高分子质量聚唾液酸生产菌株诱变筛选及其发酵优化
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摘要
为获得产高分子质量聚唾液酸(polysialic acid,PSA)菌株,分步采用常温常压等离子体(atmospheric and room temperature plasma,ARTP)和硫酸二乙酯(diethyl sulfate,DES)对大肠杆菌K235进行诱变。结果表明,通过ARTP诱变筛选得到1株产PSA分子质量较初始菌株提高了36. 84%的E. coli K235 4B31。对E. coli K235 4B31进行DES诱变,获得1株产PSA分子质量较初始菌株提高了78. 18%的突变菌株E. coli K235 6E61。通过三阶段搅拌转速控制策略最终使得突变菌株E. coli K235 6E61发酵产物PSA分子质量达到430. 5 k Da,为目前已报道的最高分子质量PSA。
In order to produce polysialic acid( PSA) with high molecular weight,Escherichia coli K235 was treated by atmospheric and room temperature plasma( ARTP) and diethyl sulfate( DES). Mutant 4 B31 obtained by ARTP mutagenesis produced PSA with 36. 84% higher molecular weight than that of the parent strain. Mutant 6 E61 obtained from mutant 4 B31 by DES mutagenesis produced PSA with 78. 18% higher molecular weight. By using a threestage agitation speed fermentation,the maximal molecular weight of PSA produced by mutant 6 E61 was 430. 5 kDa,which was the highest value reported up to date.
In order to produce polysialic acid( PSA) with high molecular weight,Escherichia coli K235 was treated by atmospheric and room temperature plasma( ARTP) and diethyl sulfate( DES). Mutant 4 B31 obtained by ARTP mutagenesis produced PSA with 36. 84% higher molecular weight than that of the parent strain. Mutant 6 E61 obtained from mutant 4 B31 by DES mutagenesis produced PSA with 78. 18% higher molecular weight. By using a threestage agitation speed fermentation,the maximal molecular weight of PSA produced by mutant 6 E61 was 430. 5 kDa,which was the highest value reported up to date.
引文
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[16] MONDAL S,HARIDAS N,LETHA S S,et al. Development of injectable high molecular weight hyaluronic acid hydrogels for cartilage regeneration[J]. Journal of Macromolecular Science,2016,53(8):507-514.
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[23] PETERSON D C,ARAKERE G,VIONNET J,et al.Characterization and acceptor preference of a soluble meningococcal group C polysialyltransferase[J]. J Bacteriol,2011,193(7):1 576-1 582.
[24] FERRERO M A,APARICIO L R. Biosynthesis and production of polysialic acids in bacteria[J]. Appl Microbiol Biotechnol,2010,86(6):1 621-1 635.
[25] BORK K,WEIDEMANN W,BERNECK B,et al. The expression of sialyltransferases is regulated by the bioavailability and biosynthesis of sialic acids[J]. Gene Expr Patterns,2017(23-24):52-58.
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[27] BARRIE F C,BLANK L M,RICHARD M,et al. Microbial hyaluronic acid production[J]. 2005,66(4):341-351.
[28] RAO Y M,SURESHKUMAR G K. Improvement in bioreactor productivities using free radicals:HOCl-induced overproduction of xanthan gum from Xanthomonas campestris and its mechanism[J]. Biotechnol Bioeng,2001,72(1):62-68.
[29]汤栋,荣绍丰,管世敏,等.氧化胁迫联合UV、DES诱变选育高产、高分子质量透明质酸菌株[J].食品工业科技,2015,36(11):136-140.
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[2] RUTISHAUSER U. Polysialic acid and the regulation of cell interactions[J]. Current Opinion in Cell Biology,1996,8(5):679-684.
[3] MCGUIRE E J,BINKLEY S. The structure and chemistry of colominic acid[J]. Biochemistry,1964,3(2):247-251.
[4]付浩,冯昌雨,詹晓北,等.聚唾液酸-透明质酸接枝聚合物的合成及其在药物缓释载体中的应用[J].合成化学,2016,24(7):570-575; 581.
[5] MURTHY R V,BHARATE P,GADE M,et al. Effect of transition metals on polysialic acid structure and functions[J]. Chem Med Chem,2016,11(7):667-673.
[6] GREGORIADIS G,JAIN S,PAPAIOANNOU I,et al. Improving the therapeutic efficacy of peptides and proteins:A role for polysialic acids[J]. Int J Pharm,2005,300(1-2):125-130.
[7] LIN B X,QIAO Y,SHI B,et al. Polysialic acid biosynthesis and production in Escherichia coli:Current state and perspectives[J]. Appl Microbiol Biotechnol,2016,100(1):1-8.
[8]郑志永,詹晓北,朱德强,等.聚唾液酸和唾液酸寡糖的生物合成及其在营养食品中的应用前景[J].食品科学,2013,34(15):361-368.
[9] COLLEY K J,KITAJIMA K,SATO C. Polysialic acid:Biosynthesis,novel functions and applications[J]. Crit Rev Biochem Mol Biol,2014,49(6):498-532.
[10] POLA R,HEINRICH A K,MUELLER T,et al. Passive tumor targeting of polymer therapeutics:In vivo imaging of both the polymer carrier and the enzymatically cleavable drug model[J]. Macromol Biosci,2016,16(11):1 577-1 582.
[11] LIU Rui,WANG Yan,MA Yudan,et al. Effects of the molecular weight of PLGA on degradation and drug release in vitro from an m PEG-PLGA nanocarrier[J]. Chemical Research in Chinese Universities,2016,32(5):848-853.
[12] ZHENG Z Y,WANG S Z,LI G S,et al. A new polysialic acid production process based on dual-stage p H control and fed-batch fermentation for higher yield and resulting high molecular weight product[J]. Appl Microbiol Biotechnol,2013,97(6):2 405-2 412.
[13] KUFELT O,EL-TAMER A,SEHRING C,et al. Hyaluronic acid based materials for scaffolding via two-photon polymerization[J]. Biomacromolecules,2014,15(2):650-659.
[14] BORKE T,NAJBERG M,ILINA P,et al. Hyaluronic acid graft copolymers with cleavable arms as potential intravitreal drug delivery vehicles[J]. Macromol Biosci2018,18(1):1-13.
[15]杨俊龙,王艳娉,张源,等.透明质酸治疗膝骨关节炎相关机制的研究进展[J].中国疼痛医学杂志,2017,23(6):451-454.
[16] MONDAL S,HARIDAS N,LETHA S S,et al. Development of injectable high molecular weight hyaluronic acid hydrogels for cartilage regeneration[J]. Journal of Macromolecular Science,2016,53(8):507-514.
[17]于军华.聚唾液酸生产菌种的选育及其发酵工艺的研究[D].无锡:江南大学,2002.
[18] JR O T,COOPER K M J A B. An improved automated periodate-resorcinol method for the determination of sialic acid[J]. 1983,133(1):233-238.
[19]李国顺,郑志永,李丹,等.端基法测定聚唾液酸平均聚合度[J].工业微生物,2012,42(5):19-23.
[20] WILLIS L M,STUPAK J,RICHARDS M R,et al. Conserved glycolipid termini in capsular polysaccharides synthesized by ATP-binding cassette transporter-dependent pathways in gram-negative pathogens[J]. Proc Natl Acad Sci U S A,2013,110(19):7 868-7 873.
[21] DESZO E L,STEENBERGEN S M,FREEDBERG D I,et al. Escherichia coli K1 polysialic acid O-acetyltransferase gene,neu O,and the mechanism of capsule form variation involving a mobile contingency locus[J]. Proc Natl Acad Sci U S A,2005,102(15):5 564-5 569.
[22]刘金龙.微生物发酵法制备聚唾液酸的研究[D].无锡:江南大学,2011.
[23] PETERSON D C,ARAKERE G,VIONNET J,et al.Characterization and acceptor preference of a soluble meningococcal group C polysialyltransferase[J]. J Bacteriol,2011,193(7):1 576-1 582.
[24] FERRERO M A,APARICIO L R. Biosynthesis and production of polysialic acids in bacteria[J]. Appl Microbiol Biotechnol,2010,86(6):1 621-1 635.
[25] BORK K,WEIDEMANN W,BERNECK B,et al. The expression of sialyltransferases is regulated by the bioavailability and biosynthesis of sialic acids[J]. Gene Expr Patterns,2017(23-24):52-58.
[26] JOSEPH D D A,JIAO W,PARKER E J. Arg314 is essential for catalysis by N-acetyl neuraminic acid synthase from Neisseria meningitidis[J]. Biochemistry,2013,52(15):2 609-2 619.
[27] BARRIE F C,BLANK L M,RICHARD M,et al. Microbial hyaluronic acid production[J]. 2005,66(4):341-351.
[28] RAO Y M,SURESHKUMAR G K. Improvement in bioreactor productivities using free radicals:HOCl-induced overproduction of xanthan gum from Xanthomonas campestris and its mechanism[J]. Biotechnol Bioeng,2001,72(1):62-68.
[29]汤栋,荣绍丰,管世敏,等.氧化胁迫联合UV、DES诱变选育高产、高分子质量透明质酸菌株[J].食品工业科技,2015,36(11):136-140.
[30]陈奕涵,钱悦,侯永泰,等.复合诱变选育大分子质量透明质酸高产菌株[J].中国酿造,2012,31(9):98-101.
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