产D-海因酶微生物的筛选及其催化特性
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摘要
为获得高活性和立体选择性海因酶生产菌株,首先采用氨基酸关环法合成了数种5'-单取代海因衍生物及N-氨甲酰-氨基酸,建立了相关液相色谱检测方法。然后以实验室保藏的野生菌株出发,利用底物诱导的方法筛选到6株具有D-海因酶活性的野生菌,其中荧光假单胞菌产生的D-海因酶具有较好的底物谱,较高的活性和立体选择性。经优化后,其最适产酶温度为30℃,培养时间为12 h,发酵活力达到92.1 U/L。该菌催化D-海因水解的最适反应温度为60℃,pH 8.5。在50 m L体系中,利用0.25 g菌体(干重)可对映选择性水解20 mmol/L对羟基苯海因底物,反应3 h转化率达到98%。
Hydantoinases are important enzymes with industrial potential in producing D-amino acids. In this study,6 strains with hydantoinase activity were selected from 23 wildtype microbial strains using 5'-monosubstituted hydantoin derivatives as induce substrates. Four 5'-monosubstituted hydantoins were chemically synthesized and verified with 1H-NMR. From 6 hydantoinase-producing strains,strain Pseudomonas fluorescenswas successfully identified with the highest enantioselectivity,desired substrate specificity and high activity in the hydrolysis of 5'-monosubstituted hydantoins andwas selected for further study. After induction with hydantoin substrates at 30 ℃ for 12 h,the D-hydantoinase activity could reach 92.1 U/L. The reaction condition was optimized to be 60 ℃ and pH 8.5,which was in favor of the spontaneous racemization of substrate. In a 50 m L whole-cell biocatalytic system,20 mmol/L D,L-p-hydroxyphenyl hydantoin was enantioselectively hydrolyzed by 0.25 g dry cells with a conversion ratioof 98% within 3 hours.
Hydantoinases are important enzymes with industrial potential in producing D-amino acids. In this study,6 strains with hydantoinase activity were selected from 23 wildtype microbial strains using 5'-monosubstituted hydantoin derivatives as induce substrates. Four 5'-monosubstituted hydantoins were chemically synthesized and verified with 1H-NMR. From 6 hydantoinase-producing strains,strain Pseudomonas fluorescenswas successfully identified with the highest enantioselectivity,desired substrate specificity and high activity in the hydrolysis of 5'-monosubstituted hydantoins andwas selected for further study. After induction with hydantoin substrates at 30 ℃ for 12 h,the D-hydantoinase activity could reach 92.1 U/L. The reaction condition was optimized to be 60 ℃ and pH 8.5,which was in favor of the spontaneous racemization of substrate. In a 50 m L whole-cell biocatalytic system,20 mmol/L D,L-p-hydroxyphenyl hydantoin was enantioselectively hydrolyzed by 0.25 g dry cells with a conversion ratioof 98% within 3 hours.
引文
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[13]王培,曹建华,宋德贵,等.一株产海因酶菌种的筛选与鉴定[J].广西师范大学学报:自然科学版,2013,31(1):119-124.WANG Pei,CAO Jianhua,SONG Zonggui,et al.Screening and identification of a strain of hydantoinase-producing microorganism[J].Journal of Guangxi Normal University:Natural Science Edition,2013,31(1):119-124.(in Chinese)
[14]LAZARUS R A.Chemical racemization of 5-benzylhydantoin[J].The Journal of Organic Chemistry,1990,55(15):4755-4757.
[15]MARTINEZ-RODRIGUEZ S,MARTINEZ-GOMEZ A I,Clemente-Jiménez J M,et al.Structure of dihydropyrimidinase from Sinorhizobium meliloti CECT4114:New features in an amidohydrolase family member[J].Journal of Structural Biology,2010,169(2):200-208.
[16]李隽,余江河.化学酶法制备D-苯甘氨酸[J].中国药科大学学报,2000,31(4):294-296.LI Jun,YU Jianghe.Chemoenzymatic synthesis of D-phenylglycine[J].Journal of China Pharmaceutical University,2000,31(4):294-296.(in Chinese)
[2]LATACZ G,Kiec-Kononowicz K.Biotransformation of new racemic(R,S)-5-benzylhydantoin derivatives by D-hydantoinases from adzuki bean[J].Biocatalysis and Biotransformation,2014,32(2):117-124.
[3]MOTHET J P,SNYDER S H.Brain d-amino acids:a novel class of neuromodulators[J].Amino Acids,2012,43(5):1809-1810.
[4]HERAS-VAZQUEZ F J L,Clemente-Jimenez J M,Martinez-Rodriguez S,et al.Optically pureα-amino acids production by the“Hydantoinase Process”[J].Recent Patents on Biotechnology,2008,2(1):35-46.
[5]KO Y M,CHEN C I,CHANG H C,et al.Exploring the complex effects of metal ions on d-hydantoinase purification with an immobilized metal affinity membrane[J].Journal of the Taiwan Institute of Chemical Engineers,2011,42(5):735-740.
[6]董妍玲,郑月,潘学武,等.豆类来源D-海因酶的提取及特性研究[J].化学与生物工程,2010,27(5):59-61.DONG Yanling,ZHENG Yue,PAN Xuewu,et al.Extraction and analysis of D-hydantoinase from beans[J].Chemistry and Bioengineering,2010,27(5):59-61.(in Chinese)
[7]DURR R,VIELHAUER O,BURTON S G,et al.Distribution of hydantoinase activity in bacterial isolates from geographically distinct environmental sources[J].Journal of Molecular Catalysis B:Enzymatic,2006,39(1):160-165.
[8]ZHANG J,CAI Z.Efficient and cost-effective production of D-p-hydroxyphenylglycine by whole-cell bioconversion[J].Biotechnology and Bioprocess Engineering,2014,19(1):76-82.
[9]于荣华,谭娇颖,乔浩,等.化学酶法制备D-丝氨酸[J].精细化工,2013,30(5):510-512.YU Ronghua,TAN Jiaoying,QIAO Hao,et al.Chemo-enzymatic synthesis of D-serine[J].Fine Chemicals,2013,30(5):510-512.(in Chinese)
[10]WARE E.The chemistry of the hydantoins[J].Chemical Reviews,1950,46(3):403-470.
[11]韦萍.D-氨基酸的制备研究[D].南京:南京工业大学,2002.
[12]孙婧.D-海因酶产生菌的筛选、性质及固定化研究[D].上海:上海师范大学,2010.
[13]王培,曹建华,宋德贵,等.一株产海因酶菌种的筛选与鉴定[J].广西师范大学学报:自然科学版,2013,31(1):119-124.WANG Pei,CAO Jianhua,SONG Zonggui,et al.Screening and identification of a strain of hydantoinase-producing microorganism[J].Journal of Guangxi Normal University:Natural Science Edition,2013,31(1):119-124.(in Chinese)
[14]LAZARUS R A.Chemical racemization of 5-benzylhydantoin[J].The Journal of Organic Chemistry,1990,55(15):4755-4757.
[15]MARTINEZ-RODRIGUEZ S,MARTINEZ-GOMEZ A I,Clemente-Jiménez J M,et al.Structure of dihydropyrimidinase from Sinorhizobium meliloti CECT4114:New features in an amidohydrolase family member[J].Journal of Structural Biology,2010,169(2):200-208.
[16]李隽,余江河.化学酶法制备D-苯甘氨酸[J].中国药科大学学报,2000,31(4):294-296.LI Jun,YU Jianghe.Chemoenzymatic synthesis of D-phenylglycine[J].Journal of China Pharmaceutical University,2000,31(4):294-296.(in Chinese)