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1 响应面法优化重组大肠杆菌生物合成别藻蓝蛋白 (holo-apca) 的发酵条件周孙林 1, 陈华新 2, 姜鹏 2, 李富超 2 1, 3, 唐东山 (1., ; 2., ; 3., ) 摘要 : 采用响应面法对重组大肠杆菌生物合成别藻蓝蛋白 (holo-apca) 的发酵条件进行优化, 提高了蛋白的表达量以对重组别藻蓝蛋白 α 亚基在大肠杆菌中表达量影响较大的几种因素用于中心组合设计 ; 中心组合设计试验结果表明诱导温度培养基初始 ph 和诱导时机对诱导结果影响显著 ; 经 Design Expert 8.0 软件优化后的最佳诱导条件为 : 诱导温度 28, 培养基初始 ph 8.5, IPTG 终浓度 0.1 mmol/l, 以及诱导时机 3 h 最后验证试验得到的蛋白表达量在已有文献报道结果的基础上提高了 70%~580% 中心组合设计 - 响应面法能够在有限的实验次数下, 对影响生物过程的因子进行优化及对其交互作用进行评价, 蛋白表达量达 20.4 mg/l; IPTG 用量由原来的 1 mmol/l 减少至现在的 0.1 mmol/l, 降低了发酵成本关键词 : 重组别藻蓝蛋白 α 亚基 ; 诱导表达 ; 优化 ; 响应面法 ; 中心组合设计中图分类号 : S188 文献标识码 : A 文章编号 : (2015) doi: /hykx ,,,,,,,,, [1], [1-4] (Synechococcus elongatus BP-1) α pcdfduet-1, Ho1 PcyA pcdfduet-1, pcdf-apca-ho1-pcya; apca CpcS CpcU prsfduet-1, prsf-cpcscpcu,, α [2], 56 / 2015 / 39 / 4,, [5],, (One-factor-at-a-time design),,,,,,,,, [6-9], (Response : ; : : ( ); 863 (2014AA093505, 2014AA039501); ( ) : (1990-),,,,, saeling@sina.cn;,, chenhx001@126.com;,, xhtds@sohu.com

2 surface methodology, RSM),,,,,, [6, 9-10] α,, 2.3, [12] 70 %~580 %, IPTG, [12] (IPTG 0.5 mm), - 1 材料与方法 1.1 菌株培养基和试剂 : E. coli BL21(DE3)/pCDFapcA-Ho1-pcyA/pRSF-cpcU-cpcS, α 5 : LB ( 10 g/l, 5 g/l, NaCl 10 g/l; ph 7.4) : TB ( 12 g/l, 12 g/l, (72 mmol/l K 2 HPO 4 ; 17 mmol/l KH 2 PO 4 ), 4 ) : Oxoid,, IPTG Solarbio, 1.2 试验方法 (RSM) [5] 4 ( ph IPTG ) 4 5, 30, 6 ( ), 3 ( 90 ) 1 表 1 中心组合设计试验因素与水平 Tab. 1 Factors and levels of central composition design A(, ) B( ph) C(IPTG, mmol/l) D(, min) % (100 μg/ml) (50 μg/ml) 50 ml LB, 37, 200 r/min 15 h 4%, ph 25 ml TB, 37 (200 r/min), IPTG, (8 h) F-4500 (Hitachi, Japan), 600~700 nm, 590 nm, 240 nm/s, 1000, 1000, (CCD), 0~500 a.u.,, (mg/l)=( )/22.781, Design-Expert 8.0,, (holo-apca), 2 结果与分析 2.1 中心组合设计试验优化重组别藻蓝蛋白 (holo-apca) 的发酵条件 Design-Expert Marine Sciences / Vol. 39, No. 4 /

3 表 2 中心组合试验设计及结果 Tab. 2 Coded and real values of variables in central composition design ( ) ph IPTG (mmol/l) (min) (au) (mg/l) 表 3 响应面二次模型回归方程的方差分析结果 Tab. 3 Analysis of variances for full quadratic model F P rob >F < : C V %= 29.84; R 2 = ; R Adj 2 = ; R Pred 2 = , 4 A( ) B( ph) C(IPTG ) D( ) R : R= A+15.08B 6.08C+31.40D+5.79AB 7.60AC+11.85AD 2.52BC+10.02BD 1.38CD 22.01A B C D 2 (1) Design expert 8.0, 3 4 3, F 9.37, P rob >F <0.0001,, 0.01% ; P rob > F = >0.05,, ; CV%= 29.84,, ; R , R 2 Adj = , 89.74% 4, A( ) B (ph) D( 58 / 2015 / 39 / 4

4 表 4 各编码因素水平方差分析 Tab. 4 Analysis of variances for holo-apca production in coded level variables P A B C D < AB AC AD BC BD CD A < B < C < D < : P>0.05, P<0.05, P<0.01 ) P 0.05, 3, D P <0.0001, ; C(IPTG ) P =0.1766>0.05,, : D>A>B>C;, AD P 0.05, ; A 2 D 2 P 0.05,, A 2 P <0.0001,, 2.1.3, 2,, ;,, 1,, (a) ph (d) IPTG ph,,, ; (b, c, e, f),,, 2.2 验证试验, Design-Expert : 28, ph 8.5, IPTG 0.1 mmol/l, 3 h 269.5, 21.2 mg/l,, 3, au, 20.4 mg/l, 4 %,, 3 讨论,,,, [5, 11],,,, - Design-expert 8.0 : 28 ph h IPTG 0.1 mmol/l, (holo-apca) 20.4 mg/l, 4 %,,, ( 9 mg/l), 2.3, α 3~12 mg/l [12], Marine Sciences / Vol. 39, No. 4 /

5 60 海洋科学 / 2015 年 / 第 39 卷 / 第 4 期

Fig. 1 图 1 各因素交互作用及其对别藻蓝蛋白表达量影响的响应面图和等高线图 Response surface curves and contour plot of factor interaction effecting on holo-apca production 表 5 最优化发酵条件验证试验结果 Table.

6 Fig. 1 图 1 各因素交互作用及其对别藻蓝蛋白表达量影响的响应面图和等高线图 Response surface curves and contour plot of factor interaction effecting on holo-apca production 表 5 最优化发酵条件验证试验结果 Table. 5 Validation of the model 菌液荧光(au) 蛋白表达量(mg/L) 项目诱导温度( ) ph IPTG (mmol/l) 诱导时机(min) 预测值实际值实际值未优化优化提高了 70 %~580 % 该发酵条件与原发酵条件相比通透性的增大, 从而有利于诱导剂 IPTG 进入到细胞主要不同在于发酵培养基初始 ph 由原来的 7.4 增大内部与阻遏蛋白结合, 起到诱导效果由于 IPTG 是到现在的 8.5, IPTG 使用终浓度由原来的 1 mmol/l 一种作用极强的诱导剂, 不被细菌代谢而十分稳定, 减少到现在的 0.1 mmol/l 因此少量的 IPTG 在较高通透性的细胞间可自由出发酵培养基初始 ph 对菌体的生长和产物的表入, 对多个细胞起到诱导作用而由响应面图 1 可以达均存在一定的影响, 因为乙酸是大肠杆菌的主要看出, IPTG 终浓度过高反而会导致表达量的减少, 副产物, 它对细菌生长和产物的表达都有抑制作由于 IPTG 对工程菌的生长有着明显的抑制作用[18-19], 用 [13-15], 刘永军等 [16] 通过试验得到大肠杆菌的最适菌体量不足固然导致蛋白表达量的降低生长酸度为 7.82, 较高的初始 ph 对菌体生长过程中通过响应面模型分析得出影响因素 A(诱导温度) 产生的乙酸存在一定的调节作用, 促进菌体的生长与 D(诱导时机)间存在明显的交互作用交互作用是与产物的表达培养基的 ph 还将影响酶分子和底物两种或两种以上因子的综合效果 [20], 在统计学上的分子的带电状况, 从而影响酶的合成, 使代谢和细定义为: 如果因素 A 的数值和水平发生变化, 试验胞透性发生变化 [17] 分析 ph 的增加可能导致细胞膜指标随因素 B 变化的规律发生变化; 或反之, 若因 Marine Sciences / Vol. 39, No. 4 /

7 B, A AD( ) : AD( ) :,,, 37, 28, 3 h,,, ( 1 ~3 h) ( ),,,,,, ;,,, ( 1 ~3 h) ( ),,,,,,,, IPTG, : [1] Zhang W J, Guan X Y, Yang Y, et al. Biosynthesis of fluorescent allophycocyanin α-subunits by autocatalysis in Escherichia coli[j]. Biotechnology and Applied Biochemistry, 2009, 52: [2]. [D]. :, [3] Ge B S H, Sun H X, Feng Y, et al. Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli[j]. Journal of Bioscience and Bioengineering, 2009, 107 (3): [4] Liu S H F, Chen Y J, Lu Y D, et al. Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli[j]. Photosynth Res, 2010, 105: [5],,,. [J]., 2009, 33(9): [6],,. [J]., 2010, 37(6): [7],,,. γ-pga [J]., 2008, 6(3): [8],. [J]., 2010, 2 (6): [9],,,. [J]. ( ), 2002, 41(Suppl): [10] He J, Zhen Q W, Qiu N, et al. Medium optimization for the production of a novel bioflocculant from Halomonas sp V3a using response surface methodology[j]. Bioresource Technology, 2009, 100(23): [11],. [J]., 2005, 29(11): [12] Biswas A, Vasquez Y M, Dragomani T M, et al. Biosynthesis of cyanobacterial phycobiliproteins in Escherichia coli: chromophorylation efficiency and specificity of all bilin lyases from Synechococcus sp. Strain PCC 7002[J]. Applied and Environmental Microbiology, 2010, 76(9): [13] Konstantinor K, Kishimoto M, Seki T, et al. A balanced DO-stat and its application to the control of acetic acid excretion by recombinant Escherichia coli[j]. Biotech Bioeng, 1990, 36(7): [14],. [J]., 2000, 20(2), [15],,. YK537/pSB-TK RP-HPLC [J]., 1998, 28(3): [16],,,. [J]., 1997, 13(7): [17],. ph [J]., 62 / 2015 / 39 / 4

8 2001, 31(4): [18],,,. α- [J]., 2007, 25(3): [19],,,. (rh.endostatin) [J]., 2005, 21(4): [20]. SPSS [J]. ( ), 2009, 12(4): Optimization of fermentation conditions for production of Holo-α-allophycocyanin (holo-apca) by recombinant Escherichia coli using response surface methodology ZHOU Sun-lin 1, CHEN Hua-xin 2, JIANG Peng 2, LI Fu-chao 2, TANG Dong-shan 1, 3 (1. Key Laboratory of pollution control and resource reuse of University of South China, college and university of Hunan province, Hunan , China; 2. Institute of Oceanology, Chinese Academy of Sciences, Qingdao , China; 3. Institute of environmental protection and safety projects, University of South China, Hunan , China) Received: Jun., 28, 2014 Key words: Holo-α-allophycocyanin (holo-apca); induce and express condition; optimization response surface methodology; central composite design Abstract: Response surface methodology was employed to optimize the fermentation conditions for holo-α-allophycocyanin (holo-apca) production in recombinant metabolically engineered Escherichia coli, by which the expression level of protein indeed increased. In the design of response surface experiment, four variables, i.e. initial ph of culture medium, IPTG concentration, post-induction temperature, and induction start time, were selected for investigation of the individual and interactive effects between them and on holo-apca production by E. coli. The experimental results showed that post-induction temperature, initial ph of culture medium, and induction start time had significant individual effects on holo-apca production, and the best possible induction condition optimized by Design Expert 8.0 software is: a post-induction temperature of 28, ph of culture medium of 8.5, IPTG concentration of 0.1 mmol/l, and induction start time of 3 h after starting cultivation. The protein expression level of final verification experiment was increased 70%~580% based on the reported results. The response surface methodology was used to evaluate the interactions among the factors under a limited number of trials. An expression level of 20.4 mg/l was obtained under optimized conditions. The IPTG consumption was decreased from 1 mmol/l to 0.1 mmol/l, which reduced the costs of fermentation. ( 本文编辑 : 梁德海 ) Marine Sciences / Vol. 39, No. 4 /

: 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