一株产几丁质脱乙酰酶海洋细菌的筛选、鉴定及发酵优化
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摘要
目的:从海洋样品中筛选产几丁质脱乙酰酶细菌,对菌株进行鉴定和发酵条件优化。方法:利用显色平板从黄海海州湾燕尾港海域的海泥中筛选几丁质脱乙酰酶产生细菌。利用形态学、生理生化特性以及16S r DNA序列分析对菌株进行鉴定。利用单因素实验和正交实验优化菌株发酵条件。结果:筛选获得几丁质脱乙酰酶产生菌MCDA01,经形态学特征、生理生化特性以及16S r DNA序列分析将菌株鉴定为天目不动杆菌(Acinetobacter schindleri)。菌株最佳生长条件为30℃,p H8.0,Na Cl 4%。最佳产酶发酵条件为:发酵时间60 h,发酵温度20℃,培养基起始p H7,装液量20%,诱导剂几丁质浓度1%。在此条件下,产酶量达到201.37 U/m L,比优化前提高了4.14倍。结论:几丁质脱乙酰酶产生菌MCDA01产酶量达到201.37 U/m L,有工业应用潜力。
Objective: Screen chitin-deacetylase-producing strains from marine samples,identify the strain and optimize its fermentation conditions.Methods: Chitin-deacetylase-producing strains were screened by color culture medium from the marine mud of Yanwei Harbor. The strains were identified by morphological,physiological and biochemical characteristics and16 S r DNA sequence analysis. The single factor and orthogonal experimental design were used to optimize the fermentation conditions.Results: Strain MCDA01 producing chitin deacetylase was screened,and it was identified as Acinetobacter schindleri through morphological,biochemical characteristics,and 16 S r DNA sequence analysis. The optimal culture conditions of strain MCDA01 was obtained as follows: 30 ℃,p H8.0,and Na Cl 4%.The optimal fermentation conditions was obtained as follows: the fermentation time was 60 h,the fermentation temperature was 20 ℃,the culture medium starts p H was 7,the liquid loading amount was 20%,and the inducer chitin concentration was 1%. Under this condition,production of enzyme was up to201.37 U/m L,which was increased by 4.14 times. Conclusion: The maximum of enzyme production were optimized to201.37 U/m L,and have industrial application potential.
Objective: Screen chitin-deacetylase-producing strains from marine samples,identify the strain and optimize its fermentation conditions.Methods: Chitin-deacetylase-producing strains were screened by color culture medium from the marine mud of Yanwei Harbor. The strains were identified by morphological,physiological and biochemical characteristics and16 S r DNA sequence analysis. The single factor and orthogonal experimental design were used to optimize the fermentation conditions.Results: Strain MCDA01 producing chitin deacetylase was screened,and it was identified as Acinetobacter schindleri through morphological,biochemical characteristics,and 16 S r DNA sequence analysis. The optimal culture conditions of strain MCDA01 was obtained as follows: 30 ℃,p H8.0,and Na Cl 4%.The optimal fermentation conditions was obtained as follows: the fermentation time was 60 h,the fermentation temperature was 20 ℃,the culture medium starts p H was 7,the liquid loading amount was 20%,and the inducer chitin concentration was 1%. Under this condition,production of enzyme was up to201.37 U/m L,which was increased by 4.14 times. Conclusion: The maximum of enzyme production were optimized to201.37 U/m L,and have industrial application potential.
引文
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[17]王皓,吴丽,朱小花,等.甲壳素脱乙酰酶的研究概况及展望[J].中国生物工程杂志,2015,35(1):96-103.
[18]布坎南R E,吉本斯N E.伯杰氏细菌鉴定手册[D].第8版.北京:科学出版社,1984:474-482.
[19]Kuwahara T,Norimatsu L,Nakayama H,et al.Genetic variation in 16S-23S r DNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of bacteroides species[J].Microbiology&Immunology,2001,45(3):191.
[2]王小红,马建标.甲壳素,壳聚糖及其衍生物的应用[J].功能高分子学报,1999(2):197-202.
[3]严俊.甲壳素的化学和应用[J].化学通报,1984(11):28-33.
[4]屠洁,刘冠卉,程曦.壳聚糖的抑菌效果研究及其与苯甲酸钠的比较[J].食品工业科技,2008(5):83-85.
[5]Amorim R V,Melo E S,Carneirodacunha M G,et al.Chitosan from Syncephalastrum racemosum used as a film support for lipase immobilization[J].Bioresource Technology,2003,89(1):35-39.
[6]Araki Y,Ito E.A Pathway of Chitosan Formation in Mucor rouxii[J].Biochemical&Biophysical Research Communications,2008,55(1):71-78.
[7]Magorzata M Jaworska,Jolanta Bryjak,Jolanta Liesiene.A search of an optimal carrier for immobilization of chitin deacetylase[J].Cellulose,2009,16(2):261-270.
[8]陈东辉,陈亮,胡家震.壳聚糖制备方法的比较研究[J].化学研究与应用,2002,14(1):56-58.
[9]张立彦,曾庆孝.酶法在低聚壳聚糖制备上的研究现状及展望[J].广东海洋大学学报,2000,20(4):72-78.
[10]Araki Y,Ito E.A pathway of chitosan formation in Mucor rouxii Enzymatic deacetylation of chitin[J].European Journal of Biochemistry,1975,55(1):71-78.
[11]张菁菁,董文宾,缑敬轩.几丁质脱乙酰酶菌株的筛选鉴定及酶学性质[J].食品工业科技,2012,33(6):244-246.
[12]Cai J,Yang J,Du Y,et al.Purification and characterization of chitin deacetylase from Scopulariopsis brevicaulis[J].Carbohydrate Polymers,2006,65(2):211-217.
[13]Sun Y,Zhang J,Wu S,et al.Statistical optimization for production of chitin deacetylase from Rhodococcus erythropolis HG05[J].Automobile Technology,2014,102(1):649-652.
[14]Yong Z,Ro-Dong P,Muzzarelli R A A.Chitin Deacetylases:Properties and Applications[J].Marine Drugs,2010,8(1):24-46.
[15]Srinivasan V R.Biotransformation of chitin to chitosan:U S,Patent 5739015[P].1998-4-14.
[16]Chatterjee S,Chatterjee S,Chatterjee B P,et al.Enhancement of growth and chitosan production by Rhizomes oryzae in whey medium by plant growth hormones[J].International Journal of Biological Macromolecules,2008,42(2):120-126.
[17]王皓,吴丽,朱小花,等.甲壳素脱乙酰酶的研究概况及展望[J].中国生物工程杂志,2015,35(1):96-103.
[18]布坎南R E,吉本斯N E.伯杰氏细菌鉴定手册[D].第8版.北京:科学出版社,1984:474-482.
[19]Kuwahara T,Norimatsu L,Nakayama H,et al.Genetic variation in 16S-23S r DNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of bacteroides species[J].Microbiology&Immunology,2001,45(3):191.