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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 陈磊等 / 基于体外分子进化技术提高弯曲芽孢杆菌 CCTCC β- 淀粉酶的热稳定性 Feb. 25, 2018, 34(2): Chin J Biotech, All rights reserved 工业生物技术 255 CCTCC β- 陈磊 1,2, 陈晟 1,2, 吴敬 1,2, 吴丹 1, ,,,. CCTCC β-., 2018, 34(2): Chen L, Chen S, Wu J, et al. Enhancing thermostability of β-amylase from Bacillus flexus CCTCC based on in vitro molecular evolution. Chin J Biotech, 2018, 34(2): 摘要 : 运用体外分子进化技术易错 PCR 方法, 高通量筛选热稳定性提高的弯曲芽孢杆菌 Bacillus flexus CCTCC β- 淀粉酶突变体 利用 LB 琼脂淀粉板显色 96- 孔板 DNS 法测酶活和酶标仪检测等, 最终筛选到了一株热稳定性显著提高的突变体 D476N 野生型和突变体 D476N 分别纯化后, 酶学性质测定表明 : 突变体 D476N 的最适 ph 为 6.5, 与野生型相比降低了 0.5 突变体 D476N 和野生型的最适温度均为 55, 突变体 D476N 在 55 下的半衰期为 35 min, 比野生型提高了 95% 突变体 D476N 的 T 50 值比野生型提高 4 突变体 D476N 的 K m 值为 µmol/l, 是野生型 (85.86 µmol/l) 1.14 倍 ; 突变体稳定性提高的同时, 催化活力相对于野生型有略微下降 通过 SWISS-MODEL 同源模拟野生型和突变体 D476N 的三维结构, 并通过 PyMol 软件分析, 发现突变后的氨基酸残基 Asn476 位于蛋白质表面的 loop 环上, 通过 MOE 软件计算,D476N 的分子自由能 (ΔG) 为 kcal/mol, 比野生酶降低 10.3%, 这一结果与蛋白质分子自由能和热稳定性呈负相关的理论相符 : 分子进化, 易错 PCR,β- 淀粉酶, 热稳定性 Received: May 26, 2017; Accepted: August 8, 2017 Supported by: National Natural Science Foundation of China (No ), National Outstanding Youth Fund (No ), Jiangsu University Outstanding Scientific and Technological Innovation Team Project, 111 Plan (No ), Central University Basic Scientific Research Business Special Funds (No. JUSRP51706A). Corresponding author: Dan Wu. Tel/Fax: ; wudan@jiangnan.edu.cn (No ) (No ) 111 (No ) (No. JUSRP51706A)

2 256 ISSN CN /Q Chin J Biotech Enhancing thermostability of β-amylase from Bacillus flexus CCTCC based on in vitro molecular evolution Lei Chen 1,2, Sheng Chen 1,2, Jing Wu 1,2, and Dan Wu 1,2 1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi , Jiangsu, China 2 Key Laboratory of Industrial Biotechnology Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi , Jiangsu, China Abstract: We used in vitro molecular evolution technology by error-prone PCR and high-throughput screening to improve thermostability of Bacillus flexus CCTCC β-amylase. Mutant D476N with significant thermostability increase was selected by LB agar starch plate colorimetric assay and 96-well plate enzyme activity assay. The optimum ph was 6.5 for the mutant D476N, compared to 7.0 of the wild type. The optimal temperature was 55 for both mutant D476N and the wild type. The T 50 value of the mutant D476N was 4 higher than that of the wild type. The half-life of mutant D476N at 55 was 35 min, 95% higher than that of the wild type. The K m of the mutant D476N was µmol/l, 1.14 times of that of the wild type (85.86 µmol/l). The thermostability of the mutant D476N was slightly lower than that of the wild type. The three-dimensional structure of wild type and mutant D476N was simulated by SWISS-MODEL and analyzed by PyMol software. The mutated amino acid residue Asn476 was located on the loop of protein surface. The molecular free energy(δg) of D476N was calculated by MOE software was kcal/mol, reduced by 10.3% compared to the wild enzyme. These results were consistent with the theory that the protein molecular free energy and thermostability were negatively correlated. Keywords: molecular evolution, error-prone PCR, β-amylase, thermostability β- (β-amylase EC ) β-1,4- α-1,4- β- β Higashihara [1] Bacillus megaterium β- Bacillus cereus [2-3] Bacillus circulans [4] Bacillus polymyxa [5] Thermoanaerobacterium Thermeoactinomyces sp. [6] β- β- β- β- PCR PCR [7] β- β- [8]

3 陈磊等 / 基于体外分子进化技术提高弯曲芽孢杆菌 CCTCC β- 淀粉酶的热稳定性 257 β- PCR β- LB 96 DNS 1 材料与方法 1.1 材料 Escherichia coli JM109 E. coli BL21(DE3) pet-24a(+) pmd18-t β- Bacillus flexus CCTCC (bfa) (CCTCC) LB 5.0 g/l 10.0 g/l NaCl 10.0 g/l LB LB 1.0% 2.0% (W/V) TB 24.0 g/l 5.0 g/l 12.0 g/l K 2 HPO 4 3H 2 O g/l KH 2 PO g/l SOB 20.0 g/l 5.0 g/l MgCl g/l NaCl 0.5 g/l KCl g/l LB LB 1 g/l rtaq T4 DNA Primer Star TM HS DNA DL-2000 DL DNA Marker TaKaRa DNA PCR (SDS-PAGE) ( ) (Kan) -β-d- (IPTG) Oxoid 1.2 方法 PCR Bacillus flexus CCTCC (bfa) β- (5 -AACCATGGCGGTAAATGGACA GTCGTT-3 ) (5 -AACTCGAGTTACCA ATTATT CGTATACGTTG-3 ) NcoⅠ XhoⅠ ( ) (50 µl) 2 mmol/l MgCl mmol/l MnCl mmol/l dntps DNA 50 ng 5 µl 10 Mg 2+ (-) rtaq DNA 5 U ddh 2 O 50 µl PCR 94 4 min s s 72 1 min 40 s min 4 PCR 1% (W/V) PCR bfa pmd-18t JM109 NcoⅠ XhoⅠ bfa-pmd-18t bfa pet24a(+) pet-bfa BL21 (DE3) LB (Kan 30 µg/ml) (Molecular Devices cjb@im.ac.cn

4 258 ISSN CN /Q Chin J Biotech Qpix 2) LB (Kan) BFA 600 µl LB (Kan 30 µg/ml) r/min 8 10 h 50 µl 600 µl TB 96 2 h 0.05 mmol/l IPTG [9] h r/min 4 20 min 200 µl LB BFA gridding BFA % (W/V) β- BFA DNS BFA β- pet-bfa r/min LB (Kan 30 µg/ml) 8 10 h 2.5 ml 50 ml TB r/min 2 h 0.05 mmol/l IPTG r/min 48 h [10] r/min 4 20 min 45% (W/V) r/min 20 min 20 mmol/l - (ph 6.5) 20 mmol/l - Superdex TM /300 GL 0.5 ml/min (SDS-PAGE 12% ) [11] β- 0.5 ml 2% (W/V) 0.4 ml 50 mmol/l - (ph 7.0) 1 ml min 1 ml 1 ml 3,5- (DNS) 5 min 12 ml 540 nm 1 µmol/l 1 (U) β- 55 ph 7.0 (1 30 mg/ml) DNS GraphPad Prism 6.0 Michaelis-Menten K m k cat k cat /K m β- (30 70 ) β- 100% β- 55 β-

5 陈磊等 / 基于体外分子进化技术提高弯曲芽孢杆菌 CCTCC β- 淀粉酶的热稳定性 (ph 7.0) (10 70 min) 10 min 10 min 50% T 50 DNS 100% β β- ph ph β- 100% β- ph SWISS-MODEL ( org/swissmod/) BFA β- (1j10.1.A PDB) [12-13] PyMol ( 2 结果与分析 2.1 突变体文库的筛选 E. coli BL21 (DE3)/ pet-bfa β- PCR β- 60% β- [14] min 96- β- BFA 476 (Asp476Asn) [15] 2.2 野生型和突变体 β- 淀粉酶的诱导表达和分离纯化 β mmol/l IPTG E. coli BL21(DE3) 48 h β- (NH 4 ) 2 SO 4 - (20 mmol/l ph 6.5) Superdex TM /300 GL ( 1 1) Bradford [16] 2 D476N U/mg 18.76% 2.3 野生型和突变体 D476N 的酶学性质分析 β- 55 (1 30 mg/ml) D476N ( 2) D476N K m µmol/l 1.14 D476N k cat s % k cat /K m 71.01% D476N β D476N β β D476N 75% 44% 37% 23% cjb@im.ac.cn

6 260 ISSN CN /Q Chin J Biotech 表 1 重组野生型和突变体 β- 淀粉酶的纯化过程参数 Table 1 Parameters of purification step of the recombinant wild type and mutant β-amylase Wild type Purification steps Total protein (mg) Total activity (U) Specific activity (U/mg) Purified fold Recovery rate (%) Crude enzyme Salt precipitation Superdex TM /300 GL D476N Crude enzyme Salt precipitation Superdex TM /300 GL 图 1 SDS-PAGE 分析纯化后的野生型和突变体 D476N β- 淀粉酶 (M: 中分子量标准蛋白 ;1: 野生型 ; 2: 突变体 D476N) Fig. 1 SDS-PAGE analysis of purified wild type and mutant D476N β-amylases. M: marker; 1: wild type; 2: mutant D476N. 图 2 野生型和突变体 D476N 的最适温度 Fig. 2 The optimum temperature of wild type and mutant D476N. 表 2 野生酶和热稳定性提高突变体 D476N 的动力学参数 Table 2 Kinetic parameters of wild type and mutant D476N enzymes Enzyme k cat (s 1 ) K m (µmol/l) k cat /K m (L/(µmol s)) Wild type ± ± ±6.19 D476N ± ± ±4.59 Enzyme reactions were measured at 55, ph 7.0, using different concentrations of soluble starch as the substrate. 图 3 野生型和突变体 D476N 的热稳定性 Fig. 3 The temperature stability of wild type and mutant D476N.

7 陈磊等 / 基于体外分子进化技术提高弯曲芽孢杆菌 CCTCC β- 淀粉酶的热稳定性 min 50% T 50 D476N T T D476N [17-18] ( 3) 3 D476N min 18 min ph D476N β- ph 4 4 ph 7.0 D476N ph 6.5 ph β- 70% β- D476N ph 0.5 [19] 3 讨论 PCR BFA D476N E. coli BL21(DE3)/pET-D476N E. coli BL21(DE3) [20] β- D476N D476N D476N [21-22] D476N D476N SWISSMODEL (1j10.1A PDB) D476N loop [23] - PCR 7 5 loop PyMol D476N Asn476 loop ( 5) MOE 图 4 野生型和突变体 D476N 的最适 ph Fig. 4 The optimum ph of wild type and mutant D476N. 图 5 热稳定性提高的突变体 D476N β- 淀粉酶的三维结构和突变氨基酸定位 Fig. 5 Three-dimensional structure of the mutant D476N β-amylase with increased thermostability and the location of mutated acid Asn476. The figure was made using PyMol cjb@im.ac.cn

8 262 ISSN CN /Q Chin J Biotech MOE D476N PDB D476N [24] (ΔG) β- D476N kcal/mol kcal/mol (ΔG) β- 476 β kcal/mol 10.3% loop REFERENCES [1] Rani RR, Jana SC, Nanda G. Saccharification of indigenous starches by β-amylase of Bacillus megaterium. World J Microbiol Biotechnol, 1994, 10(6): [2] Nanmori T, Shinke R, Aoki K, et al. Purification and characterization of β-amylase from Bacillus cereus BQ10-S1 Spo II. Agric Biol Chem, 1983, 47(5): [3] Yamaguchi T, Matsumoto Y, Shirakawa M, et al. Cloning, sequencing, and expression of a β-amylase gene from Bacillus cereus var. mycoides and characterization of its products. Biosci Biotechnol Biochem, 1996, 60(8): [4] Siggens KW. Molecular cloning and characterization of the beta-amylase gene from Bacillus circulans. Mol Microbiol, 1987, 1(3): [5] Murao S, Ohyama K, Arai M. β-amylases from Bacillus polymyxa No. 72. Agric Biol Chem, 1979, 43(4): [6] Kitamoto N, Yamagata H, Kato T, et al. Cloning and sequencing of the gene encoding thermophilic β-amylase of Clostridium thermosulfurogenes. J Bacteriol, 1988, 170(12): [7] Saha BC, Zeikus JG. Microbial glucoamylases: biochemical and biotechnological features. Starch- Stärke, 1989, 41(2): [8] Higashihara M, Okada S. Studies on β-amylase of Bacillus megaterium strain No. 32. Agric Biol Chem, 1974, 38(5): [9] Zhou BY, Wang Z, Kong DY, et al. Selection of high β-amylase producing strain and its fermentation conditions. Chin J Biotech, 1994, 10(3): (in Chinese).,,,. β-., 1994, 10(3): [10] Totsuka A, Fukazawa C. Expression and mutation of soybean β-amylase in Escherichia coli. Eur J Biochem, 1993, 214(3): [11] Sohn CB, Lee SM, Kim MH, et al. Purification and characterization of β-amylase from Bacillus polymyxa No J Food Sci, 1996, 61(1): [12] Nanmori T, Numata Y, Shinke R. Isolation and characterization of a Bacillus cereus mutant strain hyperproductive of exo-β-amylase. Appl Environ Microbiol, 1987, 53(4): [13] Nanmori T, Shinke R, Aoki K, et al. β-amylase production by a rifampin-resistant, asporogenous mutant from Bacillus cereus BQ10-Sl. Agric Biol Chem, 1983, 47(3): [14] Wu J, Zhang SZ. Cloning, expression and the characterization of β-amylase from a Bacillus megaterium WS06. Chin J Biotech, 2008, 24(10): (in Chinese).,. β-., 2008, 24(10): [15] Nanmori T, Nagai M, Shimizu Y, et al. Cloning of the β-amylase gene from Bacillus cereus and characteristics of the primary structure of the enzyme. Appl Environ Microbiol, 1993, 59(2): [16] Lee JS, Wittchen KD, Stahl C, et al. Cloning, expression, and carbon catabolite repression of the bamm gene encoding β-amylase of Bacillus megaterium DSM319. Appl Microbiol Biotechnol, 2001, 56(1/2): [17] Wijma HJ, Floor RJ, Jekel PA, et al. Computationally designed libraries for rapid enzyme stabilization.

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