实施例6:
右旋樟脑25g、异丙醇100mL加至装有800mL pH=6.5,0.1M PBS缓冲液的2000mL反应器中搅拌均匀。依次加入酮还原酶粉(采购自苏州引航生物科技有限公司:商品编号YH2067)1g,NADP+50mg。在30℃搅拌24h,HPLC监测反应转化率>99.5%,右旋龙脑:异龙脑>99.8:0.2。反应完毕,反应体系用二氯甲烷萃取,合并有机相,干燥,脱溶,得到右旋龙脑粗品24.8克。
上述实施例中,所使用的酮还原酶YH2022、YH2067、YH2069分别具有SEQ ID No.1、SEQ ID No.2和SEQ ID No.3所示的DNA序列和SEQ ID No.4、SEQ ID No.5和SEQ ID No.6所示的蛋白质序列:
SEQ ID No.1:
ATGGTTAAGCAAGAATTCTTTAAGCTGAACAATGGCCATGAGATGCCCGGTGTGGCCATCGTTGGTACCGGTACCAAGTGGCACAAGGTTAATGAGACAGATGAGAACTTCTCACAGACACTGGTAGATCAATTGAAATACGCTCTGTCTCTGCCAGGTGTTGTTCATCTGGATGCCGCTGAATTTTATATGACCTATAGAGAGGTCGGACGTGCACTAGCTGAGACTTCGAAGCCCCGTGATGAGATATTCATCACTGATAAATACTGGACTTTGAGCAAAGTTACTGAGAATCCCATTGTAGGTTTAGAGACTGGGTTGAAAAGACTCGGATTAGAGTACGTAGACTTGTACCTGTTGCATAGTCCCTTTATCTCTAAGGAAACCAACGGGTTCTCATTAGAGGAGGCCTGGGGTATGATGGAGGAATTATATCACAGTGGTAAAGCCAAGAATATTGGCGTCTCCAACTTTGCTAAGGAGGACTTAGAGAGAGTTTTGAAGGTCTGCAAAGTCAAGCCACAGGTCAATCAAATTGAGTTTAATGCCTTCTTACAAAACCAAACTCCTGGTATCTACAACTTTTGCAAGCAAAATGACATTCAATTAGCTGCATATTCTCCTCTAGGTCCTTTACAGAAAAAGCCAGCCGACGGTAATTCACAACCATTTTATAGCTATATCAACAAGCTGGCACAGCACTACAACAAAACCCCCGGTCAGGTTTTGTTGAGATGGGTTACCAAGCGTGGTGTCGTTGCTGTTACCACTTCTGAGAAGAAGGAGAGAATTAAACAAGCTCAGGAGATATTTGAATTTGATTTGAAAGATGATGAAGTTACTGAAATTACTAAACTAGGTCTAGACCACGAGCCTTTGAGACTGTATTGGCATGATCAATACAATAAGTACAACTCAGAGTCCCAGAAGGCCTAA
SEQ ID No.2:
ATGTCTAACGGTAAAACCTTCACCCTGTCTAACGGTGTTAAAATCCCGGGTGTTGGTTTCGGTACCTTCGCTTCTGAAGGTTCTAAAGGTGAAACCTACACCGCTGTTACCACCGCTCTGAAAACCGGTTACCGTCACCTGGACTGCGCTTGGTACTACCTGAACGAAGGTGAAGTTGGTGAAGGTATCCGTGACTTCCTGAAAGAAAACCCGTCTGTTAAACGTGAAGACATCTTCGTTTGCACCAAAGTTTGGAACCACCTGCACCGTTACGAAGACGTTCTGTGGTCTATCGACGACTCTCTGAAACGTCTGGGTCTGGACTACGTTGACATGTTCCTGGTTCACTGGCCGATCGCTGCTGAAAAAAACGGTCAGGGTGAACCGAAAATCGGTCCGGACGGTAAATACGTTATCCTGAAAGACCTGACCGAAAACCCGGAACCGACCTGGCGTGCTATGGAAAAAATCTACGAAGACCGTAAAGCTCGTTCTATCGGTGTTTCTAACTGGACCATCGCTGACCTGGAAAAAATGTCTAAATTCGCTAAAGTTATGCCGCACGCTAACCAGATCGAAATCCACCCGTTCCTGCCGAACGAAGAACTGGTTCAGTACTGCTTCTCTAAAAACATCATGCCGGTTGCTTACTCTCCGCTGGGTTCTCAGAACCAGGTTCCGACCACCGGTGAACGTGTTTCTGAAAACAAAACCCTGAACGAAATCGCTGAAAAAGGTGGTAACACCCTGGCTCAGGTTCTGATCGCTTGGGGTCTGCGTCGTGGTTACGTTGTTCTGCCGAAATCTTCTAACCCGAAACGTATCGAATCTAACTTCAAATCTATCGAACTGTCTGACGCTGACTTCGAAGCTATCAACGCTGTTGCTAAAGGTCGTCACTTCCGTTTCGTTAACATGAAAGACACCTTCGGTTACGACGTTTGGCCGGAAGAAACCGCTAAAAACCTGTCTGCTTAA
SEQ ID No.3:
ATGACCAACCGcCTGCAGGGTAAAGTCGCACTGGTTACCGGTGGTGCTTCCGGTGTGGGCCTGGAAGTAGTGAAACTGCTCCTGGGTGAAGGCGCGAAAGTAGCGTTCAGCGACATCAACGAAGCAGCAGGGCAGCAGTTAGCAGCTGAGCTGGGCGAACGTAGTATGTTCGTGCGTCATGATGTCAGCAGCGAGGCAGATTGGACCCTGGTTATGGCAGCAGTACAGCGTCGCCTGGGTACCCTGAACGTGCTGGTGAACAACGCGGGTATTCTGCTGCCGGGCGACATGGAGACCGGTCGCCTGGAGGATTTTTCGCGCCTGCTGAAAATCAACACCGAAAGCGTGTTCATCGGTTGCCAGCAGGGCATTGCGGCCATGAAAGAAACTGGTGGTTCAATCATCAACATGGCTTCTGTTTCGTCTTGGCTGCCAATCGAACAATATGCAGGCTACAGCGCCTCAAAAGCAGCTGTCAGCGCCCTGACTCGCGCCGCAGCGCTTTCGTGTCGTAAACAAGGTTACGCTATCCGTGTTAACTCGATCCACCCGGATGGTATCTACACCCCGATGATGCAGGCGTCCCTGCCAAAAGGCGTGTCTAAGGAAATGGTGTTGCACGACCCAAAGCTGAACCGCGCGGGTCGTGCTTACATGCCGGAACGTATCGCGCAGCTGGTTCTGTTCCTTGCAAGCGACGAATCCAGCGTGATGTCCGGGAGTGAGCTGCACGCGGATAACAGCATTCTGGGTATGGGGCTGTAA
SEQ ID No.4:
MVKQEFFKLNNGHEMPGVAIVGTGTKWHKVNETDENFSQTLVDQLKYALSLPGVVHLDAAEFYMTYREVGRALAETSKPRDEIFITDKYWTLSKVTENPIVGLETGLKRLGLEYVDLYLLHSPFISKETNGFSLEEAWGMMEELYHSGKAKNIGVSNFAKEDLERVLKVCKVKPQVNQIEFNAFLQNQTPGIYNFCKQNDIQLAAYSPLGPLQKKPADGNSQPFYSYINKLAQHYNKTPGQVLLRWVTKRGVVAVTTSEKKERIKQAQEIFEFDLKDDEVTEITKLGLDHEPLRLYWHDQYNKYNSESQKA*
SEQ ID No.5:
MSNGKTFTLSNGVKIPGVGFGTFASEGSKGETYTAVTTALKTGYRHLDCAWYYLNEGEVGEGIRDFLKENPSVKREDIFVCTKVWNHLHRYEDVLWSIDDSLKRLGLDYVDMFLVHWPIAAEKNGQGEPKIGPDGKYVILKDLTENPEPTWRAMEKIYEDRKARSIGVSNWTIADLEKMSKFAKVMPHANQIEIHPFLPNEELVQYCFSKNIMPVAYSPLGSQNQVPTTGERVSENKTLNEIAEKGGNTLAQVLIAWGLRRGYVVLPKSSNPKRIESNFKSIELSDADFEAINAVAKGRHFRFVNMKDTFGYDVWPEETAKNLSA*
SEQ ID No.6:
MTNRLQGKVALVTGGASGVGLEVVKLLLGEGAKVAFSDINEAAGQQLAAELGERSMFVRHDVSSEADWTLVMAAVQRRLGTLNVLVNNAGILLPGDMETGRLEDFSRLLKINTESVFIGCQQGIAAMKETGGSIINMASVSSWLPIEQYAGYSASKAAVSALTRAAALSCRKQGYAIRVNSIHPDGIYTPMMQASLPKGVSKEMVLHDPKLNRAGRAYMPERIAQLVLFLASDESSVMSGSELHADNSILGMGL*
上述实施例只为说明本发明的技术构思及特点,其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。
序列表
<110> 苏州引航生物科技有限公司
<120> 一种制备右旋龙脑的方法
<130> 2018
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 936
<212> DNA
<213> 人工序列(未知)
<400> 1
atggttaagc aagaattctt taagctgaac aatggccatg agatgcccgg tgtggccatc 60
gttggtaccg gtaccaagtg gcacaaggtt aatgagacag atgagaactt ctcacagaca 120
ctggtagatc aattgaaata cgctctgtct ctgccaggtg ttgttcatct ggatgccgct 180
gaattttata tgacctatag agaggtcgga cgtgcactag ctgagacttc gaagccccgt 240
gatgagatat tcatcactga taaatactgg actttgagca aagttactga gaatcccatt 300
gtaggtttag agactgggtt gaaaagactc ggattagagt acgtagactt gtacctgttg 360
catagtccct ttatctctaa ggaaaccaac gggttctcat tagaggaggc ctggggtatg 420
atggaggaat tatatcacag tggtaaagcc aagaatattg gcgtctccaa ctttgctaag 480
gaggacttag agagagtttt gaaggtctgc aaagtcaagc cacaggtcaa tcaaattgag 540
tttaatgcct tcttacaaaa ccaaactcct ggtatctaca acttttgcaa gcaaaatgac 600
attcaattag ctgcatattc tcctctaggt cctttacaga aaaagccagc cgacggtaat 660
tcacaaccat tttatagcta tatcaacaag ctggcacagc actacaacaa aacccccggt 720
caggttttgt tgagatgggt taccaagcgt ggtgtcgttg ctgttaccac ttctgagaag 780
aaggagagaa ttaaacaagc tcaggagata tttgaatttg atttgaaaga tgatgaagtt 840
actgaaatta ctaaactagg tctagaccac gagcctttga gactgtattg gcatgatcaa 900
tacaataagt acaactcaga gtcccagaag gcctaa 936
<210> 2
<211> 978
<212> DNA
<213> 人工序列(未知)
<400> 2
atgtctaacg gtaaaacctt caccctgtct aacggtgtta aaatcccggg tgttggtttc 60
ggtaccttcg cttctgaagg ttctaaaggt gaaacctaca ccgctgttac caccgctctg 120
aaaaccggtt accgtcacct ggactgcgct tggtactacc tgaacgaagg tgaagttggt 180
gaaggtatcc gtgacttcct gaaagaaaac ccgtctgtta aacgtgaaga catcttcgtt 240
tgcaccaaag tttggaacca cctgcaccgt tacgaagacg ttctgtggtc tatcgacgac 300
tctctgaaac gtctgggtct ggactacgtt gacatgttcc tggttcactg gccgatcgct 360
gctgaaaaaa acggtcaggg tgaaccgaaa atcggtccgg acggtaaata cgttatcctg 420
aaagacctga ccgaaaaccc ggaaccgacc tggcgtgcta tggaaaaaat ctacgaagac 480
cgtaaagctc gttctatcgg tgtttctaac tggaccatcg ctgacctgga aaaaatgtct 540
aaattcgcta aagttatgcc gcacgctaac cagatcgaaa tccacccgtt cctgccgaac 600
gaagaactgg ttcagtactg cttctctaaa aacatcatgc cggttgctta ctctccgctg 660
ggttctcaga accaggttcc gaccaccggt gaacgtgttt ctgaaaacaa aaccctgaac 720
gaaatcgctg aaaaaggtgg taacaccctg gctcaggttc tgatcgcttg gggtctgcgt 780
cgtggttacg ttgttctgcc gaaatcttct aacccgaaac gtatcgaatc taacttcaaa 840
tctatcgaac tgtctgacgc tgacttcgaa gctatcaacg ctgttgctaa aggtcgtcac 900
ttccgtttcg ttaacatgaa agacaccttc ggttacgacg tttggccgga agaaaccgct 960
aaaaacctgt ctgcttaa 978
<210> 3
<211> 765
<212> DNA
<213> 人工序列(未知)
<400> 3
atgaccaacc gcctgcaggg taaagtcgca ctggttaccg gtggtgcttc cggtgtgggc 60
ctggaagtag tgaaactgct cctgggtgaa ggcgcgaaag tagcgttcag cgacatcaac 120
gaagcagcag ggcagcagtt agcagctgag ctgggcgaac gtagtatgtt cgtgcgtcat 180
gatgtcagca gcgaggcaga ttggaccctg gttatggcag cagtacagcg tcgcctgggt 240
accctgaacg tgctggtgaa caacgcgggt attctgctgc cgggcgacat ggagaccggt 300
cgcctggagg atttttcgcg cctgctgaaa atcaacaccg aaagcgtgtt catcggttgc 360
cagcagggca ttgcggccat gaaagaaact ggtggttcaa tcatcaacat ggcttctgtt 420
tcgtcttggc tgccaatcga acaatatgca ggctacagcg cctcaaaagc agctgtcagc 480
gccctgactc gcgccgcagc gctttcgtgt cgtaaacaag gttacgctat ccgtgttaac 540
tcgatccacc cggatggtat ctacaccccg atgatgcagg cgtccctgcc aaaaggcgtg 600
tctaaggaaa tggtgttgca cgacccaaag ctgaaccgcg cgggtcgtgc ttacatgccg 660
gaacgtatcg cgcagctggt tctgttcctt gcaagcgacg aatccagcgt gatgtccggg 720
agtgagctgc acgcggataa cagcattctg ggtatggggc tgtaa 765
<210> 4
<211> 311
<212> PRT
<213> 人工序列(未知)
<400> 4
Met Val Lys Gln Glu Phe Phe Lys Leu Asn Asn Gly His Glu Met Pro
1 5 10 15
Gly Val Ala Ile Val Gly Thr Gly Thr Lys Trp His Lys Val Asn Glu
20 25 30
Thr Asp Glu Asn Phe Ser Gln Thr Leu Val Asp Gln Leu Lys Tyr Ala
35 40 45
Leu Ser Leu Pro Gly Val Val His Leu Asp Ala Ala Glu Phe Tyr Met
50 55 60
Thr Tyr Arg Glu Val Gly Arg Ala Leu Ala Glu Thr Ser Lys Pro Arg
65 70 75 80
Asp Glu Ile Phe Ile Thr Asp Lys Tyr Trp Thr Leu Ser Lys Val Thr
85 90 95
Glu Asn Pro Ile Val Gly Leu Glu Thr Gly Leu Lys Arg Leu Gly Leu
100 105 110
Glu Tyr Val Asp Leu Tyr Leu Leu His Ser Pro Phe Ile Ser Lys Glu
115 120 125
Thr Asn Gly Phe Ser Leu Glu Glu Ala Trp Gly Met Met Glu Glu Leu
130 135 140
Tyr His Ser Gly Lys Ala Lys Asn Ile Gly Val Ser Asn Phe Ala Lys
145 150 155 160
Glu Asp Leu Glu Arg Val Leu Lys Val Cys Lys Val Lys Pro Gln Val
165 170 175
Asn Gln Ile Glu Phe Asn Ala Phe Leu Gln Asn Gln Thr Pro Gly Ile
180 185 190
Tyr Asn Phe Cys Lys Gln Asn Asp Ile Gln Leu Ala Ala Tyr Ser Pro
195 200 205
Leu Gly Pro Leu Gln Lys Lys Pro Ala Asp Gly Asn Ser Gln Pro Phe
210 215 220
Tyr Ser Tyr Ile Asn Lys Leu Ala Gln His Tyr Asn Lys Thr Pro Gly
225 230 235 240
Gln Val Leu Leu Arg Trp Val Thr Lys Arg Gly Val Val Ala Val Thr
245 250 255
Thr Ser Glu Lys Lys Glu Arg Ile Lys Gln Ala Gln Glu Ile Phe Glu
260 265 270
Phe Asp Leu Lys Asp Asp Glu Val Thr Glu Ile Thr Lys Leu Gly Leu
275 280 285
Asp His Glu Pro Leu Arg Leu Tyr Trp His Asp Gln Tyr Asn Lys Tyr
290 295 300
Asn Ser Glu Ser Gln Lys Ala
305 310
<210> 5
<211> 325
<212> PRT
<213> 人工序列(未知)
<400> 5
Met Ser Asn Gly Lys Thr Phe Thr Leu Ser Asn Gly Val Lys Ile Pro
1 5 10 15
Gly Val Gly Phe Gly Thr Phe Ala Ser Glu Gly Ser Lys Gly Glu Thr
20 25 30
Tyr Thr Ala Val Thr Thr Ala Leu Lys Thr Gly Tyr Arg His Leu Asp
35 40 45
Cys Ala Trp Tyr Tyr Leu Asn Glu Gly Glu Val Gly Glu Gly Ile Arg
50 55 60
Asp Phe Leu Lys Glu Asn Pro Ser Val Lys Arg Glu Asp Ile Phe Val
65 70 75 80
Cys Thr Lys Val Trp Asn His Leu His Arg Tyr Glu Asp Val Leu Trp
85 90 95
Ser Ile Asp Asp Ser Leu Lys Arg Leu Gly Leu Asp Tyr Val Asp Met
100 105 110
Phe Leu Val His Trp Pro Ile Ala Ala Glu Lys Asn Gly Gln Gly Glu
115 120 125
Pro Lys Ile Gly Pro Asp Gly Lys Tyr Val Ile Leu Lys Asp Leu Thr
130 135 140
Glu Asn Pro Glu Pro Thr Trp Arg Ala Met Glu Lys Ile Tyr Glu Asp
145 150 155 160
Arg Lys Ala Arg Ser Ile Gly Val Ser Asn Trp Thr Ile Ala Asp Leu
165 170 175
Glu Lys Met Ser Lys Phe Ala Lys Val Met Pro His Ala Asn Gln Ile
180 185 190
Glu Ile His Pro Phe Leu Pro Asn Glu Glu Leu Val Gln Tyr Cys Phe
195 200 205
Ser Lys Asn Ile Met Pro Val Ala Tyr Ser Pro Leu Gly Ser Gln Asn
210 215 220
Gln Val Pro Thr Thr Gly Glu Arg Val Ser Glu Asn Lys Thr Leu Asn
225 230 235 240
Glu Ile Ala Glu Lys Gly Gly Asn Thr Leu Ala Gln Val Leu Ile Ala
245 250 255
Trp Gly Leu Arg Arg Gly Tyr Val Val Leu Pro Lys Ser Ser Asn Pro
260 265 270
Lys Arg Ile Glu Ser Asn Phe Lys Ser Ile Glu Leu Ser Asp Ala Asp
275 280 285
Phe Glu Ala Ile Asn Ala Val Ala Lys Gly Arg His Phe Arg Phe Val
290 295 300
Asn Met Lys Asp Thr Phe Gly Tyr Asp Val Trp Pro Glu Glu Thr Ala
305 310 315 320
Lys Asn Leu Ser Ala
325
<210> 6
<211> 254
<212> PRT
<213> 人工序列(未知)
<400> 6
Met Thr Asn Arg Leu Gln Gly Lys Val Ala Leu Val Thr Gly Gly Ala
1 5 10 15
Ser Gly Val Gly Leu Glu Val Val Lys Leu Leu Leu Gly Glu Gly Ala
20 25 30
Lys Val Ala Phe Ser Asp Ile Asn Glu Ala Ala Gly Gln Gln Leu Ala
35 40 45
Ala Glu Leu Gly Glu Arg Ser Met Phe Val Arg His Asp Val Ser Ser
50 55 60
Glu Ala Asp Trp Thr Leu Val Met Ala Ala Val Gln Arg Arg Leu Gly
65 70 75 80
Thr Leu Asn Val Leu Val Asn Asn Ala Gly Ile Leu Leu Pro Gly Asp
85 90 95
Met Glu Thr Gly Arg Leu Glu Asp Phe Ser Arg Leu Leu Lys Ile Asn
100 105 110
Thr Glu Ser Val Phe Ile Gly Cys Gln Gln Gly Ile Ala Ala Met Lys
115 120 125
Glu Thr Gly Gly Ser Ile Ile Asn Met Ala Ser Val Ser Ser Trp Leu
130 135 140
Pro Ile Glu Gln Tyr Ala Gly Tyr Ser Ala Ser Lys Ala Ala Val Ser
145 150 155 160
Ala Leu Thr Arg Ala Ala Ala Leu Ser Cys Arg Lys Gln Gly Tyr Ala
165 170 175
Ile Arg Val Asn Ser Ile His Pro Asp Gly Ile Tyr Thr Pro Met Met
180 185 190
Gln Ala Ser Leu Pro Lys Gly Val Ser Lys Glu Met Val Leu His Asp
195 200 205
Pro Lys Leu Asn Arg Ala Gly Arg Ala Tyr Met Pro Glu Arg Ile Ala
210 215 220
Gln Leu Val Leu Phe Leu Ala Ser Asp Glu Ser Ser Val Met Ser Gly
225 230 235 240
Ser Glu Leu His Ala Asp Asn Ser Ile Leu Gly Met Gly Leu
245 250