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1 微生物学通报 MAR 20, 2009, 36(3): 398~403 Microbiology 研究报告 2009 by Institute of Microbiology, CAS 海洋侧孢短芽孢杆菌 Brevibacillus laterosporus Lh-1 产抗菌肽 R-1 的培养条件优化 * 郑媛王跃军孙谧 ( ) 摘要 : 采用 Plackett-Burman(PB) 设计和响应面分析 (RSM) 方法, 对一株海洋侧孢短芽孢杆菌 Lh-1 产抗菌肽 R-1 的发酵条件进行了优化研究中数据的统计和分析均使用 MINITAB 15.0 在研究中, 首先使用了 Plackett-Burman (PB) 设计对影响 Lh-1 产抗菌肽的 15 个因素进行了筛选, 得到了影响产量的显著因素为 : 葡萄糖蛋白胨和氯化钙在此基础上采用响应面法对该 3 个显著因素的最佳水平范围进行研究, 得到的最佳浓度为 g/l 葡萄糖 6.01 g/l 蛋白胨和 3.29 g/l CaCl 2, 同时 R-1 的产量从 ku/ml 提高到了 ku/ml 关键词 : Brevibacillus laterosporus Lh-1, 抗菌肽 R-1, Plackett-Burman (PB) 设计, 响应面分析 (RSM) Optimization of an Antimicrobial Peptide R-1 Production by Brevibacillus laterosporus Lh-1 ZHENG Yuan WANG Yue-Jun SUN Mi * (Laboratory of Marine Enzyme and Engineering, Yellow Sea Fisheries Research Institute, Qingdao, Shandong , China) Abstract: To improve and optimize marine antimicrobial peptide R-1 production by a newly isolated Brevibacillus laterosporus Lh-1, Plackett-Burman (PB) design and response surface methodology (RSM) using central composite design was adopted in culture conditions. MINITAB 15.0 was used for planning the experiments, data analysis, contour diagrams and response optimizations. In this study, PB design was undertaken to evaluate the effects of the fifteen factors. By the statistical regression analysis, the significant factors affecting the novel antimicrobial peptide R-1 in submerged fermentation by Br. laterosporus Lh-1 were determined as follows: glucose, peptone and CaCl 2. Then a RSM was used to optimize the above critical internal factors, and the optical concentration of the variables were determined as: g/l glucose, 6.01 g/l peptone and 3.29 g/l CaCl 2. The content of R-1 was increased from ku/ml to ku/ml. Keywords: Brevibacillus laterosporus Lh-1, Antimicrobial peptide R-1, Plackett-Burman (PB) design, Response surface methodology(rsm), plipastatin surfactin iturin fengycin subtilin subtilosin sunlancin [4],, 基金项目 : (No ); 863 (No. 2006AA10Z349, 2007AA091602) * 通讯作者 :Tel: ; : clowisme@sina.com.cn 收稿日期 : ; 接受日期 :

2 : Brevibacillus laterosporus Lh-1 R-1 399,, [1 3],, 100, [5],,,, [6 8], (Brevibacillus laterosporus Lh-1), R-1 (G + G ), [6] Plackett-Burman(PB) (Response surface methodology, RSM),, (R-1), Plackett-Burman(PB) (Response surface methodology, RSM),,, [9 14] 1 材料与方法 1.1 供试菌株 Br. laterosporus Lh-1 (Micrococcus lysodeikticus) ATCC No.4698 Sigma 1.2 培养基及培养条件 10 g/l, 5 g/l, CaCl 2 2 g/l, MgSO 4 5 g/l, 1000 ml, ph 7.2~ 7.5 Plackett-Burman RSM (g/l), 1000 ml, 30 C 200 r/min, 18 h, 8%(V/V), 25 ml(250 ml ), 30 C 200 r/min, 24 h 1.3 抗菌肽 R-1 活力的测定 [15] 3, 3, ( ) 1.4 实验设计 Plackett-Burman(PB) 设计 :, Minitab 15.0,, 响应面设计 (RSM): (RSM), Plackett- Burman (PB), RSM, R-1 2 (1) xi = ( Xi Xi )/ Δ Xi (1) x i X i, X 0 X i, ΔX i X i 2 0 i i ij i j ii i x Y = b + b x + b x x + b x + e (2) Y, R-1, b 0, b i, b ij, 20 2 结果 2.1 抗菌活力测定,,, ( [6] ), ( 1) R 影响抗菌肽 R-1 产量的关键因素 Plackett-Burman(PB) 3, 3 3,, (P=0.008) CaCl 2 (P=0.003) (P=0.008) 3 95%,,

3 400 微生物学通报 2009, Vol.36, No.3 Table 1 表 1 Plackett-Burman 实验因素水平及编码 Level and code of variables chosen for Plackett-Burman design Variable (g/l) Symbol Coded levels 1 1 NH 4 NO 3 X Glucose X KH 2 PO 4 X Sucrose X Soluble starch X MgSO 4 X CaCl 2 X Peptone X (NH 4 ) 2 SO 4 X Maltose X Beef extract X Yeast extract X ZnCl 2 X BaCl 2 X Dextrine X Table 2 表 2 实验因素水平和编码 Boundaries of experimental domain and spacing of levels expressed in coded and natural units Code unit Experimental factor Glucose (g/l, X 1 ) Peptone (g/l, X 2 ) CaCl 2 (g/l, X 3 ) Δx Table 3 表 3 抗菌肽 R-1 在 Plackett-Burman 设计中回归方程方差分析表 ANOVA for the regression equation of antimicrobial peptide R-1 in Plackett-Burman design Source Seq SS DF Adj MS F value P value Model Residual Total R 2 = 96.41% Adj R 2 = 82.93% 图 1 抗菌肽 R-1 活力标准曲线 Fig. 1 Standard activity curve of antimicrobial peptide R-1 CaCl 2 Lh-1 R-1,, 2.3 利用响应面方法优化抗菌肽 R-1 发酵培养基多元二次模型的建立以及检验 :, R-1, 20

4 : Brevibacillus laterosporus Lh-1 R Minitab , Y= X X X X X X X 1 X X 1 X X 2 X 3, Y, X 1 X 2 X 3 5,,, (R 2 =92.61%), R 响应面交互作用分析及优化 : 2~4, 2~4,, R-1, 表 4 中心复合设计以及抗菌肽 R-1 产量的实验结果 Table 4 Central composite design and values of antimicrobial peptide R-1 Yield (ku/ml) Run no. X 1 X 2 X 3 Observed value Predicted value Table 5 表 5 抗菌肽 R-1 在中心复合设计中回归方程方差分析表 ANOVA for the regression equation of antimicrobial peptide R-1 in Central composite design Source Seq SS DF Adj MS F value P value Model <0.001 Residual Total R 2 = 92.61% Adj R 2 = 85.97%

corresponding contour plot of R-1 versus glucose and peptone 图 3 葡萄糖与

3 Response surface plot and corresponding contour plot of R-1 versus

5 402 微生物学通报 2009, Vol.36, No.3 Fig. 2 图 2 葡萄糖与蛋白胨交互影响 R-1 产量的曲面图和等高线图 Response surface plot and corresponding contour plot of R-1 versus glucose and peptone 图 3 葡萄糖与 CaCl 2 交互影响 R-1 产量的曲面图和等高线图 Fig. 3 Response surface plot and corresponding contour plot of R-1 versus glucose and CaCl 2 图 4 蛋白胨与 CaCl 2 交互影响 R-1 产量的曲面图和等高线图 Fig. 4 Response surface plot and corresponding contour plot of R-1 versus peptone and CaCl 2,,,,,

6 : Brevibacillus laterosporus Lh-1 R 讨论,,,,,,, [16,17] Plackett-Burman, (response surface methodology, RSM),,,,, [9 14], Lh-1 R-1, Plackett-Burman, Lh-1 15, 3 R-1,,, 3 R-1,, g/l, 6.01 g/l, 3.29 g/l, R-1, ku/ml, ku/ml, ku/ml,, PB RSM,, 参考文献 [1] Alexander DJ, Newcastle disease and other paramyxovirus infections. In: Diseases of Poultry, 9th ed. Iowa State University Press, Ames, LA. 1991, pp [2] BW.., 1999, p.59. [3] Faulkner DJ. Marine natural products. Nat Prod Rep, 1996, 13(2): 75. [4],,,. fmbr., 2006, 27(6): [5] Kobayashy J, lshibashi M, Walchi MR, et al. AmphidinodeC: Amphidinolide C: The first 25-membered macrocyclic lactone with potent antineoplastic activity from the cultured dinoflagellate Amphidinium sp.. J Am Chem Soc, 1988, 110: 490. [6],,,. Lh-1., 2007, 47(6): [7] 琇,,,. S , 2007, 14(3): [8] Xiu Z, Yue-jun W, Mi S, et al. Purification and properties of lysozyme from a marine strain. Annals of Microbiology, 2008, 58(1): [9],,,. Lactobacillus casei Zhang., 2008, 35(4): [10],,.., 2008, 35(2): [11],,,. Ju-A10 CMCase., 2006, 33(3): [12],,,. SAS L-., 2004, 31(3): [13],.., 2004, 31(6): [14],,.., 2002, 29(5): [15],.. :, 1982, p.152. [16] Li F, Rong-ping L, Jin L, et al. Study on a protein kinases lysis activity of Bacillus subtilis HW-2: Selection of liquid fermentation conditions. Progress in Biotechnology, 1997, 17(3): [17] Qiu-ling X, Yong G. The optimization of fermentation conditions of nattokinase. Journal o f South China University of Technology: Natural Science Edition, 1999, 27(5):

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3 微生物学通报 FEB 20, 2008, 35(2) 306~30 Microbiology 2008 by Institute of Microbiology, CAS tongbao@im.ac.cn 生物实验室响应面分析法优化副溶血性弧菌生长条件刘代新宁喜斌 * 张继伦 2 (. 200090) (2. 20202) 摘要