【具体实施方式】
下面结合具体实施实例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1:获得T.brockii菌株的野生型酮还原酶母本重组质粒
通过NCBI Benbank核酸数据库获得T.brockii菌株的酮还原酶母本编码基因(GenBank:X64841.1),经密码子和酶切位点优化并委托服务商人工合成全长基因和两端的酶切位点,与pET28a(+)表达质粒分别进行酶切、切胶回收、连接和转化大肠杆菌BL21(DE3)感受态细胞,涂布于含有50mg/L硫酸卡那霉素的LB琼脂平皿中,37℃培养过夜。挑选数个单菌落至LB培养基(含50mg/L硫酸卡那霉素),37℃培养过夜后使用质粒提取试剂盒提取重组质粒,进行PCR和测序验证,获得阳性酮还原酶母本的重组质粒。
实施例2:对野生型酮还原酶母本基因进行随机突变
根据实施例1所述内容,以含T.brockii菌株野生型酮还原酶母本编码基因的重组质粒为模板,并根据T.brockii菌株的酮还原酶母本编码基因用Primer 5.0设计并合成两端引物(表1),使用易错PCR技术(物料及浓度见表2,反应条件见表3)获得含有大量碱基突变的线性基因片段,将这些PCR产物和pET28a(+)表达质粒分别进行酶切、切胶回收、连接和转化大肠杆菌BL21(DE3)感受态细胞,涂布于含有50mg/L硫酸卡那霉素的LB琼脂平皿中,37℃培养过夜。
表1随机突变引物序列
表2 50μL易错PCR物料体系
表3易错PCR反应条件:
实施例3:酮还原酶突变体的克隆与表达
为了便于酮还原酶突变体的克隆和表达及鉴定,在其基因的5’和3’末端设计了兼容的限制内切酶位点,可以采用Nde I和Xho I限制性内切酶分别将目的基因和pET28a(+)(其它可以在大肠杆菌中表达蛋白质的表达质粒也可使用)同时进行酶切和DNA切胶回收,回收后的目的基因和质粒的较大片段用T4 DNA连接酶进行连接反应,将连接产物转化到大肠杆菌BL21(DE3)感受态细胞中,然后将转化后的感受态细胞涂布于含有50mg/L硫酸卡那霉素的LB琼脂平皿中,37℃培养过夜。
挑取上述培养皿上长出的单菌落接种于含有50mg/L硫酸卡那霉素的LB液体培养基中,37℃振荡培养过夜,收集菌体进行质粒提取、PCR鉴定和双酶切鉴定后,将正确的重组质粒命名为pET28a(+)-A-Z,并将含有正确的重组质粒的大肠杆菌进行后续的诱导表达。将上述菌液转接于500mL含50mg/L硫酸卡那霉素的LB液体培养基中,37℃振荡培养至OD600=0.6~0.7时,加入IPTG至终浓度分别为0.05~0.5mM,在22~25℃下进行诱导表达12~16hr后,取出菌液,6000×g离心15min收集菌体,于-20℃冻存备用。
实施例4:酮还原酶突变体的初筛
根据实施例2和实施例3所述内容,挑取上述LB琼脂培养基上的单克隆菌落接种于96深孔板中,每孔预先加入1mL含有50mg/L硫酸卡那霉素的LB培养基,于37℃,220rpm振荡培养3hr后,加入一定量的诱导剂异丙基-β-D-硫代半乳糖苷(IPTG,终浓度为0.05mM),25℃,220rpm诱导培养16hr,4000×g离心20min收集菌细胞,倒掉离心上清液后使用相同体积的100mM磷酸盐缓冲液(pH 7.0)冲悬菌细胞。通过室温-零下80℃反复5次冻融裂解菌细胞后,10℃,8000×g离心30min获得上清液,即酮还原酶突变体粗酶液,用酶标仪进行活性初筛。向96孔板中加入25μL含有5mg/mL还原型烟酰胺腺嘌呤二核苷酸(NADH)和2mM硫酸镁的磷酸盐缓冲液(100mM,pH=7.0),125μL 5-((4S)-2-氧代-4-苯基(1,3-恶唑烷-3-基))-1-(4-氟苯基)戊烷-1,5-二酮溶液(10mg/mL溶于异丙醇),于340nm进行本底检测,然后分别向各孔中加入已经制备好的母本和突变体酶液100μL,并立即于30℃检测340nm处吸光光度值的变化。
酶活计算公式:酶活(U/mL)=(△A×60×V1)/(6.22×t×V2)
△A:反应过程中的吸光光度值变化量;
V1:反应体系的总体积;
6.22:NADH消光系数;
t:△A的检测时间;
V2:加入的酶液体积。
实施例5:酮还原酶突变体的复筛
将实施例4中酶活高于母本的突变体菌株以0.01%接种量接种于500mL含有50mg/L硫酸卡那霉素的LB培养基,于37℃,220rpm振荡培养5~6hr,加入一定量的诱导剂异丙基-β-D-硫代半乳糖苷(IPTG,终浓度为0.05mM),25℃,220rpm诱导培养16hr,7000×g离心收集菌体。使用100mM磷酸盐缓冲液(pH 7.0)冲悬菌细胞后用超声波破碎仪破碎细胞,10℃,10000×g离心20min获得上清液,即酮还原酶突变体粗酶液。向10mL反应瓶中加入0.05g主原料5-((4S)-2-氧代-4-苯基(1,3-恶唑烷-3-基))-1-(4-氟苯基)戊烷-1,5-二酮和0.5mL异丙醇,振荡至原料溶解,加入4mL磷酸盐缓冲液(100mM,pH=7.0),主原料均匀分散于缓冲液中。加入2mg NAD+,20mg甲酸铵,10mg辅酶(FDH,甲酸脱氢酶)和0.5mL上述突变体粗酶液,体系pH=7.0,并于40℃保温16hr后,薄层色谱跟踪,选取转化产品点明显、主原料点不明显的体系进行甲基叔丁基醚萃取,静置分液,取有机相进行HPLC分析。
选取催化活性优于母本的突变体进行测序,分析突变位点,复测催化活性确定突变体I140S(SEQ ID NO:2)、I140S-D205E(SEQ ID NO:3)、H125Q-I140S-N329S(SEQ ID NO:4)、I140S-L294I(SEQ ID NO:5)、I140S-D150Q(SEQ ID NO:6)和P36S-I140S(SEQ ID NO:7)的催化活性及立体选择性均比本方案母本显著提高,复筛反应结果如表4所示。从表4可见,六种酮还原酶突变体对5-((4S)-2-氧代-4-苯基(1,3-恶唑烷-3-基))-1-(4-氟苯基)戊烷-1,5-二酮的催化效率为野生型酮还原酶母本的约200倍,且产物有更高的e.e.值。
表4酮还原酶母本与突变体酶法制备(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮活性比较
SEQ ID NO: |
突变位点 |
酶量*/wt |
转化率/% |
e.e./% |
1 |
母本 |
20 |
10.2 |
78.0 |
1 |
母本 |
50 |
40.1 |
76.8 |
2 |
I140S突变体 |
1 |
99.5 |
99.7 |
3 |
I140S-D205E突变体 |
1 |
99.2 |
99.8 |
4 |
H125Q-I140S-N329S突变体 |
1 |
99.8 |
99.8 |
5 |
I140S-L294I突变体 |
1 |
99.1 |
99.8 |
6 |
I140S-D150Q突变体 |
1 |
99.6 |
99.7 |
7 |
P36S-I140S突变体 |
1 |
99.5 |
99.5 |
注:表4中的*指转化1g底物所需各酮还原酶突变体重组细胞的湿重。1wt指转化1g主原料需要1g酮还原酶突变体重组湿细胞。
实施例6:酮还原酶突变体酶粉制备
将实施例5中酶活高于母本的突变体菌株分别以0.01%接种量接种于(500mL/瓶*10瓶/突变体)含有50mg/L硫酸卡那霉素的LB培养基,于37℃,220rpm振荡培养5~6hr,加入一定量的诱导剂异丙基-β-D-硫代半乳糖苷(IPTG,终浓度为0.05mM),25℃,220rpm诱导培养16hr,7000×g离心收集菌体。所得25~30g菌细胞使用100mM磷酸盐缓冲液(pH 7.0)冲悬菌细胞后用高压均质机破碎细胞,10℃,10000×g离心20min获得上清液,使用真空冷冻干燥机-25℃将其冻干制成3~5g酶粉,即酮还原酶突变体酶粉。0.1%(w/v)各突变体酶粉溶液SDS-PAGE电泳检测见图2。
实施例7:SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7所示的酮还原酶突变体在(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮制备中的应用
向500mL反应瓶中加入20g主原料5-((4S)-2-氧代-4-苯基(1,3-恶唑烷-3-基))-1-(4-氟苯基)戊烷-1,5-二酮,100mL异丙醇,振荡至原料完全溶解,加入100mL磷酸盐缓冲液(100mM,pH 7.0,含2mM硫酸镁);加入20mg氧化型烟酰胺腺嘌呤二核苷酸(NAD+),20.6g甲酸铵,0.1g辅酶(FDH,即甲酸脱氢酶)和实例5中的0.2g酮还原酶突变体酶粉,体系pH=7.0,并于40±2℃保温16h;用200mL乙酸乙酯终止反应,并用125g硅藻土过滤体系,200mL乙酸乙酯萃取两次,静置分液,有机相经浓缩得到粗品。反应转化率和产物e.e.值检测结果见表5。
表5酮还原酶突变体酶法制备(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮
SEQ ID NO: |
突变位点 |
酶量/g |
转化率/% |
e.e./% |
2 |
I140S突变体 |
0.2 |
99.5 |
99.7 |
3 |
I140S-D205E突变体 |
0.2 |
99.2 |
99.8 |
4 |
H125Q-I140S-N329S突变体 |
0.2 |
99.8 |
99.8 |
5 |
I140S-L294I突变体 |
0.2 |
99.1 |
99.7 |
6 |
I140S-D150Q突变体 |
0.2 |
99.6 |
99.4 |
7 |
P36S-I140S突变体 |
0.2 |
99.5 |
99.5 |
表5结果表明,六条突变体(I140S突变体(SEQ ID NO:2)、I140S-D205E突变体(SEQID NO:3)、H125Q-I140S-N329S突变体(SEQ ID NO:4)、I140S-L294I突变体(SEQ ID NO:5)、I140S-D150Q突变体(SEQ ID NO:6)和P36S-I140S突变体(SEQ ID NO:7))在酶催化5-((4S)-2-氧代-4-苯基(1,3-恶唑烷-3-基))-1-(4-氟苯基)戊烷-1,5-二酮合成(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮中,即100g/L底物和1g/L酶粉的反应体系中,反应16hr,转化率即可达到99.0%以上,产物e.e值均达到99.0%以上,筛选出的六条酮还原酶突变体在酶法制备(4S)-3-[(5S)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1,3-氧氮杂环戊烷-2-酮中均表现出了极高的立体选择性和高效性。
序列表
序列表
<110> 迪沙药业集团(天津)药物研究有限公司
迪沙药业集团有限公司
<120> 一种改进的酮还原酶及其应用
<141> 2018-07-10
<160> 14
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<213> I140S-L294I突变体(Thermoanaerobacter brockii)
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130 135 140
Ala Val Met Ile Pro Ala Met Met Thr Thr Gly Pro His Gly Ala Gly
145 150 155 160
Leu Ala Ala Ile Gly Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Leu Leu Ala Gly Ala Gly
180 185 190
Ala Ile Ile Ala Val Gly Ser Ala Pro Val Cys Val Ala Ala Ala Leu
195 200 205
Thr Thr Gly Ala Thr Ala Ile Val Ala Thr Leu Ala Gly Pro Ile Gly
210 215 220
Ser Gly Ile Met Ala Leu Thr Gly Gly Leu Gly Val Ala Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Ala Ala Ala Ile Met Ala Thr Ala Val Leu Ile Val
245 250 255
Leu Pro Gly Gly Thr Ile Ala Ala Val Ala Thr Pro Gly Gly Gly Gly
260 265 270
Val Leu Pro Val Pro Ala Leu Gly Thr Gly Cys Gly Met Ala His Leu
275 280 285
Thr Ile Leu Gly Gly Ile Cys Pro Gly Gly Ala Leu Ala Met Gly Ala
290 295 300
Leu Ile Ala Leu Val Pro Thr Leu Ala Val Ala Pro Ser Leu Leu Val
305 310 315 320
Thr His Val Pro Ala Gly Pro Ala Ala Ile Gly Leu Ala Pro Met Leu
325 330 335
Met Leu Ala Leu Pro Leu Ala Leu Ile Leu Pro Val Val Ile Leu Ala
340 345 350
<210> 6
<211> 352
<212> PRT
<213> I140S-D150Q突变体(Thermoanaerobacter brockii)
<400> 6
Met Leu Gly Pro Ala Met Leu Ser Ile Gly Leu Val Gly Thr Ile Gly
1 5 10 15
Leu Gly Leu Pro Ala Pro Gly Pro Pro Ala Ala Ile Val Ala Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Ala Ile His Thr Val Pro Gly Gly Ala
35 40 45
Ile Gly Gly Ala His Ala Met Ile Leu Gly His Gly Ala Val Gly Gly
50 55 60
Val Val Gly Val Gly Ser Gly Val Leu Ala Pro Leu Pro Gly Ala Ala
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Ala Thr Ala Thr Ser Gly Val Gly
85 90 95
Ala Gly Thr His Gly His Ser Gly Gly Met Leu Ala Gly Thr Leu Pro
100 105 110
Ser Ala Val Leu Ala Gly Val Pro Gly Gly Pro Pro His Val Ala Ala
115 120 125
Ala Ala Met Ala Leu Ala His Leu Pro Leu Gly Ser Pro Leu Gly Ala
130 135 140
Ala Val Met Ile Pro Gly Met Met Thr Thr Gly Pro His Gly Ala Gly
145 150 155 160
Leu Ala Ala Ile Gly Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Leu Leu Ala Gly Ala Gly
180 185 190
Ala Ile Ile Ala Val Gly Ser Ala Pro Val Cys Val Ala Ala Ala Leu
195 200 205
Thr Thr Gly Ala Thr Ala Ile Val Ala Thr Leu Ala Gly Pro Ile Gly
210 215 220
Ser Gly Ile Met Ala Leu Thr Gly Gly Leu Gly Val Ala Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Ala Ala Ala Ile Met Ala Thr Ala Val Leu Ile Val
245 250 255
Leu Pro Gly Gly Thr Ile Ala Ala Val Ala Thr Pro Gly Gly Gly Gly
260 265 270
Val Leu Pro Val Pro Ala Leu Gly Thr Gly Cys Gly Met Ala His Leu
275 280 285
Thr Ile Leu Gly Gly Leu Cys Pro Gly Gly Ala Leu Ala Met Gly Ala
290 295 300
Leu Ile Ala Leu Val Pro Thr Leu Ala Val Ala Pro Ser Leu Leu Val
305 310 315 320
Thr His Val Pro Ala Gly Pro Ala Ala Ile Gly Leu Ala Pro Met Leu
325 330 335
Met Leu Ala Leu Pro Leu Ala Leu Ile Leu Pro Val Val Ile Leu Ala
340 345 350
<210> 7
<211> 352
<212> PRT
<213> P36S-I140S突变体(Thermoanaerobacter brockii)
<400> 7
Met Leu Gly Pro Ala Met Leu Ser Ile Gly Leu Val Gly Thr Ile Gly
1 5 10 15
Leu Gly Leu Pro Ala Pro Gly Pro Pro Ala Ala Ile Val Ala Pro Leu
20 25 30
Ala Val Ala Ser Cys Thr Ser Ala Ile His Thr Val Pro Gly Gly Ala
35 40 45
Ile Gly Gly Ala His Ala Met Ile Leu Gly His Gly Ala Val Gly Gly
50 55 60
Val Val Gly Val Gly Ser Gly Val Leu Ala Pro Leu Pro Gly Ala Ala
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Ala Thr Ala Thr Ser Gly Val Gly
85 90 95
Ala Gly Thr His Gly His Ser Gly Gly Met Leu Ala Gly Thr Leu Pro
100 105 110
Ser Ala Val Leu Ala Gly Val Pro Gly Gly Pro Pro His Val Ala Ala
115 120 125
Ala Ala Met Ala Leu Ala His Leu Pro Leu Gly Ser Pro Leu Gly Ala
130 135 140
Ala Val Met Ile Pro Ala Met Met Thr Thr Gly Pro His Gly Ala Gly
145 150 155 160
Leu Ala Ala Ile Gly Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Leu Leu Ala Gly Ala Gly
180 185 190
Ala Ile Ile Ala Val Gly Ser Ala Pro Val Cys Val Ala Ala Ala Leu
195 200 205
Thr Thr Gly Ala Thr Ala Ile Val Ala Thr Leu Ala Gly Pro Ile Gly
210 215 220
Ser Gly Ile Met Ala Leu Thr Gly Gly Leu Gly Val Ala Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Ala Ala Ala Ile Met Ala Thr Ala Val Leu Ile Val
245 250 255
Leu Pro Gly Gly Thr Ile Ala Ala Val Ala Thr Pro Gly Gly Gly Gly
260 265 270
Val Leu Pro Val Pro Ala Leu Gly Thr Gly Cys Gly Met Ala His Leu
275 280 285
Thr Ile Leu Gly Gly Leu Cys Pro Gly Gly Ala Leu Ala Met Gly Ala
290 295 300
Leu Ile Ala Leu Val Pro Thr Leu Ala Val Ala Pro Ser Leu Leu Val
305 310 315 320
Thr His Val Pro Ala Gly Pro Ala Ala Ile Gly Leu Ala Pro Met Leu
325 330 335
Met Leu Ala Leu Pro Leu Ala Leu Ile Leu Pro Val Val Ile Leu Ala
340 345 350
<210> 8
<211> 1059
<212> DNA
<213> 野生型(Thermoanaerobacter brockii)
<400> 8
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaatt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 9
<211> 1059
<212> DNA
<213> I140S突变体(Thermoanaerobacter brockii)
<400> 9
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 10
<211> 1059
<212> DNA
<213> I140S-D205E突变体(Thermoanaerobacter brockii)
<400> 10
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagaagctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 11
<211> 1059
<212> DNA
<213> H125Q-I140S-N329S突变体(Thermoanaerobacter brockii)
<400> 11
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcaagtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacagtatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 12
<211> 1059
<212> DNA
<213> I140S-L294I突变体(Thermoanaerobacter brockii)
<400> 12
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagc 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcggga tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 13
<211> 1059
<212> DNA
<213> I140S-D150Q突变体(Thermoanaerobacter brockii)
<400> 13
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagt 420
ccattggaag ctgcagttat gattccccaa atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 14
<211> 1059
<212> DNA
<213> P36S-I140S突变体(Thermoanaerobacter brockii)
<400> 14
atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60
gctcctggcc catttgatgc tattgtaaga cctctagctg tggcctcttg cacttcggac 120
attcataccg tttttgaagg cgccattggc gaaagacata acatgatact cggtcacgaa 180
gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240
gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300
cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360
ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaagc 420
ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc taagctcgtc 960
actcacgttt tccggggatt tgacaatatt gaaaaagcct ttatgttgat gaaagacaaa 1020
ccaaaagacc taatcaaacc tgttgtaata ttagcataa 1059