Optimization of fermentation conditions for chitinase production by the Pseudoalteromonas sp. DL-6 using the response surface methodology
-
摘要: 采用响应面法对交替假单胞菌发酵产几丁质酶的培养基进行了优化。首先利用Plackett Burman实验设计筛选出影响产酶的3个主要因素,即胶体几丁质、发酵温度和pH。在此基础上用最陡爬坡路径逼近最大响应区域,再利用Box-Behnken实验设计及响应面分析法确定最佳条件。结果表明,胶体几丁质8.95g/L,蛋白胨2g/L,转速130r/min,接种量2%,发酵温度20℃,pH6.18,发酵时间96h,几丁质酶最大理论酶活为57.95U/mL。经3次实验验证,实际平均酶活与预测酶活相近,比优化前几丁质酶酶活提高了23.77%。Abstract: Response surface methodology was used to optimize the fermentation conditions of chitinase by Pseudoalteromonas sp.DL-6. Firstly, the important factors influencing chitinase production were identified by the method of Plackett-Burman as colloidal chitin, fermentation temperature, and pH. The path of steepest ascent was applied to approach the optimal region of the three significant factors. Box-Behnken design and response surface analysis were used to further investigate the optimal central levels and relationships of these factors. By using the quadratic regression model equation and analyzing the response surface of chitinase production, the optimal fermentation conditions were determined as followed:colloidal chitin 8.95g/L, peptone2g/L, shaking speed 130r/min, inoculums dose 2%, fermentation temperature 20℃, and pH 6.18, respectively.Under these conditons, the maximumtheoretic activity of chitinase was 57.95U/mL. After three verifications, the maximum theoretic value was consistent with mean value of verification test and the chitinase production was increased by 23.77% comparing to that before optimization.
-
Keywords:
- Pseudoalteromonas /
- chitinase /
- fermentation /
- response surface analysis
-
[1] Souza CP, Almeida BC, Colwell RR, et al.The importance of chitin in the marine environment[J].Mar Biotechnol, 2011, 13 (5) :823-830.
[2] AL Svitil, S Chadhain, JA Moore, et al.Chitin Degradation Proteins Produced by the Marine Bacterium Vibrio harveyi Growing on Different Forms of Chitin[J].Applied and Environmental Microbiology, 1997, 63 (2) :408-413.
[3] 王海东, 陈飚, 伦镜盛, 等.产几丁质酶菌株SWCH-6的筛选、鉴定及其产酶条件的优化研究[J].微生物学通报, 2008, 35 (5) :705-711. [4] Bhattacharya D, A Nagpure, RK Gupta.Bacterial Chitinases:Properties and Potential[J].Critical Reviews in Biotechnology, 2007, 27 (1) :21-28.
[5] T Ohno, S Armand, T Hata, et al.A modular family 19chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037[J].Journal of Bacteriology, 1996, 178 (17) :5065-5070.
[6] 肖湘, 周樱, 王凤平, 等.高效几丁质降解茵CBl01的分离鉴定及其几丁质酶系的研究[J].海洋学报, 2003, 25 (1) :138-142. [7] McCreath KJ, Gooday GW.A rapid and sensitive microassay for determination of chitinolytic activity[J].J Microbiol Methods, 1992 (14) :229-237.
[8] Horn SJ, Sikorski P, Cederkvist JB, et al.Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides[J].Proceedings of the National Academy of Sciences of the United States of America, 2006, 103 (48) :18089-18094.
[9] Vincent GH Eijsink, Gustav Vaaje-Kolstad, Kjell M V覽rum, et al.Towards new enzymes for biofuels:lessons from chitinase research[J].Trends Biotechnol, 2008, 26 (5) :228-235. [10] Tsujibo H, Orikoshi H, Shiotani K, et al.Characterization of chitinase C from a marine bacterium, Alteromonas sp.strain O-7, and its corresponding gene and domain structure[J].Appl Environ Microbiol, 1998, 64 (2) :472-478.
[11] Hideyuki Orikoshi, Nao Baba, Shigenari Nakayama, et al.Molecular analysis of the gene encoding a novel cold-adapted chitinase (ChiB) from a marine bacterium, Alteromonas sp.strain O-7[J].J Bacteriol, 2003, 185 (4) :1153-1160.
[12] Hideyuki Orikoshi, Shigenari Nakayama, Katsushiro Miyamoto, et al.Roles of four chitinases (chia, chib, chic, and chid) in the chitin degradation system of marine bacterium Alteromonas sp.strain O-7[J].Appl Environ Microbiol, 2005, 71 (4) :1811-1185.
[13] Xie B B, Shu YL, Qin QL.Genome Sequences of Type Strains of Seven Species of the Marine Bacterium Pseudoalteromonas[J].Journal of Bacteriology, 2012, 194 (10) :2746-2747.
[14] SC Hsu, JK Lockwood.Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil[J].Appl Microbiol, 1975, 29 (3) :422-466.
[15] Imoto T, Yagisshita K.A simple activity measurement of lysozyme[J].Agric Biol Chem, 1971, 35 (7) :1154-1156.
[16] Miller A, Sittter RR.Using the folded-over 12-run plackettburman design to consider interactions[J].Technometrics, 2001 (3) :44-54.
[17] Ambat P, Ayyanna C.Optimizing medium constituents and fermentation conditions for citric production from palmyra jaggery using response surface methods[J].World Journal of Microbiology and Biotechnology, 2001 (17) :331-335.
[18] Ratnam BVV, Narasimha RM, Dmodar RM, et al.Optimization of fermentation conditions for the production of ethanol fromsago starch using response surface methodology[J].World Journal of Microbiology and Biotechnology, 2003, 19 (5) :523-526.
[19] 孟彦羽, 岳敏, 张建平, 等.海南土地杆菌Pedobacter hainanensis 13-Q发酵生产κ-卡拉胶酶的条件优化[J].中国酿造, 2013 (7) :20-23.
计量
- 文章访问数: 123
- HTML全文浏览量: 15
- PDF下载量: 102
下载:
