No.15	SEQID No.16
				a9	脱硫杆菌(Desulfobacterales bacterium)	SEQID No.17	SEQID No.18
a10	假丝酵母亚细菌(Candidatus Abyssubacteria)	SEQID No.19	SEQID No.20
				a11	硝化螺旋菌(Nitrospirales bacterium)	SEQID No.21	SEQID No.22
a12	硝化螺旋菌(Nitrospirales bacterium)	SEQID No.23	SEQID No.24
				a13	硝化螺旋菌(Nitrospira sp.)	SEQID No.25	SEQID No.26
a14	扁平菌科细菌(Planctomycetaceae bacterium)	SEQID No.27	SEQID No.28
				a15	厌氧绳菌(Anaerolineales bacterium)	SEQID No.29	SEQID No.30
b1	弓形脱硫菌(Desulfallas arcticus)	SEQID No.31	SEQID No.32
				b2	马钱子脱硫弧菌(Desulfonatronovibrio magnus)	SEQID No.33	SEQID No.34

实施例2醇脱氢酶或其突变体的表达及粗酶制备

将实施例1构建的醇脱氢酶或突变体的重组表达载体转入大肠杆菌BL21(DE3)感受态细胞中，37℃培养12-16h，待转化子长出，将单个转化子接种到含有卡纳霉素(50μg/mL)的5mL LB培养基中，37℃、220rmp培养过夜(12-16h)。然后按照1％(体积百分含量)的比例接种到LB培养基中，37℃、220rmp培养到OD600nm为0.6左右，添加终浓度为0.1mM的IPTG，25℃诱导培养16h，于4℃，6000rpm离心20min收集菌体，用50mM，pH值为7.0的磷酸盐缓冲重悬清洗一次，之后超声破菌并制备酶冻干粉。

实施例3利用醇脱氢酶或突变体的粗酶制备R-1,3-丁二醇

在反应体系中依次加入浓度为50mM，pH值为7.0的磷酸盐缓冲液、终浓度为500mM的4-羟基-2-丁酮、终浓度为5mM的氧化型辅酶I(即NAD+)、终浓度为10％(v/v)的异丙醇和终浓度为10g/L的醇脱氢酶冻干粉或酶液组成反应体系。将该反应体系在25℃的条件下反应24h。

反应结束后，反应液中加入等体积乙酸乙酯萃取，涡旋振荡10min，离心(14000rpm，2min)。取上相过0.22μm膜，进行气相色谱检测。气相检测条件为：手性柱supelcoβ-120(250*2.5mM)，柱温：140℃，进样口温度：250℃，FID检测器温度：300℃。

气相色谱检测结果如图2～3所示。图2为4-羟基-2-丁酮和R-1,3-丁二醇标准品气相检测峰图，4-羟基-2-丁酮保留时间在7.6min左右，R-1,3-丁二醇的保留时间在8.3min左右；图3为醇脱氢酶或突变体粗酶制备R-1,3-丁二醇的气相检测图，表2为醇脱氢酶或其突变体粗酶制备R-1,3-丁二醇的结果。

表2醇脱氢酶或其突变体粗酶制备R-1,3-丁二醇的结果

实施例4利用醇脱氢酶或突变体的全细胞制备R-1,3-丁二醇

在反应体系中依次加入浓度为50mM，pH值为7.5的磷酸盐缓冲液、终浓度为500mM的4-羟基-2-丁酮、终浓度为5mM的氧化型辅酶I(即NAD+)、终浓度为10％(v/v)的异丙醇和终浓度为100g/L的醇脱氢酶全细胞组成反应体系。将该反应体系在25℃的条件下反应24h。

反应结束后，反应液中加入等体积乙酸乙酯萃取，涡旋振荡10min，离心(14000rpm，2min)。取上相过0.22μm膜，进行气相色谱检测。气相检测条件为：手性柱supelcoβ-120(250*2.5mM)，柱温：140℃，进样口温度：250℃，FID检测器温度：300℃。表3为醇脱氢酶或其突变体全细胞制备R-1,3-丁二醇的结果。

表3醇脱氢酶或其突变体全细胞制备R-1,3-丁二醇的结果

以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

序列表

<110> 河北省科学院生物研究所

<120> 一种双功能酶生物催化剂及其制备方法和应用

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180 185 190

Ile Ile Thr Val Glu Thr Asn Pro Lys Arg Gln Glu Leu Ser Arg Lys

195 200 205

Phe Gly Ala Asp Val Val Val Asp Phe Lys Ala Val Asp Ala Val Gln

210 215 220

Glu Ile Phe Asn Leu Thr Gly Gly Val Gly Val Asp Ser Ala Ile Glu

225 230 235 240

Cys Leu Gly Ser Gln Ile Thr Phe Glu Asn Cys Ile Lys Ala Thr Arg

245 250 255

Pro Gly Gly Thr Ile Ser Asn Val Gly Tyr His Gly Glu Gly Glu Tyr

260 265 270

Leu Met Ile Pro Arg Ala Glu Trp Gly Val Gly Met Ser Asp Lys Thr

275 280 285

Ile Arg Thr Gly Leu Cys Pro Gly Gly Arg Glu Arg Met Glu Arg Leu

290 295 300

Leu Arg Leu Ile Glu Thr Gly Arg Ile Asp Pro Arg Pro Leu Thr Thr

305 310 315 320

His Thr Phe Lys Phe Ala Glu Ile Glu Lys Ala Phe Asn Met Met Ala

325 330 335

Thr Lys Glu Asp Gly Ile Ile Lys Pro Met Ile Leu Phe Asp

340 345 350

<210> 7

<211> 1050

<212> DNA

<213> 嗜热厌氧菌(Thermoanaerobacteraceae bacterium)

<400> 7

atgaaagctt ttgttatgca tggaattaat aaagttggct ttatggaaaa gccaattccc 60

gaacccggac caaatgaggt cctgattaag actaccgcat gcttggtatg tacttccgac 120

gtccataccg taagtggcgc aattggagaa agaaaggatc tgactcttgg ccacgaagcc 180

gtgggaacta tagccaagtt aggtagtgct gttgaaggtt ttacgatcgg acagagagtt 240

gccgttaatg ccattacccc ttgttaccag tgtgaaaatt gccagcgcgg gtacacttct 300

caatgcacgc aaatgttagg gggatggcgt tttgccaatg ttaaagacgg tacctttgcc 360

gagtattttc tggttaacga cgcaaaggcc aacctcgcac ctatcccgga aactattact 420

gatgaacagg ccttatacac aaccgatatg atgagcaccg gttttatggg agccgaaaac 480

gcggatatcc ccttaggcgg gtcagtagcc atatttgccc agggggctgt cggtctaatg 540

gctactgccg gtgcgaggct tttaggagcc ggattaataa tagccgtaga atccgtgccc 600

gaacgcaaaa aactggcaaa acattttggt gctgattacg tgcttgattt taacgaagtt 660

gatgttattg aagaaattaa caagctgact gacggtcaag gagttgactc gtccatagaa 720

gcgctgggat cccaggtaac ttttgaaaat tgcataaagg taacccggcc tggcggaacc 780

atatctaata ttggctatca tggtgagggg gaatatctga aaataccgcg tttggactgg 840

ggcgttggca tgggtgaaaa gactatcaaa accggtctct gtcccggcgg ttctgaaaga 900

atgcgcaggt taatgcgcct aatcgcaaat ggccgcattg acccaactcc actgacaact 960

cacaagtttt catttgatga agtagataaa gcatttgagt atatgaaata taaaaaagat 1020

aacattatta aaccgctgat tactttttaa 1050

<210> 8

<211> 349

<212> PRT

<213> 嗜热厌氧菌(Thermoanaerobacteraceae bacterium)

<400> 8

Met Lys Ala Phe Val Met His Gly Ile Asn Lys Val Gly Phe Met Glu

1 5 10 15

Lys Pro Ile Pro Glu Pro Gly Pro Asn Glu Val Leu Ile Lys Thr Thr

20 25 30

Ala Cys Leu Val Cys Thr Ser Asp Val His Thr Val Ser Gly Ala Ile

35 40 45

Gly Glu Arg Lys Asp Leu Thr Leu Gly His Glu Ala Val Gly Thr Ile

50 55 60

Ala Lys Leu Gly Ser Ala Val Glu Gly Phe Thr Ile Gly Gln Arg Val

65 70 75 80

Ala Val Asn Ala Ile Thr Pro Cys Tyr Gln Cys Glu Asn Cys Gln Arg

85 90 95

Gly Tyr Thr Ser Gln Cys Thr Gln Met Leu Gly Gly Trp Arg Phe Ala

100 105 110

Asn Val Lys Asp Gly Thr Phe Ala Glu Tyr Phe Leu Val Asn Asp Ala

115 120 125

Lys Ala Asn Leu Ala Pro Ile Pro Glu Thr Ile Thr Asp Glu Gln Ala

130 135 140

Leu Tyr Thr Thr Asp Met Met Ser Thr Gly Phe Met Gly Ala Glu Asn

145 150 155 160

Ala Asp Ile Pro Leu Gly Gly Ser Val Ala Ile Phe Ala Gln Gly Ala

165 170 175

Val Gly Leu Met Ala Thr Ala Gly Ala Arg Leu Leu Gly Ala Gly Leu

180 185 190

Ile Ile Ala Val Glu Ser Val Pro Glu Arg Lys Lys Leu Ala Lys His

195 200 205

Phe Gly Ala Asp Tyr Val Leu Asp Phe Asn Glu Val Asp Val Ile Glu

210 215 220

Glu Ile Asn Lys Leu Thr Asp Gly Gln Gly Val Asp Ser Ser Ile Glu

225 230 235 240

Ala Leu Gly Ser Gln Val Thr Phe Glu Asn Cys Ile Lys Val Thr Arg

245 250 255

Pro Gly Gly Thr Ile Ser Asn Ile Gly Tyr His Gly Glu Gly Glu Tyr

260 265 270

Leu Lys Ile Pro Arg Leu Asp Trp Gly Val Gly Met Gly Glu Lys Thr

275 280 285

Ile Lys Thr Gly Leu Cys Pro Gly Gly Ser Glu Arg Met Arg Arg Leu

290 295 300

Met Arg Leu Ile Ala Asn Gly Arg Ile Asp Pro Thr Pro Leu Thr Thr

305 310 315 320

His Lys Phe Ser Phe Asp Glu Val Asp Lys Ala Phe Glu Tyr Met Lys

325 330 335

Tyr Lys Lys Asp Asn Ile Ile Lys Pro Leu Ile Thr Phe

340 345

<210> 9

<211> 1056

<212> DNA

<213> 消化球菌(Peptococcaceae bacterium BRH_c4b)

<400> 9

atgaaagcat tcgtaatgca tgaaatcggc aaggtcggca tcatggacaa gccaatccct 60

gaagcgggtc ccaatgatgc ggttattaaa actaccgcag ctttcgtatg cacatctgac 120

gtacataccg ttcatggcgc attagggcca agggaagacc ggaccctggg tcacgaagca 180

gtaggaatca tctacaaact gggcagtgcc gtacataatc taaaagtagg ggatagagta 240

gcagtaaatg ctatcactcc ttgctataag tgtgaaaatt gccagcgtgg atttacttcc 300

cagtgtaaag gtatgttggg aggttacaag tttaccaact tagtggaagg aaatatggct 360

gagtatttcc acgtaaatga cgccgaggca aatgtcgttc caattcccga atcggtatcc 420

gacgaggaag ctctgtatac tgtagatatg atgactaccg gtttcatggg agcagaacac 480

gccagtattc ctcttggagg caccgtggca atttttggtc agggaccggt cggcctgatg 540

gctactgtag gcgcgcgtct tctgggagcc ggcctgatta taactgttga aacagatcca 600

aaacgacagg agttgtccag aaagtacggg gctgatatag tggttgattt taaagcggtc 660

gacgcagtcc aggaaataat gaatataact ggtggaatag gcgtagattc agctattgaa 720

tgcctgggcg ctcagataac ctttgaaaac tgtattaaag tgacaaggcc ggggggcact 780

atttccaatg tggggtatca cggtggtgac gctgaatata tgttggtacc gagacttgat 840

tggggagtgg gcatgagcga caaaactatc cgcaccggtc tatgcccggg aggaagggag 900

cgtatggttc gcttactccg ccttattgaa acaggccgcg tagatccaaa gccattaaca 960

acacacacct ttaaatttgc cgatgttgaa aaagccttcc agttaatgga atcaaaggaa 1020

gacggtataa ttaaaccaat ggtactgttt gattag 1056

<210> 10

<211> 351

<212> PRT

<213> 消化球菌(Peptococcaceae bacterium BRH_c4b)

<400> 10

Met Lys Ala Phe Val Met His Glu Ile Gly Lys Val Gly Ile Met Asp

1 5 10 15

Lys Pro Ile Pro Glu Ala Gly Pro Asn Asp Ala Val Ile Lys Thr Thr

20 25 30

Ala Ala Phe Val Cys Thr Ser Asp Val His Thr Val His Gly Ala Leu

35 40 45

Gly Pro Arg Glu Asp Arg Thr Leu Gly His Glu Ala Val Gly Ile Ile

50 55 60

Tyr Lys Leu Gly Ser Ala Val His Asn Leu Lys Val Gly Asp Arg Val

65 70 75 80

Ala Val Asn Ala Ile Thr Pro Cys Tyr Lys Cys Glu Asn Cys Gln Arg

85 90 95

Gly Phe Thr Ser Gln Cys Lys Gly Met Leu Gly Gly Tyr Lys Phe Thr

100 105 110

Asn Leu Val Glu Gly Asn Met Ala Glu Tyr Phe His Val Asn Asp Ala

115 120 125

Glu Ala Asn Val Val Pro Ile Pro Glu Ser Val Ser Asp Glu Glu Ala

130 135 140

Leu Tyr Thr Val Asp Met Met Thr Thr Gly Phe Met Gly Ala Glu His

145 150 155 160

Ala Ser Ile Pro Leu Gly Gly Thr Val Ala Ile Phe Gly Gln Gly Pro

165 170 175

Val Gly Leu Met Ala Thr Val Gly Ala Arg Leu Leu Gly Ala Gly Leu

180 185 190

Ile Ile Thr Val Glu Thr Asp Pro Lys Arg Gln Glu Leu Ser Arg Lys

195 200 205

Tyr Gly Ala Asp Ile Val Val Asp Phe Lys Ala Val Asp Ala Val Gln

210 215 220

Glu Ile Met Asn Ile Thr Gly Gly Ile Gly Val Asp Ser Ala Ile Glu

225 230 235 240

Cys Leu Gly Ala Gln Ile Thr Phe Glu Asn Cys Ile Lys Val Thr Arg

245 250 255

Pro Gly Gly Thr Ile Ser Asn Val Gly Tyr His Gly Gly Asp Ala Glu

260 265 270

Tyr Met Leu Val Pro Arg Leu Asp Trp Gly Val Gly Met Ser Asp Lys

275 280 285

Thr Ile Arg Thr Gly Leu Cys Pro Gly Gly Arg Glu Arg Met Val Arg

290 295 300

Leu Leu Arg Leu Ile Glu Thr Gly Arg Val Asp Pro Lys Pro Leu Thr

305 310 315 320

Thr His Thr Phe Lys Phe Ala Asp Val Glu Lys Ala Phe Gln Leu Met

325 330 335

Glu Ser Lys Glu Asp Gly Ile Ile Lys Pro Met Val Leu Phe Asp

340 345 350

<210> 11

<211> 1053

<212> DNA

<213> 磺基磺酸盐脱硫弧菌(Desulfovibrio sulfodismutans)

<400> 11

atgaaggcat tcgtcatgct aggcattgga aaggttggca tcatcgacaa gcccattcct 60

gagccaggtc caaatgatgt cattctcaag acgacaagcg cgctcatttg cacctccgat 120

gtacacaccg tcgggggagc catcggcgac agaaaaaatc tcaccttggg acatgaagcc 180

ggtggcgtcg tgtacaagat cggcagtgcg gtgaccgggt tcaaggttgg cgaccgttgt 240

gtggtcaacg ccatcacccc gtgctacaaa tgcgagaact gcctgcgcgg ctttacgtcc 300

cagtgcggtg aggcctgcgg cggctggaaa tacgccaaca tcaaggacgg ttccttcgcg 360

gaatatttcc atgtcaatga cgccatcgcc aacctggtca aggtgcccga cgacgtcacc 420

gacgaggcgg ccctgtacac cacggacatg atggccacgg gatttatggg cgcggaacac 480

ggcaacaccc ccctgggcgg cagcgtggcc gtcttcggcc agggcccggt ggggcttatg 540

gccaccgccg gggcgcggct tttgggcgcg ggactgatca tcgccgtgga aagcgtgccc 600

cagcggcagg agttggccaa gtactacggc gcggacgtga tcgtggactt cagcaaggtc 660

gacgccgtgg ccgagatcaa gcgcctgacc ggcggccagg gcgtggacac cgccatcgaa 720

tgtctgggca tgcaggccac cttcgagaac tgcgtcaagg ccacccggcc cggcggcgtg 780

atttccaact gcggctacca tggcaagggc gagtacgtga agattccccg cgtcgaatgg 840

ggctacggca tggccgacaa gaccatccgc accgggcttt gccccggcgg cagcgagcgc 900

atgggccgtc tgctgcggct catccagacc ggacgcattg atcccacgaa gttgaccacg 960

catcgcctgc ccttcgccga catcgaaaag ggcttccgca tcatggccaa caaggaagac 1020

aacgtgatca agccgatgat catcttctcc taa 1053

<210> 12

<211> 350

<212> PRT

<213> 磺基磺酸盐脱硫弧菌(Desulfovibrio sulfodismutans)

<400> 12

Met Lys Ala Phe Val Met Leu Gly Ile Gly Lys Val Gly Ile Ile Asp

1 5 10 15

Lys Pro Ile Pro Glu Pro Gly Pro Asn Asp Val Ile Leu Lys Thr Thr

20 25 30

Ser Ala Leu Ile Cys Thr Ser Asp Val His Thr Val Gly Gly Ala Ile

35 40 45

Gly Asp Arg Lys Asn Leu Thr Leu Gly His Glu Ala Gly Gly Val Val

50 55 60

Tyr Lys Ile Gly Ser Ala Val Thr Gly Phe Lys Val Gly Asp Arg Cys

65 70 75 80

Val Val Asn Ala Ile Thr Pro Cys Tyr Lys Cys Glu Asn Cys Leu Arg

85 90 95

Gly Phe Thr Ser Gln Cys Gly Glu Ala Cys Gly Gly Trp Lys Tyr Ala

100 105 110

Asn Ile Lys Asp Gly Ser Phe Ala Glu Tyr Phe His Val Asn Asp Ala

115 120 125

Ile Ala Asn Leu Val Lys Val Pro Asp Asp Val Thr Asp Glu Ala Ala

130 135 140

Leu Tyr Thr Thr Asp Met Met Ala Thr Gly Phe Met Gly Ala Glu His

145 150 155 160

Gly Asn Thr Pro Leu Gly Gly Ser Val Ala Val Phe Gly Gln Gly Pro

165 170 175

Val Gly Leu Met Ala Thr Ala Gly Ala Arg Leu Leu Gly Ala Gly Leu

180 185 190

Ile Ile Ala Val Glu Ser Val Pro Gln Arg Gln Glu Leu Ala Lys Tyr

195 200 205

Tyr Gly Ala Asp Val Ile Val Asp Phe Ser Lys Val Asp Ala Val Ala

210 215 220

Glu Ile Lys Arg Leu Thr Gly Gly Gln Gly Val Asp Thr Ala Ile Glu

225 230 235 240

Cys Leu Gly Met Gln Ala Thr Phe Glu Asn Cys Val Lys Ala Thr Arg

245 250 255

Pro Gly Gly Val Ile Ser Asn Cys Gly Tyr His Gly Lys Gly Glu Tyr

260 265 270

Val Lys Ile Pro Arg Val Glu Trp Gly Tyr Gly Met Ala Asp Lys Thr

275 280 285

Ile Arg Thr Gly Leu Cys Pro Gly Gly Ser Glu Arg Met Gly Arg Leu

290 295 300

Leu Arg Leu Ile Gln Thr Gly Arg Ile Asp Pro Thr Lys Leu Thr Thr

305 310 315 320

His Arg Leu Pro Phe Ala Asp Ile Glu Lys Gly Phe Arg Ile Met Ala

325 330 335

Asn Lys Glu Asp Asn Val Ile Lys Pro Met Ile Ile Phe Ser

340 345 350

<210> 13

<211> 1056

<212> DNA

<213> 斯氏假丝酵母(Candidatus Stahlbacteria)

<400> 13

atgaaagcgt ttgtgatgaa aggcatcggg aaggtcggat tcatggagaa acccattcct 60

gaagatccag gaccaaatgg tgccatcatt aagacaacta aagcccttgt ctgcacctct 120

gatacccata cagttgctgg ggcaatcggc gacaggaaag acctcacgct tggtcatgaa 180

gcggttggga ttgtttacaa actgggtagc gaagtgaaag ggataaagga aggtgatcgc 240

gttgcagtta acgcaataac accttgctat aagtgcgaaa actgtttacg aggttacacg 300

tcgcaatgtc agcaaatgct tggtggatgg aagttcgcca acattaagga tggggtcttc 360

gcagaatatt ttcacgtgaa cgatgctgag gcgaatctgg cactaattcc agattccatt 420

cccgatgagg cagctgtata cactacagac atgatgtcca cgggctttat gggtgcagaa 480

catgcgaata tccctctagg gggaactgtt gcaatttttg ctcagggtcc gatagggctc 540

atgtgtaccg tgggtgcacg cctgctgggt gcaggtctcg ttattgcggt tgaaagcatg 600

tcgaaacgaa aggagcttgc taagcatttt ggggctgaca cagttgtgga tttcacaaaa 660

gtagatcctg taaaggatat tctccgattg actgagggca aaggtgttga ctcagccata 720

gaggccttgg gagctcaaga gacctttgaa gcttgcatca aagtaactcg cccaggtggg 780

acgatctcag tagtgggata ctttggcaaa ggagattatg tcaaaattcc aaggcttgaa 840

tggggcgttg gaatgagtga taagaccatc aggacaggac tctgtccggg tggtaaagag 900

cgcatgcagc ggttgcttat gttgattaaa acaggccggg ttgatcctac acctctgacc 960

actcacacat tcaagttcga tgaactcgaa agagccttcc acatgatgga aactaaagag 1020

gacgggataa taaagcccct cataatcttt gactaa 1056

<210> 14

<211> 351

<212> PRT

<213> 斯氏假丝酵母(Candidatus Stahlbacteria)

<400> 14

Met Lys Ala Phe Val Met Lys Gly Ile Gly Lys Val Gly Phe Met Glu

1 5 10 15

Lys Pro Ile Pro Glu Asp Pro Gly Pro Asn Gly Ala Ile Ile Lys Thr

20 25 30

Thr Lys Ala Leu Val Cys Thr Ser Asp Thr His Thr Val Ala Gly Ala

35 40 45

Ile Gly Asp Arg Lys Asp Leu Thr Leu Gly His Glu Ala Val Gly Ile

50 55 60

Val Tyr Lys Leu Gly Ser Glu Val Lys Gly Ile Lys Glu Gly Asp Arg

65 70 75 80

Val Ala Val Asn Ala Ile Thr Pro Cys Tyr Lys Cys Glu Asn Cys Leu

85 90 95

Arg Gly Tyr Thr Ser Gln Cys Gln Gln Met Leu Gly Gly Trp Lys Phe

100 105 110

Ala Asn Ile Lys Asp Gly Val Phe Ala Glu Tyr Phe His Val Asn Asp

115 120 125

Ala Glu Ala Asn Leu Ala Leu Ile Pro Asp Ser Ile Pro Asp Glu Ala

130 135 140

Ala Val Tyr Thr Thr Asp Met Met Ser Thr Gly Phe Met Gly Ala Glu

145 150 155 160

His Ala Asn Ile Pro Leu Gly Gly Thr Val Ala Ile Phe Ala Gln Gly

165 170 175

Pro Ile Gly Leu Met Cys Thr Val Gly Ala Arg Leu Leu Gly Ala Gly

180 185 190

Leu Val Ile Ala Val Glu Ser Met Ser Lys Arg Lys Glu Leu Ala Lys

195 200 205

His Phe Gly Ala Asp Thr Val Val Asp Phe Thr Lys Val Asp Pro Val

210 215 220

Lys Asp Ile Leu Arg Leu Thr Glu Gly Lys Gly Val Asp Ser Ala Ile

225 230 235 240

Glu Ala Leu Gly Ala Gln Glu Thr Phe Glu Ala Cys Ile Lys Val Thr

245 250 255

Arg Pro Gly Gly Thr Ile Ser Val Val Gly Tyr Phe Gly Lys Gly Asp

260 265 270

Tyr Val Lys Ile Pro Arg Leu Glu Trp Gly Val Gly Met Ser Asp Lys

275 280 285

Thr Ile Arg Thr Gly Leu Cys Pro Gly Gly Lys Glu Arg Met Gln Arg

290 295 300

Leu Leu Met Leu Ile Lys Thr Gly Arg Val Asp Pro Thr Pro Leu Thr

305 310 315 320

Thr His Thr Phe Lys Phe Asp Glu Leu Glu Arg Ala Phe His Met Met

325 330 335

Glu Thr Lys Glu Asp Gly Ile Ile Lys Pro Leu Ile Ile Phe Asp

340 345 350

<210> 15

<211> 912

<212> DNA

<213> 硫氧化还原脱硫弧菌(Desulfolutivibrio sulfoxidireducens)

<400> 15

atgaaacaca cgcaatgcct cattgtcggc gcggggccag ccgggctttc ggcggccatc 60

tataccgcca gggccggcgt ggacaccctg gccctgggct gcaatcccaa ggtcgcgggc 120

gactacgaca tcgacaacta ttttggtttc ccggagaccg tcaccggccg tgagctcatc 180

gagcgcggca tggcccaggc caaacgtttc ggggccacgc tgcggtgcga gcgggtgatg 240

gccgtgcacc acggcgaaaa cggaaacttt gtggtcaaga gcgatacgga cgaatatgcg 300

gcggacgcgg tgatcatcgc cgccggggtg gcaagggtgc gaccgggcat cgccaacctc 360

gcggactacg agggcaaggg cgtgtcctac tgcgtcagtt gcgacggttt tttctaccgg 420

ggcaaatcgg tcctggtcct cggcgaaggg gactacgccg ccaaccaggc cctggaactg 480

accaccttca ccccccaggt ggccatctac acccagggca aggccccggt catatccgaa 540

ggcttcgcgg cgcgcctctc ccaggcggga atcccggtga tcgaacagac cgtggccacc 600

ctggccggcg aaccggccct ggccgcggcg cgtctggcca acggcaccga actccccgtg 660

gacggcatct tcatcgccat gggccaggcc tcggccctgg atttcgccaa gaccctgggt 720

ttgccgcttc gcggggcgtt catccaggcc gaccacgacc agaaaaccgc cctgcccggg 780

gtgttcgcgg ccggagactg cgtggggcgg tttctccaga tcagcgtggc cgtgggcgaa 840

ggggccaagg ccggaaaatc ggccatcgcc tatctcaaac aggcgggcgc ggcccataca 900

aagacatcgt ga 912

<210> 16

<211> 350

<212> PRT

<213> 硫氧化还原脱硫弧菌(Desulfolutivibrio sulfoxidireducens)

<400> 16

Met Lys Ala Phe Val Met His Gly Ile Gly Lys Val Gly Ile Val Asp

1 5 10 15

Lys Pro Ile Pro Glu Pro Gly Pro Asn Asp Val Ile Leu Lys Thr Thr

20 25 30

Ser Ala Leu Ile Cys Thr Ser Asp Val His Thr Val Gly Gly Ala Ile

35 40 45

Gly Asp Arg Lys Asn Leu Thr Leu Gly His Glu Ala Gly Gly Ile Val

50 55 60

Tyr Lys Ile Gly Ser Ala Val Thr Gly Phe Lys Glu Gly Asp Arg Cys

65 70 75 80

Val Val Asn Ala Ile Thr Pro Cys Tyr Lys Cys Gln Asn Cys Leu Arg

85 90 95

Gly Phe Pro Ser Gln Cys Gly Glu Ala Cys Gly Gly Trp Lys Tyr Ala

100 105 110

Asn Ile Lys Asp Gly Ser Phe Ala Glu Tyr Phe His Val Asn Asp Ala

115 120 125

Ile Ala Asn Leu Val Lys Val Pro Asp Asp Val Thr Asp Glu Ala Ala

130 135 140

Leu Tyr Thr Thr Asp Met Met Ala Thr Gly Phe Met Gly Ala Glu His

145 150 155 160

Gly Asn Thr Pro Leu Gly Gly Ser Val Ala Val Phe Gly Gln Gly Pro

165 170 175

Val Gly Leu Met Ala Thr Ala Gly Ala Arg Leu Leu Gly Ala Gly Leu

180 185 190

Ile Ile Ala Val Glu Ser Val Pro Lys Arg Gln Glu Leu Ala Lys Tyr

195 200 205

Tyr Gly Ala Asp Val Ile Val Asp Phe Ser Lys Val Asp Ala Val Ala

210 215 220

Glu Ile Lys Arg Leu Thr Gly Gly Glu Gly Val Asp Thr Ala Ile Glu

225 230 235 240

Ala Leu Gly Met Gln Pro Thr Phe Glu Asn Cys Val Lys Ala Thr Arg

245 250 255

Pro Gly Gly Thr Ile Ser Asn Cys Gly Tyr His Gly Lys Gly Glu Tyr

260 265 270

Val Lys Ile Pro Arg Val Glu Trp Gly Phe Gly Met Ala Asp Lys Thr

275 280 285

Ile Arg Thr Gly Leu Cys Pro Gly Gly Ser Glu Arg Met Gly Arg Leu

290 295 300

Leu Arg Leu Ile Gln Asn Gly Arg Ile Asp Pro Thr Lys Leu Thr Thr

305 310 315 320

His Arg Met Pro Phe Ser Asp Ile Glu Lys Gly Phe Arg Ile Met Ala

325 330 335

Asn Lys Glu Asp Gly Val Ile Lys Pro Met Ile Leu Phe Ser

340 345 350

<210> 17

<211> 1050

<212> DNA

<213> 脱硫杆菌(Desulfobacterales bacterium)

<400> 17

atgaaagcat tcgttatgca gggtattggt aaggtaggta tcgtggataa acccattccg 60

gaacccggcc caaatgatgc cgtcatcaaa accaccacag cacttgtctg tacatctgac 120

gttcatactg tcgctggtgc gattggtgac agatacaacc tcacacttgg tcacgaaggt 180

atgggtgttg tttacaaatt ggggagcgaa gtgaaggggt ttaaggaagg cgaccgcgtt 240

gtgatcaatg cgattacccc gtgttaccag tgcacaaact gtcagcgggg gttcacctcc 300

caatgtgagg aagccttagg gggctggaag tatgcgaaca ttaaagatgg ttgctttgcc 360

gaatactttc atgtcaacga cgccaaggcc aatatggtca aggttcctga cgatgttacg 420

gatgaacagg cgctctacac aacagacatg atgtccacag gctttatggg ggctgagcac 480

gcaaacatcc ccttgggtgg cacggttgcc gtttttggac agggtcctgt tggcctgatg 540

gccacagcag gtgcaaaact tttaggcgcg ggtttgatta tcggtgttga atgcgtgcct 600

gaacgccaaa agctgtccag acaatttggt gcagacgaaa tagtggattt tacaaaggtt 660

gacaccattg aagaaatcat gcgcctcact gacggagaag gcgttgactc tgccatagaa 720

gccttgggtt cacaaattac ttttgaaaat tgtgttaagg ccactcgtcc tggtggcacc 780

atttccaatg cgggatatca tggtgatggc gactatgtca aaatacctcg cgcagaatgg 840

ggcgtaggta tggcggataa gacaattcga accggcctgt gccctggtgg aagcgaaaga 900

atggggcgtc tcctccgact cttgcaacgg aatcggattg atccgacggc catgaccaca 960

catcggttcc cctttgacga gatcgaaaaa ggatatcttc tcatgaagac caaagaagat 1020

aatgtattga aaccacttat agaattctag 1050

<210> 18

<211> 349

<212> PRT

<213> 脱硫杆菌(Desulfobacterales bacterium)

<400> 18

Met Lys Ala Phe Val Met Gln Gly Ile Gly Lys Val Gly Ile Val Asp

1 5 10 15

Lys Pro Ile Pro Glu Pro Gly Pro Asn Asp Ala Val Ile Lys Thr Thr

20 25 30

Thr Ala Leu Val Cys Thr Ser Asp Val His Thr Val Ala Gly Ala Ile

35 40 45

Gly Asp Arg Tyr Asn Leu Thr Leu Gly His Glu Gly Met Gly Val Val

50 55 60

Tyr Lys Leu Gly Ser Glu Val Lys Gly Phe Lys Glu Gly Asp Arg Val

65 70 75 80

Val Ile Asn Ala Ile Thr Pro Cys Tyr Gln Cys Thr Asn Cys Gln Arg

85 90 95

Gly Phe Thr Ser Gln Cys Glu Glu Ala Leu Gly Gly Trp Lys Tyr Ala

100 105 110

Asn Ile Lys Asp Gly Cys Phe Ala Glu Tyr Phe His Val Asn Asp Ala

115 120 125

Lys Ala Asn Met Val Lys Val Pro Asp Asp Val Thr Asp Glu Gln Ala

130 135 140

Leu Tyr Thr Thr Asp Met Met Ser Thr Gly Phe Met Gly Ala Glu His

145 150 155 160

Ala Asn Ile Pro Leu Gly Gly Thr Val Ala Val Phe Gly Gln Gly Pro

165 170 175

Val Gly Leu Met Ala Thr Ala Gly Ala Lys Leu Leu Gly Ala Gly Leu

180 185 190

Ile Ile Gly Val Glu Cys Val Pro Glu Arg Gln Lys Leu Ser Arg Gln

195 200 205

Phe Gly Ala Asp Glu Ile Val Asp Phe Thr Lys Val Asp Thr Ile Glu

210 215 220

Glu Ile Met Arg Leu Thr Asp Gly Glu Gly Val Asp Ser Ala Ile Glu

225 230 235 240

Ala Leu Gly Ser Gln Ile Thr Phe Glu Asn Cys Val Lys Ala Thr Arg

245 250 255

Pro Gly Gly Thr Ile Ser Asn Ala Gly Tyr His Gly Asp Gly Asp Tyr

260 265 270

Val Lys Ile Pro Arg Ala Glu Trp Gly Val Gly Met Ala Asp Lys Thr

275 280 285

Ile Arg Thr Gly Leu Cys Pro Gly Gly Ser Glu Arg Met Gly Arg Leu

290 295 300

Leu Arg Leu Leu Gln Arg Asn Arg Ile Asp Pro Thr Ala Met Thr Thr

305 310 315 320

His Arg Phe Pro Phe Asp Glu Ile Glu Lys Gly Tyr Leu Leu Met Lys

325 330 335

Thr Lys Glu Asp Asn Val Leu Lys Pro Leu Ile Glu Phe

340 345

<210> 19

<211> 1050

<212> DNA

<213> 假丝酵母亚细菌(Candidatus Abyssubacteria bacterium SURF_5)

<400> 19

atgagagcct ttgtgatgaa agggatcggg gaggtgggca ttgttgaaaa gccgattccc 60

gaggatcccg gcccgaatgg ggcgatcata agaacgacca gggcgcttat ttgcacgtcg 120

gatgcgcaca cggtgttggg cggtatcgcc gagcgcaggg atctcaccct cggccatgag 180

gccgttggca tagtgcatcg gctcggcagc gaggtcaggc acgtcaaaga aggcgaccgc 240

gtcgccgtca atgcgattac gccgtgttat cactgtgaaa actgtcttcg cggctacact 300

tcacagtgca ccacgatgct cggcggttgg aagttcgcaa acacgaaaga cggcgtgttt 360

tccgacttct ttcatgtgaa tgacgccgaa gccaaccttg cgcctattcc cgattccgtt 420

cccgacgagg cggccgttta cacgtgcgac atgatgtcga caggcttcat gggcgccgag 480

aacgcgaata tccctgtcgg gggcattgtg gcggtattcg cgcaaggtcc ggtggggctg 540

atggcgaccg cgggtgcaaa gcttttggga gcgggactga tcatcactgt agaatcgatc 600

ccccggcgca aagagctttc caaacgatac ggagccgatt tggtgatcga cttcaaggat 660

cgtgatcccg tcaaggcgat acttgatctt accgatggca gcggagtgga ttcgagcatc 720

gaatcgctag gatcgcaagc gacgtttgag gcctgcataa aagtgacccg cccgggcggc 780

accatttcct cgatcgggta ctacggcaaa ggagactacg tcaagattcc gcgcgtcgaa 840

tggggggttg ggatgggtga taaaaccatc cggaccggcc tgtgcccggg cggcaaggag 900

cggatgaagc ggctgttgag gatgatcgag aacgggcgaa tcgacccgac gcctttgact 960

tcgcacacgt tcggttttga cgagatcgag aaggcgttcc acttgatgga aacgaaaggc 1020

gataacatca taaagccgct tatcatcttt 1050

<210> 20

<211> 351

<212> PRT

<213> 假丝酵母亚细菌(Candidatus Abyssubacteria bacterium SURF_5)

<400> 20

Met Arg Ala Phe Val Met Lys Gly Ile Gly Glu Val Gly Ile Val Glu

1 5 10 15

Lys Pro Ile Pro Glu Asp Pro Gly Pro Asn Gly Ala Ile Ile Arg Thr

20 25 30

Thr Arg Ala Leu Ile Cys Thr Ser Asp Ala His Thr Val Leu Gly Gly

35 40 45

Ile Ala Glu Arg Arg Asp Leu Thr Leu Gly His Glu Ala Val Gly Ile

50 55 60

Val His Arg Leu Gly Ser Glu Val Arg His Val Lys Glu Gly Asp Arg

65 70 75 80

Val Ala Val Asn Ala Ile Thr Pro Cys Tyr His Cys Glu Asn Cys Leu

85 90 95

Arg Gly Tyr Thr Ser Gln Cys Thr Thr Met Leu Gly Gly Trp Lys Phe

100 105 110

Ala Asn Thr Lys Asp Gly Val Phe Ser Asp Phe Phe His Val Asn Asp

115 120 125

Ala Glu Ala Asn Leu Ala Pro Ile Pro Asp Ser Val Pro Asp Glu Ala

130 135 140

Ala Val Tyr Thr Cys Asp Met Met Ser Thr Gly Phe Met Gly Ala Glu

145 150 155 160

Asn Ala Asn Ile Pro Val Gly Gly Ile Val Ala Val Phe Ala Gln Gly

165 170 175

Pro Val Gly Leu Met Ala Thr Ala Gly Ala Lys Leu Leu Gly Ala Gly

180 185 190

Leu Ile Ile Thr Val Glu Ser Ile Pro Arg Arg Lys Glu Leu Ser Lys

195 200 205

Arg Tyr Gly Ala Asp Leu Val Ile Asp Phe Lys Asp Arg Asp Pro Val

210 215 220

Lys Ala Ile Leu Asp Leu Thr Asp Gly Ser Gly Val Asp Ser Ser Ile

225 230 235 240

Glu Ser Leu Gly Ser Gln Ala Thr Phe Glu Ala Cys Ile Lys Val Thr

245 250 255

Arg Pro Gly Gly Thr Ile Ser Ser Ile Gly Tyr Tyr Gly Lys Gly Asp

260 265 270

Tyr Val Lys Ile Pro Arg Val Glu Trp Gly Val Gly Met Gly Asp Lys

275 280 285

Thr Ile Arg Thr Gly Leu Cys Pro Gly Gly Lys Glu Arg Met Lys Arg

290 295 300

Leu Leu Arg Met Ile Glu Asn Gly Arg Ile Asp Pro Thr Pro Leu Thr

305 310 315 320

Ser His Thr Phe Gly Phe Asp Glu Ile Glu Lys Ala Phe His Leu Met

325 330 335

Glu Thr Lys Gly Asp Asn Ile Ile Lys Pro Leu Ile Ile Phe Asp

340 345 350

<210> 21

<211> 1053

<212> DNA

<213> 硝化螺旋菌(Nitrospirales bacterium)

<400> 21

atgaaagcct ttgtgatgaa acggatcggc gaggtgggcg tgatggagaa gccaataccc 60

gatccgggtc ccaatgacgc aatcgttaag accactgccg cactcgtctg tacatcagac 120

atccatacgg tagctggttc cattggggaa cgggggaatt taactctcgg gcatgaagcg 180

gtcggagtcg tacacaaact cggccatgcg gtaaagggat ttcgagaagg tgatcgggtc 240

gtcgtgaatg ccattacccc atgttacctc tgtgagaatt gtttgcgagg gtacacctcc 300

caatgcaccg agatgcttgg aggttggaaa ttcgccaatg tgaaagatgg caacatggcg 360

gaatattttc atgtcaacag cgcgcaagcc aatctggcgc ccatcccggc tagtttgacg 420

gatgagcagg cactctattg cacggacatg atgtcgaccg gcttcatggg cgccgaacac 480

gccaacattc ccatcggtgg aagcgtcgcc atttttgcgc agggacccgt cggattgatg 540

gcgacagtcg gtgcaaaatt gttaggagca ggtcttatca tcgccgtaga aacagtgccg 600

catcggcaag aactggccaa gcggtttggt gcagatgtgg tcatcgattt caaaaaccaa 660

gaccctgtca ccgcgattct tgatctaacg gatgggctag gtgtggatgc ggctattgag 720

gcactgggac tccaggtaag tttcgagggc tgtatcaagg cgacgcgacc tgggggaacg 780

atttcaaaca ttggctatca cggcgatggg gagtttgtgg agattccaag ggcagcctgg 840

ggggtcggga tgggtgataa gaccattcga accgggcttt gtccgggagg agctgagcgg 900

atgaaacgac tcatgcgact ccttgtgatg ggtcgggtgg atccaacccc gctcacgact 960

catcgcttca acttcagcga ggtggaaaag gctttctcca tgatgaaaac caaagaggat 1020

ggcatgctga aacccttgat ccttttcgac taa 1053

<210> 22

<211> 350

<212> PRT

<213> 硝化螺旋菌(Nitrospirales bacterium)

<400> 22

Met Lys Ala Phe Val Met Lys Arg Ile Gly Glu Val Gly Val Met Glu

1 5 10 15

Lys Pro Ile Pro Asp Pro Gly Pro Asn Asp Ala Ile Val Lys Thr Thr

20 25 30

Ala Ala Leu Val Cys Thr Ser Asp Ile His Thr Val Ala Gly Ser Ile

35 40 45

Gly Glu Arg Gly Asn Leu Thr Leu Gly His Glu Ala Val Gly Val Val

50 55 60

His Lys Leu Gly His Ala Val Lys Gly Phe Arg Glu Gly Asp Arg Val

65 70 75 80

Val Val Asn Ala Ile Thr Pro Cys Tyr Leu Cys Glu Asn Cys Leu Arg

85 90 95

Gly Tyr Thr Ser Gln Cys Thr Glu Met Leu Gly Gly Trp Lys Phe Ala

100 105 110

Asn Val Lys Asp Gly Asn Met Ala Glu Tyr Phe His Val Asn Ser Ala

115 120 125

Gln Ala Asn Leu Ala Pro Ile Pro Ala Ser Leu Thr Asp Glu Gln Ala

130 135 140

Leu Tyr Cys Thr Asp Met Met Ser Thr Gly Phe Met Gly Ala Glu His

145 150 155 160

Ala Asn Ile Pro Ile Gly Gly Ser Val Ala Ile Phe Ala Gln Gly Pro

165 170 175

Val Gly Leu Met Ala Thr Val Gly Ala Lys Leu Leu Gly Ala Gly Leu

180 185 190

Ile Ile Ala Val Glu Thr Val Pro His Arg Gln Glu Leu Ala Lys Arg

195 200 205

Phe Gly Ala Asp Val Val Ile Asp Phe Lys Asn Gln Asp Pro Val Thr

210 215 220

Ala Ile Leu Asp Leu Thr Asp Gly Leu Gly Val Asp Ala Ala Ile Glu

225 230 235 240

Ala Leu Gly Leu Gln Val Ser Phe Glu Gly Cys Ile Lys Ala Thr Arg

245 250 255

Pro Gly Gly Thr Ile Ser Asn Ile Gly Tyr His Gly Asp Gly Glu Phe

260 265 270

Val Glu Ile Pro Arg Ala Ala Trp Gly Val Gly Met Gly Asp Lys Thr

275 280 285

Ile Arg Thr Gly Leu Cys Pro Gly Gly Ala Glu Arg Met Lys Arg Leu

290 295 300

Met Arg Leu Leu Val Met Gly Arg Val Asp Pro Thr Pro Leu Thr Thr

305 310 315 320

His Arg Phe Asn Phe Ser Glu Val Glu Lys Ala Phe Ser Met Met Lys

325 330 335

Thr Lys Glu Asp Gly Met Leu Lys Pro Leu Ile Leu Phe Asp

340 345 350

<210> 23

<211> 1053

<212> DNA

<213> 硝化螺旋菌(Nitrospirales bacterium)

<400> 23

atgaaagcct ttgtgatgaa acggatcggc gaggtgggcg tgatggagaa accaataccc 60

gatccgggtc ccaatgacgc aatcgttaag accactgccg cactcgtctg tacatcagac 120

atccatacgg tagctggttc cattgggcaa cgggggaatt taactctcgg gcatgaagcg 180

gtcggagtcg tacacaaact cggccatgcg gtaaagggat ttcgagaagg tgatcgggtc 240

gtcgtgaatg ccattacccc atgttacctc tgtgagaatt gtttgcgagg gtacacctcc 300

caatgcaccg agatgcttgg aggttggaaa ttcgccaatg tgaaagatgg caacatggcg 360

gaatattttc atgtcaacag cgcgcaagcc aatctggcgc ccatcccggc tagtttgacg 420

gatgagcagg cactctattg cacggacatg atgtcgaccg gcttcatggg cgccgaacac 480

gccaacattc ccatcggtgg aagcgtcgcc atttttgcgc agggacccgt cggattgatg 540

gcgacagtcg gtgcaaaatt gttaggagca ggtcttatca tcgccgtaga aacagtgccg 600

catcggcaag aactggccaa gcggtttggt gcagatgtgg tcatcgattt caaaaaccaa 660

gaccctgtca ccgcgattct tgatctaacg gatgggctag gtgtggatgc ggctattgag 720

gcactgggac tccaggtaag tttcgagggc tgtatcaagg cgacgcgacc tgggggaacg 780

atttcaaaca ttggctatca cggcgatggg gagtttgtgg agattccaag ggcagcctgg 840

ggggtcggga tgggtgataa gaccattcga accgggcttt gtccgggagg agctgagcgg 900

atgaaacgac tcatgcgact ccttgtgatg ggtcgggtgg atccaacccc gctcacgact 960

catcgcttca acttcagcga ggtggaaaag gctttctcca tgatgaaaac caaagaggat 1020

ggcatgctga aacccttgat ccttttcgac taa 1053

<210> 24

<211> 350

<212> PRT

<213> 硝化螺旋菌(Nitrospirales bacterium)

<400> 24

Met Lys Ala Phe Val Met Lys Arg Ile Gly Glu Val Gly Val Met Glu

1 5 10 15

Lys Pro Ile Pro Asp Pro Gly Pro Asn Asp Ala Ile Val Lys Thr Thr

20 25 30

Ala Ala Leu Val Cys Thr Ser Asp Ile His Thr Val Ala Gly Ser Ile

35 40 45

Gly Gln Arg Gly Asn Leu Thr Leu Gly His Glu Ala Val Gly Val Val

50 55 60

His Lys Leu Gly His Ala Val Lys Gly Phe Arg Glu Gly Asp Arg Val

65 70 75 80

Val Val Asn Ala Ile Thr Pro Cys Tyr Leu Cys Glu Asn Cys Leu Arg

85 90 95

Gly Tyr Thr Ser Gln Cys Thr Glu Met Leu Gly Gly Trp Lys Phe Ala

100 105 110

Asn Val Lys Asp Gly Asn Met Ala Glu Tyr Phe His Val Asn Ser Ala

115 120 125

Gln Ala Asn Leu Ala Pro Ile Pro Ala Ser Leu Thr Asp Glu Gln Ala

130 135 140

Leu Tyr Cys Thr Asp Met Met Ser Thr Gly Phe Met Gly Ala Glu His

145 150 155 160

Ala Asn Ile Pro Ile Gly Gly Ser Val Ala Ile Phe Ala Gln Gly Pro

165 170 175

Val Gly Leu Met Ala Thr Val Gly Ala Lys Leu Leu Gly Ala Gly Leu

180 185 190

Ile Ile Ala Val Glu Thr Val Pro His Arg Gln Glu Leu Ala Lys Arg

195 200 205

Phe Gly Ala Asp Val Val Ile Asp Phe Lys Asn Gln Asp Pro Val Thr

210 215 220

Ala Ile Leu Asp Leu Thr Asp Gly Leu Gly Val Asp Ala Ala Ile Glu

225 230 235 240

Ala Leu Gly Leu Gln Val Ser Phe Glu Gly Cys Ile Lys Ala Thr Arg

245 250 255

Pro Gly Gly Thr Ile Ser Asn Ile Gly Tyr His Gly Asp Gly Glu Phe

260 265 270

Val Glu Ile Pro Arg Ala Ala Trp Gly Val Gly Met Gly Asp Lys Thr

275 280 285

Ile Arg Thr Gly Leu Cys Pro Gly Gly Ala Glu Arg Met Lys Arg Leu

290 295 300

Met Arg Leu Leu Val Met Gly Arg Val Asp Pro Thr Pro Leu Thr Thr

305 310 315 320

His Arg Phe Asn Phe Ser Glu Val Glu Lys Ala Phe Ser Met Met Lys

325 330 335

Thr Lys Glu Asp Gly Met Leu Lys Pro Leu Ile Leu Phe Asp

340 345 350

<210> 25

<211> 1065

<212> DNA

<213> 硝化螺旋菌(Nitrospirales bacterium)

<400> 25

atgacccgga caatgaaagc ctttgtaatg cggaagctcg gctcggtggg agtgatggag 60

aagccgattc ctgatcctgg accgaatgat gcgatcgtca aaaccacggc cgcgctcatc 120

tgcacctcgg atgtccatac cgttgacggc gcgatcggag aacggaccaa cctgaccctg 180

ggccatgaag ccgtcggcgt catttacaaa ctcggaagcg ccgtcaaggg gcttcgtgag 240

ggagatcggg tcggagtcaa cgccattact ccctgctatc agtgcgagaa ttgcctgcgc 300

ggctacacct cgcagtgtca ggagatgctc ggcggctgga agttcgccaa cgtcaaggat 360

ggaaacctcg cggaatattt ccatgtcaat agcgcgcaag cgaacctggc gccgattcca 420

acgggtctta ccgacgaaca agtcgcgtac tgcgcggaca tgatgtccac cgggttcatg 480

ggggctgagc atgcgaacat tcctgttggg ggctcagtgg ccgtctttgc gcagggacca 540

gtcggactga tggccaccgt gggggcccgg ttactgggag ccggcttggt catcgcagtg 600

gaggccgtcc cgcagcgcaa gaaactggcg aaagagttcg gggcggatgt ggtgatcgac 660

ttcaaggaac aggatcccgt cgaggtcatt ctcggtttga cgggcggaca aggcgtggat 720

tcgtccatcg aagccctcgg ggctcaggcg acgttcgaag cctgtatcaa agcgacacgg 780

cccggcggga ccatctctaa cgtgggctat cacggagagg gagaatacgt gcagatgcct 840

cgtaaagaat ggggcgtcgg gatgagcgac aaggtcatcc gaacggggct gtgtcccggc 900

ggagcggaac ggatgaaacg gctgatgcgg ctcttagaaa ccgggcgcgt caatccactc 960

cctctgacga cccatcgatt caatttccac gatgtggaga aggcctttga gttgatgcga 1020

acgaaggcag acggcatgtt gaagccgctc atcacgtttg cgtga 1065

<210> 26

<211> 354

<212> PRT

<213> 硝化螺旋菌(Nitrospirales bacterium)

<400> 26

Met Thr Arg Thr Met Lys Ala Phe Val Met Arg Lys Leu Gly Ser Val

1 5 10 15

Gly Val Met Glu Lys Pro Ile Pro Asp Pro Gly Pro Asn Asp Ala Ile

20 25 30

Val Lys Thr Thr Ala Ala Leu Ile Cys Thr Ser Asp Val His Thr Val

35 40 45

Asp Gly Ala Ile Gly Glu Arg Thr Asn Leu Thr Leu Gly His Glu Ala

50 55 60

Val Gly Val Ile Tyr Lys Leu Gly Ser Ala Val Lys Gly Leu Arg Glu

65 70 75 80

Gly Asp Arg Val Gly Val Asn Ala Ile Thr Pro Cys Tyr Gln Cys Glu

85 90 95

Asn Cys Leu Arg Gly Tyr Thr Ser Gln Cys Gln Glu Met Leu Gly Gly

100 105 110

Trp Lys Phe Ala Asn Val Lys Asp Gly Asn Leu Ala Glu Tyr Phe His

115 120 125

Val Asn Ser Ala Gln Ala Asn Leu Ala Pro Ile Pro Thr Gly Leu Thr

130 135 140

Asp Glu Gln Val Ala Tyr Cys Ala Asp Met Met Ser Thr Gly Phe Met

145 150 155 160

Gly Ala Glu His Ala Asn Ile Pro Val Gly Gly Ser Val Ala Val Phe

165 170 175

Ala Gln Gly Pro Val Gly Leu Met Ala Thr Val Gly Ala Arg Leu Leu

180 185 190

Gly Ala Gly Leu Val Ile Ala Val Glu Ala Val Pro Gln Arg Lys Lys

195 200 205

Leu Ala Lys Glu Phe Gly Ala Asp Val Val Ile Asp Phe Lys Glu Gln

210 215 220

Asp Pro Val Glu Val Ile Leu Gly Leu Thr Gly Gly Gln Gly Val Asp

225 230 235 240

Ser Ser Ile Glu Ala Leu Gly Ala Gln Ala Thr Phe Glu Ala Cys Ile

245 250 255

Lys Ala Thr Arg Pro Gly Gly Thr Ile Ser Asn Val Gly Tyr His Gly

260 265 270

Glu Gly Glu Tyr Val Gln Met Pro Arg Lys Glu Trp Gly Val Gly Met

275 280 285

Ser Asp Lys Val Ile Arg Thr Gly Leu Cys Pro Gly Gly Ala Glu Arg

290 295 300

Met Lys Arg Leu Met Arg Leu Leu Glu Thr Gly Arg Val Asn Pro Leu

305 310 315 320

Pro Leu Thr Thr His Arg Phe Asn Phe His Asp Val Glu Lys Ala Phe

325 330 335

Glu Leu Met Arg Thr Lys Ala Asp Gly Met Leu Lys Pro Leu Ile Thr

340 345 350

Phe Ala

<210> 27

<211> 1053

<212> DNA

<213> 扁平菌科细菌(Planctomycetaceae bacterium)

<400> 27

atgaaagcat ttgtcatgaa gaagctcggc tcggtgggga tgatggagaa gccaattcct 60

gatcccggac caaacgatgc cgttgtcaag acgaccgccg cactcgtctg tacgtccgat 120

gtccatacgg tcggcggggc gatcggtgaa cgcaccaacc tgaccctggg ccatgaggcc 180

gtgggggtca tttacaaatt gggcagcgcc gtgaagggat ttcgtgaggg agatcgagtc 240

gcagtcaacg ccatcacgcc ctgttatcag tgcgagaact gtttgcgcgg ttacacctct 300

caatgtaccg agatgctcgg cgggtggaag ttcgcgaacg tgaaggatgg gaatctcgcc 360

gaatatttcc atgtaaacag cgcgcaagcg aacctggcga cgattcctcc gagtctcaca 420

gatgagcagg tggcttactg cacggacatg atgtcgaccg ggttcatggg agctgagcat 480

gcgaacattc ccgttggggg cgcagtggct gtcttcgcgc agggaccagt cggactgatg 540

gccaccgtgg gggcccggtt attgggggcc ggcttggtca ttgcggtgga ggtggtcccg 600

cagcgcaaga agctagcgaa agagttcggg gccgatgtcg tgctggactt caaggaacag 660

gatccggtca gggccattct ggatttgacc ggcggacagg gggtggactc gtccatcgag 720

gccttggggg ctcaggagac cttcgaggcc tgcgtcaagg ccacgcgtcc gggcgggacg 780

atctcgaata ttggatacca tggcgaagga gattatgtgc agatgcctcg caaggagtgg 840

ggggtcggga tgggcgataa ggtgatcagg acgggtctct gccccggggg agccgaacgg 900

atgaagcggc tgatgcgact cttggaaacc ggccgggtca atccactccc actgacgacg 960

catcggttcc gattcaatga catcgaaaag gccttcgacc tgatgagaac gaaggcagac 1020

ggcatattga agccgctcat cacgtttatg tga 1053

<210> 28

<211> 350

<212> PRT

<213> 扁平菌科细菌(Planctomycetaceae bacterium)

<400> 28

Met Lys Ala Phe Val Met Lys Lys Leu Gly Ser Val Gly Met Met Glu

1 5 10 15

Lys Pro Ile Pro Asp Pro Gly Pro Asn Asp Ala Val Val Lys Thr Thr

20 25 30

Ala Ala Leu Val Cys Thr Ser Asp Val His Thr Val Gly Gly Ala Ile

35 40 45

Gly Glu Arg Thr Asn Leu Thr Leu Gly His Glu Ala Val Gly Val Ile

50 55 60

Tyr Lys Leu Gly Ser Ala Val Lys Gly Phe Arg Glu Gly Asp Arg Val

65 70 75 80

Ala Val Asn Ala Ile Thr Pro Cys Tyr Gln Cys Glu Asn Cys Leu Arg

85 90 95

Gly Tyr Thr Ser Gln Cys Thr Glu Met Leu Gly Gly Trp Lys Phe Ala

100 105 110

Asn Val Lys Asp Gly Asn Leu Ala Glu Tyr Phe His Val Asn Ser Ala

115 120 125

Gln Ala Asn Leu Ala Thr Ile Pro Pro Ser Leu Thr Asp Glu Gln Val

130 135 140

Ala Tyr Cys Thr Asp Met Met Ser Thr Gly Phe Met Gly Ala Glu His

145 150 155 160

Ala Asn Ile Pro Val Gly Gly Ala Val Ala Val Phe Ala Gln Gly Pro

165 170 175

Val Gly Leu Met Ala Thr Val Gly Ala Arg Leu Leu Gly Ala Gly Leu

180 185 190

Val Ile Ala Val Glu Val Val Pro Gln Arg Lys Lys Leu Ala Lys Glu

195 200 205

Phe Gly Ala Asp Val Val Leu Asp Phe Lys Glu Gln Asp Pro Val Arg

210 215 220

Ala Ile Leu Asp Leu Thr Gly Gly Gln Gly Val Asp Ser Ser Ile Glu

225 230 235 240

Ala Leu Gly Ala Gln Glu Thr Phe Glu Ala Cys Val Lys Ala Thr Arg

245 250 255

Pro Gly Gly Thr Ile Ser Asn Ile Gly Tyr His Gly Glu Gly Asp Tyr

260 265 270

Val Gln Met Pro Arg Lys Glu Trp Gly Val Gly Met Gly Asp Lys Val

275 280 285

Ile Arg Thr Gly Leu Cys Pro Gly Gly Ala Glu Arg Met Lys Arg Leu

290 295 300

Met Arg Leu Leu Glu Thr Gly Arg Val Asn Pro Leu Pro Leu Thr Thr

305 310 315 320

His Arg Phe Arg Phe Asn Asp Ile Glu Lys Ala Phe Asp Leu Met Arg

325 330 335

Thr Lys Ala Asp Gly Ile Leu Lys Pro Leu Ile Thr Phe Met

340 345 350

<210> 29

<211> 1053

<212> DNA

<213> 厌氧绳菌(Anaerolineales bacterium)

<400> 29

atgaaagcat tcgtcatgaa gaaaattggt gaagtaggtt ttaccgaaaa accaatccct 60

gaaccgggac ctaatgatgc tgtcataaaa acaaccaaag cgttggtttg cacctcggat 120

gttcataccg ttaaaggcgc aatcggtgaa agagagaacc aaacccttgg gcatgaggct 180

gttggtgtcg cttataaact agggagcgaa gtaaacgagg tgaaagaagg cgaccgcgtt 240

gccgtaaatg ctattacacc atgctataaa tgtgaaaact gcatacgagg ttttacatct 300

caatgtcaac aactgttagg tggttggaaa tttgctaata tcaaagatgg ggtcttttca 360

gaatatttcc atgtcaacga tgctgaagcc aatcttacgt taattccaga tagtgtccct 420

gatgaagcag cagtctacgc tgccgatatg ctctctactg gttttatggg agctgaaaat 480

gctgatatac ctatgggtgg cagcgttgct gtttttgcac aaggtccagt tgggcttatg 540

gctactgtag gcgcacgtct tcaaggagca agtctagtaa ttgcagtcga caccattccc 600

aggagaatgg aattggcaaa acaatacgga gctgattttg tgatcgattt tcgaaaagct 660

gaacccgtgg aagaaattct tcgtttgact gatggaaaag gcgtagattc tacaatagaa 720

gctttaggcg ctcaaatcac attcgaagca tgtattaaag taactaaacc tggaggcaca 780

atctcgatca ccggttactt cggcgaaggt gattatgtaa aaattcccag actcgaatgg 840

ggagtaggga tgggtgataa gaccatccgc accggtcttt gtccaggagg caaagtacgc 900

atgcagaggt tgctacggct gatcgaaaat ggtcgcgtag atcccactct gatgactacc 960

cataaattca aattcgatga ggtggataaa gcttttcatc tgatggatac caaagatgat 1020

gatatattaa aaccccttat cctattcgat tga 1053

<210> 30

<211> 350

<212> PRT

<213> 厌氧绳菌(Anaerolineales bacterium)

<400> 30

Met Lys Ala Phe Val Met Lys Lys Ile Gly Glu Val Gly Phe Thr Glu

1 5 10 15

Lys Pro Ile Pro Glu Pro Gly Pro Asn Asp Ala Val Ile Lys Thr Thr

20 25 30

Lys Ala Leu Val Cys Thr Ser Asp Val His Thr Val Lys Gly Ala Ile

35 40 45

Gly Glu Arg Glu Asn Gln Thr Leu Gly His Glu Ala Val Gly Val Ala

50 55 60

Tyr Lys Leu Gly Ser Glu Val Asn Glu Val Lys Glu Gly Asp Arg Val

65 70 75 80

Ala Val Asn Ala Ile Thr Pro Cys Tyr Lys Cys Glu Asn Cys Ile Arg

85 90 95

Gly Phe Thr Ser Gln Cys Gln Gln Leu Leu Gly Gly Trp Lys Phe Ala

100 105 110

Asn Ile Lys Asp Gly Val Phe Ser Glu Tyr Phe His Val Asn Asp Ala

115 120 125

Glu Ala Asn Leu Thr Leu Ile Pro Asp Ser Val Pro Asp Glu Ala Ala

130 135 140

Val Tyr Ala Ala Asp Met Leu Ser Thr Gly Phe Met Gly Ala Glu Asn

145 150 155 160

Ala Asp Ile Pro Met Gly Gly Ser Val Ala Val Phe Ala Gln Gly Pro

165 170 175

Val Gly Leu Met Ala Thr Val Gly Ala Arg Leu Gln Gly Ala Ser Leu

180 185 190

Val Ile Ala Val Asp Thr Ile Pro Arg Arg Met Glu Leu Ala Lys Gln

195 200 205

Tyr Gly Ala Asp Phe Val Ile Asp Phe Arg Lys Ala Glu Pro Val Glu

210 215 220

Glu Ile Leu Arg Leu Thr Asp Gly Lys Gly Val Asp Ser Thr Ile Glu

225 230 235 240

Ala Leu Gly Ala Gln Ile Thr Phe Glu Ala Cys Ile Lys Val Thr Lys

245 250 255

Pro Gly Gly Thr Ile Ser Ile Thr Gly Tyr Phe Gly Glu Gly Asp Tyr

260 265 270

Val Lys Ile Pro Arg Leu Glu Trp Gly Val Gly Met Gly Asp Lys Thr

275 280 285

Ile Arg Thr Gly Leu Cys Pro Gly Gly Lys Val Arg Met Gln Arg Leu

290 295 300

Leu Arg Leu Ile Glu Asn Gly Arg Val Asp Pro Thr Leu Met Thr Thr

305 310 315 320

His Lys Phe Lys Phe Asp Glu Val Asp Lys Ala Phe His Leu Met Asp

325 330 335

Thr Lys Asp Asp Asp Ile Leu Lys Pro Leu Ile Leu Phe Asp

340 345 350

<210> 31

<211> 1059

<212> DNA

<213> 弓形脱硫菌(Desulfotomaculum arcticum)

<400> 31

atgaaagctt ttgtaatgca cggaataggg cgcgtgggca tcatggaaaa gccgctgccg 60

gcagatccgg gtccaaacga cgcgatcatt aaaaccaccg cggctttggt ttgtacctcg 120

gacgttcaca ccgtgcacgg cgcgattggt gagaaggtgg atctgaccct gggccatgaa 180

gcgttgggaa tcatccacaa gctgggttcc gcggtcgaga gcctgaaagt tggcgaccgc 240

gtcgttgtga acgccatcac cccgtgttat aagtgcgaaa actgccagcg tggttttacc 300

agccaatgta ccaccatgct gggcgggtgg aaattcgcta atatcaagga cggctgcttc 360

gccgaatatt tccacgtaaa cgacgcggat gcaaatctgg tcccggttcc gtcgagcgtg 420

agcgacgagg cggcgctcta cgctacggat atgatgtcaa ccggtttcat gggtgctgag 480

cacgccggta ttccgctggg cggtacggtt gcaatcttcg gtcaaggtcc ggttggcctg 540

atggcgactg cgggctctcg cctgttgggc gctggtctca ttattgcggt tgagacagtg 600

ccgcatcgtc agcagctgag caaatactac ggcgcagaca tcatcctgga ttttaagaaa 660

gtggatgttg tggaggaaat tttgcgcatc accaatggcc aaggtgtcga cagcgcgata 720

gaagcgttag gcacgcaggt taccttcgag aactgcatta aggcaacccg tcctggtggc 780

acgatcagca acattggtta tcatggcgag ggtgattacc tgaaaattcc gcgtgtggac 840

tggggtgtgg gtatgtccga caaaaccatc cgtaccggtc tgtgcccggg cggtcgtgaa 900

cgtatgagtc gtcttctgcg tttgatcgaa tctggtagaa ttgatccgac tgcgttgact 960

acgcataaat tccgctttga tgaaatcgag aaggcctttg agctgatggc tatcaagggt 1020

gataatgtta ttaaaccact tattctgttt gaagattaa 1059

<210> 32

<211> 352

<212> PRT

<213> 弓形脱硫菌(Desulfotomaculum arcticum)

<400> 32

Met Lys Ala Phe Val Met His Gly Ile Gly Arg Val Gly Ile Met Glu

1 5 10 15

Lys Pro Leu Pro Ala Asp Pro Gly Pro Asn Asp Ala Ile Ile Lys Thr

20 25 30

Thr Ala Ala Leu Val Cys Thr Ser Asp Val His Thr Val His Gly Ala

35 40 45

Ile Gly Glu Lys Val Asp Leu Thr Leu Gly His Glu Ala Leu Gly Ile

50 55 60

Ile His Lys Leu Gly Ser Ala Val Glu Ser Leu Lys Val Gly Asp Arg

65 70 75 80

Val Val Val Asn Ala Ile Thr Pro Cys Tyr Lys Cys Glu Asn Cys Gln

85 90 95

Val Gly Phe Thr Ser Gln Cys Thr Thr Met Leu Gly Gly Trp Lys Phe

100 105 110

Ala Asn Ile Lys Asp Gly Cys Phe Ala Glu Tyr Phe His Val Asn Asp

115 120 125

Ala Asp Ala Asn Leu Val Pro Val Pro Ser Ser Val Ser Asp Glu Ala

130 135 140

Ala Leu Lys Ala Thr Asp Met Met Ser Thr Gly Phe Met Gly Ala Glu

145 150 155 160

His Ala Gly Ile Pro Leu Gly Gly Thr Val Ala Ile Phe Gly Gln Gly

165 170 175

Pro Val Gly Leu Met Ala Thr Ala Gly Ser Arg Leu Leu Gly Ala Gly

180 185 190

Leu Ile Ile Ala Val Glu Thr Val Pro His Arg Gln Gln Leu Ser Lys

195 200 205

Tyr Tyr Gly Ala Asp Ile Ile Leu Asp Phe Lys Lys Val Asp Val Val

210 215 220

Glu Glu Ile Leu Arg Ile Thr Asn Gly Gln Gly Val Asp Ser Ala Ile

225 230 235 240

Glu Ala Leu Gly Thr Gln Val Thr Phe Glu Asn Cys Ile Lys Ala Thr

245 250 255

Arg Pro Gly Gly Thr Ile Ser Asn Ile Gly Tyr His Gly Glu Gly Asp

260 265 270

Tyr Leu Lys Ile Pro Arg Val Asp Trp Gly Val Gly Met Ser Asp Lys

275 280 285

Thr Ile Arg Thr Gly Leu Cys Pro Gly Gly Arg Glu Arg Met Ser Arg

290 295 300

Leu Leu Arg Leu Ile Glu Ser Gly Arg Ile Asp Pro Thr Ala Leu Thr

305 310 315 320

Thr His Lys Phe Arg Phe Asp Glu Ile Glu Lys Ala Phe Glu Leu Met

325 330 335

Ala Ile Lys Gly Asp Asn Val Ile Lys Pro Leu Ile Leu Phe Glu Asp

340 345 350

<210> 33

<211> 1053

<212> DNA

<213> 马钱子脱硫弧菌(Desulfonatronovibrio magnus)

<400> 33

atgaaagcgt ttgttgttca cagtatcggt aaagttggaa taatggagaa accagttcct 60

gaaccagggc caaatgatgt aattgtaaag acaaccaacg ccttgatttg tacatctgat 120

gtacataccg ttgccggtgc tattggcgag aaaagcgacc tgactcttgg gcacgaaggt 180

gcaggtactg tttataagat tggcagtgcg gttaaagggt tcaaggaggg cgagagagtt 240

ctggttaatg ccataactcc ctgtttcaag tgtcacaact gccagcgcgg ctatacttca 300

caatgcggac aggccctggg tggatggaag tttgccaata ttaaggatgg ctgttttgct 360

gagtactttc atgttaatga tgctgaatcc aacttagtaa agattccaga ctcagtttct 420

gatgaagctg ctctatatac aacagatatg atgtccaccg gattcatggg ggctgagaat 480

ggaaatattc ctcttggagg gattgtagct gtttttggtc agggaccggt tggtcttatg 540

tcaacagcag gagcacgcct gttgggtgct ggtttggtca tagcagtgga aaacattcct 600

gccagacagg aactcgctaa attctacggt gctgacgtta ttgttgattt taccaaggtt 660

gatgcagtgg aagaaattat gaagctgact gatgggcagg gggtagatgc tgccattgag 720

gcactgggag ctcagatcac atttgagaac tgcattaaag tcaccaagcc tggaggtacc 780

atatccaata ttggctacca tggagaggga gattacataa aaataccgag agctgaatgg 840

ggtgtgggca tgtcggataa gactattcgg actggacttt gccctggagg cagcgaaaga 900

atgtccagac ttctgcggct tattgaaaac ggacgtattg atcccaccaa gctgaccact 960

catcgtttca gctttgatga gattgaaaaa ggatttcaca tgatggccaa taaagaggac 1020

ggagttatca aacccctggt aacattcagc tga 1053

<210> 34

<211> 350

<212> PRT

<213> 马钱子脱硫弧菌(Desulfonatronovibrio magnus)

<400> 34

Met Lys Ala Phe Val Val His Ser Ile Gly Lys Val Gly Ile Met Glu

1 5 10 15

Lys Pro Val Pro Glu Pro Gly Pro Asn Asp Val Ile Val Lys Thr Thr

20 25 30

Asn Ala Leu Ile Cys Thr Ser Asp Val His Thr Val Ala Gly Ala Ile

35 40 45

Gly Glu Lys Ser Asp Leu Thr Leu Gly His Glu Gly Ala Gly Thr Val

50 55 60

Tyr Lys Ile Gly Ser Ala Val Lys Gly Phe Lys Glu Gly Glu Arg Val

65 70 75 80

Leu Val Asn Gly Thr Thr Pro Cys Phe Lys Cys His Asn Cys Gln Arg

85 90 95

Gly Tyr Thr Ser Gln Cys Gly Gln Ala Leu Gly Gly Trp Thr Phe Ala

100 105 110

Asn Ile Lys Asp Gly Cys Phe Ala Glu Tyr Phe His Val Asn Asp Ala

115 120 125

Glu Ser Asn Leu Val Lys Ile Pro Asp Ser Val Ser Asp Glu Ala Ala

130 135 140

Leu Tyr Thr Thr Asp Met Met Ser Thr Gly Phe Met Gly Ala Glu Asn

145 150 155 160

Gly Asn Ile Pro Leu Gly Gly Ile Val Ala Val Phe Gly Gln Gly Pro

165 170 175

Val Gly Leu Met Ser Thr Ala Gly Ala Arg Leu Leu Gly Ala Gly Leu

180 185 190

Val Ile Ala Val Glu Asp Ile Pro Ala Arg Gln Glu Leu Ala Lys Phe

195 200 205

Tyr Gly Ala Asp Val Ile Val Asp Phe Thr Lys Val Asp Ala Val Glu

210 215 220

Glu Ile Met Lys Leu Thr Asp Gly Gln Gly Val Asp Ala Ala Ile Glu

225 230 235 240

Ala Leu Gly Ala Gln Ile Thr Phe Glu Asn Cys Ile Lys Val Thr Lys

245 250 255

Pro Gly Gly Thr Ile Ser Asn Ile Gly Tyr His Gly Glu Gly Asp Tyr

260 265 270

Ile Lys Ile Pro Arg Ala Glu Trp Gly Val Gly Met Ser Asp Lys Thr

275 280 285

Ile Arg Thr Gly Leu Cys Pro Gly Gly Ser Glu Arg Met Ser Arg Leu

290 295 300

Leu Arg Leu Ile Glu Asn Gly Arg Ile Asp Pro Thr Lys Leu Thr Thr

305 310 315 320

His Arg Phe Ser Phe Asp Glu Ile Glu Lys Gly Phe His Met Met Ala

325 330 335

Asn Lys Glu Asp Gly Val Ile Lys Pro Leu Val Thr Phe Ser

340 345 350

Claims

1.一种R-1,3-丁二醇的合成方法，其特征在于，包括以下步骤：

以4-羟基-2-丁酮为底物，利用醇脱氢酶的突变体进行催化反应，反应产物为R-1,3-丁二醇；

所述醇脱氢酶来源于：弓形脱硫菌(Desulfallas arcticus)、马钱子脱硫弧菌(Desulfonatronovibrio magnus)；

来源于弓形脱硫菌的醇脱氢酶的氨基酸序列如SEQ ID No.2所示；

所述醇脱氢酶的突变体为（1）对SEQ ID No . 2所示的氨基酸序列进行以下点突变：R97V和Y147N；

（2）对SEQ ID No . 4所示的氨基酸序列进行如下点突变： A84G、I85T、K110T、N198D；

在所述醇脱氢酶的N端和/或C端连接标签。

2.根据权利要求1所述的合成方法，其特征在于，所述醇脱氢酶的突变体以粗酶液、粗酶液冻干粉、纯酶或全细胞的形式加入。

3.根据权利要求1所述的合成方法，其特征在于，所述催化反应的温度为20-35℃，所述催化反应的时间为24-48h。

4.根据权利要求1或3所述的合成方法，其特征在于，当所述醇脱氢酶的突变体是以粗酶液、粗酶液冻干粉或纯酶的形式加入时，所述醇脱氢酶的突变体在反应体系中的浓度为0.1g/L-10g/L；

当所述醇脱氢酶的突变体以全细胞的形式加入时，所述全细胞的湿重为100g/L。

5.根据权利要求4所述的合成方法，其特征在于，所述催化反应在浓度为50-100mM，pH值为6.5-8.5的磷酸盐缓冲液中进行。

6.根据权利要求1或3所述的合成方法，其特征在于，当所述醇脱氢酶的突变体是以粗酶液、粗酶液冻干粉或纯酶的形式加入时，所述催化反应的反应体系中除了含有4-羟基-2-丁酮、所述醇脱氢酶的突变体和辅酶NAD⁺外，还含有异丙醇。