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1 微生物学通报 Microbiology China Feb. 20, 2017, 44(2): DOI: /j.microbiol.china 研究报告细菌麦芽糖淀粉酶在枯草芽孢杆菌中的诱导型异源表达杨韵霏李由然张梁李赢顾正华丁重阳 ( ) ( ) 石贵阳 * 摘要 : 目的实现地衣芽孢杆菌麦芽糖淀粉酶在枯草芽孢杆菌中的高效异源表达, 并研究该重组酶的酶学性质方法克隆巨大芽孢杆菌木糖异构酶基因的启动子区域及其调控蛋白, 构建一个大肠杆菌 / 芽孢杆菌穿梭型诱导表达质粒, 使用该诱导型启动子介导麦芽糖淀粉酶编码基因, 实现其在枯草芽孢杆菌中的功能表达对重组枯草芽孢杆菌的诱导条件进行优化, 提高麦芽糖淀粉酶的产量结果获得了诱导表达麦芽糖淀粉酶基因的重组枯草芽孢杆菌菌株最适诱导温度为 45 C, 最适诱导剂添加浓度为 1%, 最适添加诱导剂时间为接种培养 9 h 后重组酶蛋白分子量大小为 67 kd, 对该酶的酶学性质研究发现, 以可溶性淀粉为底物, 反应生成麦芽糖和葡萄糖, 其中麦芽糖含量为 60.42% 重组酶最适作用温度为 45 C, 最适作用 ph 为 6.5,Ca 2+ Co 2+ EDTA 对该重组麦芽糖淀粉酶具有激活作用结论通过木糖诱导表达系统可以实现麦芽糖淀粉酶在枯草芽孢杆菌中的高效诱导型表达, 酶活最高可达 U/mL 发酵液, 在工业上有着较好的应用前景关键词 : 枯草芽孢杆菌, 麦芽糖淀粉酶, 木糖诱导启动子, 诱导条件, 酶学性质 Inducible heterogenous expression of bacterial maltogenic amylase in Bacillus subtilis YANG Yun-Fei LI You-Ran ZHANG Liang LI Ying GU Zheng-Hua DING Zhong-Yang SHI Gui-Yang * (1. National Engineering Laboratory for Cereal Fermentation Technology, Wuxi, Jiangsu , China) (2. School of Biotechnology, Jiangnan University, Wuxi, Jiangsu , China) Abstract: [Objective] To achieve the efficient heterogenous expression of a bacterial maltogenic amylase in Bacillus subtilis and investigate the characterization of the recombinant enzyme. [Methods] We cloned the genes of xylose isomerase promoter region and its regulatory protein from Bacillus megatherium into an Escherichia coli / Bacillus sp. shuttle expression vector, and constructed an recombinant expression plasmid which harbored an encoding gene of maltogenic amylase from Foundation item: National Natural Science Foundation of China (No ); Autonomous Program of Jiangnan University (No. JUSRP51503) *Corresponding author: Tel: ; gyshi@jiangnan.edu.cn Received: January 27, 2016; Accepted: May 03, 2016; Published online ( May 31, 2016 基金项目 (No ); (No. JUSRP51503) * 通讯作者 :Tel gyshi@jiangnan.edu.cn 收稿日期 : 接受日期 : 优先数字出版日期 (

2 264 微生物学通报 Microbiol. China 2017, Vol.44, No.2 Bacillus licheniformis. Then we transformed the expression plasmid into Bacillus subtilis and optimized the induction conditions of the transformant to increase the production of the recombinant enzyme. [Results] The recombinant Bacillus subtilis which inductively expressed the maltogenic amylase was obtained. The optimal induction condition was as follow, that 1% inducer was added into fermentation medium after 9 h incubation at 45 C. The recombinant maltogenic amylase had a molecular size of 67 kd. From the study of the enzymatic properties of the enzyme, it was found that, with soluble starch as substrate, the reaction products were 60.42% maltose and few glucose. Furthermore, the recombinant enzyme showed an optimal activity at 45 C with ph 6.5. Ca 2+ Co 2+ and EDTA could improve the efficiency of enzyme reaction. [Conclusion] With induction of xylose, the bacterial maltogenic amylase was efficiently expressed in the recombinant Bacillus subtilis, and the maximal yield reached U/mL, which showed good application prospect in industry. Keywords: Bacillus subtilis, Maltogenic amylase, Xylose promoter, Induction conditions, Enzymatic properties (Maltogenic amylase) 1,4-α-D- α- (1,4-α-D-glucan α-maltohydrolase EC ) GH13 [1-3] α- (Bacillus subtilis) GRAS (Generally recognized as safe) Rygus 200 [4] / phy300-plk 1 材料与方法 1.1 菌种质粒和培养基 (Bacillus licheniformis) ATCC14580 (Escherichia coli) JM109 (Bacillus megaterium) 1514 (Bacillus subtilis) WB600 phy300-plk (CICIM) phybm phybmyf WB600/pHYBMYF LB (g/l) NaCl 10.0 (g/l) KH 2 PO K 2 HPO ph 7.0

3 : mg/l 30 mg/l 1.2 主要试剂和仪器 DNA ( ) T4 DNA pmd19-t Simple Vector Fermentas ( ) ( ) ( ) ( ) K20 CF16RX II HITACHI PICO17 Thermo VCX-750 Sonic & Materials V-1200 DYY-6C University Hood II Bio-Rad (HPLC) Dionex 1.3 细菌基因组 DNA 的提取 PCR 引物及条件 LB 10 h Wilson DNA [5] 1514 Pxyl01 (5 -GCCAGATCTTAA CTAACTTATAGGGGT-3 Bgl II) Pxyl02 (5 -CGC GGATCCTTGTCATTTCCCCCTTT-3 BamH I) 1514 DNA PCR 95 C 5 min 94 C 30 s 54 C 30 s 72 C 90 s C 10 min (50 μl) dntps (2 mmol/l) 5 μl 10 Buffer 5 μl /(25 μmol/l) 1 μl 1 μl HiFi (2.5 U/μL) 0.5 μl ddh 2 O 37.5 μl ATCC14580 PyvdF01 (5 -GGCAGATCTATGGAATATGCAGCG ATACA-3 Bgl II) PyvdF02 (5 -ATGCCCGGGT TAGACCGCCCCCAAAATGA-3 Sma I) ATCC14580 DNA PCR 95 C 5 min 94 C 30 s 54 C 30 s 72 C 2 min C 10 min PCR pmd19-t Simple [6] JM109 pmdbm pmdyf 1.4 重组枯草芽孢杆菌的构建 pmdbm BMxyl phy300-plk JM109 phybm pmdyf yvdf phybm JM109 phybmyf phybmyf phybm Bott [7] WB600 WB600/pHYBMYF WB600/pHYBM 1.5 重组麦芽糖淀粉酶的诱导发酵及粗酶液的制备 WB600/pHYBMYF 15 ml LB 37 C 200 r/min 1 ml 30 ml 37 C 200 r/min 8 h 1% 16 h r/min 4 C Na 2 HPO 4 -NaH 2 PO 4 (0.2 mol/l ph 6.5) OD 600

4 266 微生物学通报 Microbiol. China 2017, Vol.44, No 麦芽糖淀粉酶酶活力检测方法蛋白含量测定及 SDS-PAGE 电泳 1 ml Na 2 HPO 4 -NaH 2 PO 4 (0.2 mol/l ph 6.5) 1% 40 C 2 min 50 μl 40 C 30 min 500 μl 1.5 ml DNS 5 min 540 nm DNS (U) 40 C ph μl Bradford [8] SDS-PAGE Schägger [9] 1.7 重组表达质粒稳定性测定 WB600/pHYBMYF 30 ml 30 mg/l 37 C 200 r/min 18 h 12 h 7 OD h 96 h LB 30 mg/l A 1 A 2 =A 2 /A 1 100% 1.8 发酵诱导条件优化诱导温度优化 : WB600/pHYBMYF 37 C 200 r/min 8 h 1% 200 r/min 16 h 诱导剂含量优化 : WB600/pHYBMYF 37 C 200 r/min 8 h 45 C 200 r/min 16 h 添加诱导剂时间优化 : WB600/ phybmyf 37 C 200 r/min 1% 36 h 麦芽糖淀粉酶酶学性质研究重组麦芽糖淀粉酶的纯化 : Histrap (A ) 25 mmol/l Tris 0.5 mol/l NaCl 20 mmol/l ph 7.4 (B ) 25 mmol/l Tris 0.5 mol/l NaCl 500 mmol/l ph % 1 ml/min SDS-PAGE 麦芽糖淀粉酶水解产物检测 : Waters XBridge Amide 70% 1 ml/min 40 C 最适作用温度及温度稳定性的测定 : ph 6.5 (20 70 C) - (20 70 C) 1 h 最适作用 ph 及 ph 稳定性的测定 : 40 C ph ( ) 1% ph ph- ph 20 C

: 267 ph (4.5 8.5) 1 h ph- 1.9.5 化学试剂对酶活力和酶热稳定性的影响 : 40 C ph 6.5 2 结果与讨论 2.1 麦芽糖淀粉酶表达质粒 phybmyf 的构建 1514 DNA PCR 1.4 kb pmd19-t Simple Bgl II BamH I phy300-plk phybm ATCC14580 DNA PCR 1.

5 : 267 ph ( ) 1 h ph 化学试剂对酶活力和酶热稳定性的影响 : 40 C ph 结果与讨论 2.1 麦芽糖淀粉酶表达质粒 phybmyf 的构建 1514 DNA PCR 1.4 kb pmd19-t Simple Bgl II BamH I phy300-plk phybm ATCC14580 DNA PCR 1.8 kb pmd19-t Simple yvdf bp kd Bgl II Sma I phybm phybmyf 1A Bgl II Sma I bp phy300-plk bp yvdf 1B 图 1 麦芽糖淀粉酶重组质粒 phybmyf 的构建 Figure 1 Construction of maltogenic amylase expression plasmid phybmyf M λ DNA/Pst I marker 1 phybmyf Bgl II+Sma I. Note: M: λ DNA/Pst I marker; 1: phybmyf/bgl II+Sma I.

268 微生物学通报 Microbiol. China 2017, Vol.44, No.2 2.2 重组枯草芽孢杆菌 WB600/pHYBMYF 的构建 phybmyf phybm WB600 WB600/pHYBMYF WB600/pHYBM 2.

6 268 微生物学通报 Microbiol. China 2017, Vol.44, No 重组枯草芽孢杆菌 WB600/pHYBMYF 的构建 phybmyf phybm WB600 WB600/pHYBMYF WB600/pHYBM 2.3 重组麦芽糖淀粉酶的表达 WB600 WB600/pHYBM WB600/pHYBMYF 30 mg/l 8 h 1% WB600/pHYBMYF 16 h WB600 WB600/pHYBM PsacB 100 [10] SDS-PAGE kd yvdf 67 kd 2.4 重组表达质粒 phybmyf 的稳定性测定 phybmyf 96 h 97% phybmyf 2.5 重组枯草芽孢杆菌 WB600/pHYBMYF 发酵诱导条件优化最适诱导温度 : 37 C 200 r/min 8 h 1% C 30 C 50 C 45 C 45 C 45 C 最适诱导剂添加量 : 37 C 200 r/min 8 h 45 C 2 2 1% 1% 图 2 胞内粗酶液 SDS-PAGE 电泳图 Figure 2 SDS-PAGE of the intracellular maltogenic amylase M Marker 1 B. subtilis 2 B. subtilis/phybm 3 B. subtilis/phybmyf 4 B. subtilis/phybmyf. Note: M: Molecular marker; 1: B. subtilis; 2: B. subtilis/phybm; 3: B. subtilis/phybmyf without induction; 4: B. subtilis/phybmyf with induction.

: 269 表 1 诱导温度对产酶的影响 Table 1 Effects of inducing temperature on the production of the recombinant enzyme Inducing Enzyme activity Total protein OD 600 temperature ( C) (U/mL) (g/l) 20 17.11±0.27 3.

02 表 2 诱导剂添加量对产酶的影响 Table 2 Effects of inducer addition on the production of the recombinant enzyme Inducer Enzyme activity Total protein OD 600 concentration (%) (U/mL) (g/l) 0.0 0.00±0.00 6.62±1.

7 : 269 表 1 诱导温度对产酶的影响 Table 1 Effects of inducing temperature on the production of the recombinant enzyme Inducing Enzyme activity Total protein OD 600 temperature ( C) (U/mL) (g/l) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.02 表 2 诱导剂添加量对产酶的影响 Table 2 Effects of inducer addition on the production of the recombinant enzyme Inducer Enzyme activity Total protein OD 600 concentration (%) (U/mL) (g/l) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.02 表 3 诱导剂添加时间对产酶的影响 Table 3 Effects of inducer addition time on the production of the recombinant enzyme Inducer addition Enzyme activity OD 600 (g/l) time (h) (U/mL) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.01 operator xylr xyl [11] 2.6 重组麦芽糖淀粉酶的纯化 SDS-PAGE 67 kd ( 3) yvdf 最适添加诱导剂的时间 : 1% 37 C 200 r/min h 3 h 9 h 9 h 9 h xyl XylA XylB xyl bp XylR XylR xyl 图 3 纯化后的重组麦芽糖淀粉酶 SDS-PAGE 鉴定图谱 Figure 3 SDS-PAGE of purified recombinant maltogenic amylase M Marker 1 2. Note: M: Molecular marker; 1: Crude recombinant maltogenic amylase; 2: Purified recombinant maltogenic amylase.

270 微生物学通报 Microbiol. China 2017, Vol.44, No.2 2.7 重组枯草芽孢杆菌 WB600/pHYBMYF 所产麦芽糖淀粉酶的基本性质 2.7.1 重组麦芽糖淀粉酶水解性质 : 1% 100 μl 40 C ph 6.5 1 h 4A 4B 60.42% 39.58% 2.7.2 温度对酶活力及酶热稳定性的影响 : ph 6.

8 270 微生物学通报 Microbiol. China 2017, Vol.44, No 重组枯草芽孢杆菌 WB600/pHYBMYF 所产麦芽糖淀粉酶的基本性质重组麦芽糖淀粉酶水解性质 : 1% 100 μl 40 C ph h 4A 4B 60.42% 39.58% 温度对酶活力及酶热稳定性的影响 : ph C - 5A 45 C 50 C 1 h 5B 40 C 40 C 1 h 96.0% 45 C 1 h 27.2% 50 C 1 h 70.0% 50 C 50 C [12] 50 C (Bacillus sp.) US149 [13] (Aspergillus niger) [14] 40 C 50 C (Geobacillus thermoleovorans) 80 C 35 h 50% [15] [16] 图 4 重组酶分解淀粉产物分析 Figure 4 Product analysis of recombinant enzyme reaction with starch 图 5 温度对重组酶活力和稳定性的影响 Figure 5 Effects of temperature on the activity and stability of the recombinant enzyme

9 : ph 对酶活力及酶稳定性的影响 : 40 C ph ph- 6A ph 6.5 ph 5.0 ph C ph 1 h 6B 6B ph ph 5.0 ph 7.5 ph [17-19] 化学试剂对酶活力和酶热稳定性的影响 : 4 4 Ca 2+ Co 2+ EDTA EDTA Zn 2+ Mn 2+ Cu 2+ Fe 3+ SDS Tween mmol/l Cu 2+ SDS Ca 2+ Co 2+ EDTA 3 Ca 2+ Co 2+ α-ca 2+ α-ca mmol/l EDTA [20] Li + K + (Geobacillus sp.) [19] (Thermus sp.) 10 mmol/l EDTA [21] 重组酶的底物特异性 : 1 ml 2% β- 1% 40 C ph μl 1 h 2% 100% 5 5 β- β- Figure 6 图 6 ph 对重组酶活力和稳定性的影响 Effects of ph on the activity and stability of the recombinant enzyme

10 272 微生物学通报 Microbiol. China 2017, Vol.44, No.2 表 4 化学试剂对重组酶酶活力的影响 Table 4 Effects of chemical reagents on the activity of the recombinant enzyme Chemical reagents Concentration (mmol/l) Relative activity (%) Li ± ±1.23 K ± ±1.68 Mg ± ±0.60 Zn ± ±0.63 Ca ± ±1.62 Mn ± ±0.92 Cu Co ± ±0.63 Fe ± ±2.88 EDTA SDS Triton-X 114 Tween-80 Table ± ± ± ± ± ± ± ±1.40 表 5 重组酶底物特异性关系 The substrate specificity of the recombinant enzyme Substrate Relative activity (%) Soluble starch 100 Potato starch ±2.39 Maltodextrin 20.05±0.48 β- β-cyclodextrin ±4.43 Glycogen 0 Amylopectin 0 3 结论 / phybm U/mL U/mL [22] 30.2% 参考文献 [1] Lee HS, Auh JH, Yoon HG, et al. Cooperative action of α-glucanotransferase and maltogenic amylase for an improved process of isomaltooligosaccharide (IMO) production[j]. Journal of Agricultural and Food Chemistry, 2002, 50(10): [2] Igarashi K, Ara K, Saeki K, et al. Nucleotide sequence of the gene that encodes a neopullulanase from an alkalophilic Bacillus[J]. Bioscience, Biotechnology, and Biochemistry, 1992, 56(3): [3] Oguma T, Matsuyama A, Kikuchi M, et al. Cloning and sequence analysis of the cyclomaltodextrinase gene from Bacillus sphaericus and expression in Escherichia coli cells[j]. Applied Microbiology and Biotechnology, 1993, 39(2): [4] Rygus T, Scheler A, Allmansberger R, et al. Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization[j]. Archives of Microbiology, 1991, 155(6): [5] Wilson GA, Young FE. Isolation of a sequence-specific endonuclease (Bam I) from Bacillus amyloliquefaciens H[J]. Journal of Molecular Biology, 1975, 97(1): [6] Hanahan D. Studies on transformation of Escherichia coli with plasmids[j]. Journal of Molecular Biology, 1983, 166(4): [7] Bott KF, Wilson GA. Development of competence in the Bacillus subtilis transformation system[j]. Journal of Bacteriology, 1967, 94(3): [8] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[j]. Analytical Biochemistry, 1976, 72(1/2): [9] Schägger H, Von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kd[j]. Analytical Biochemistry, 1987, 166(2): [10] Zukowski MM, Miller L. Hyperproduction of an intracellular heterologous protein in a sacu h mutant of Bacillus subtilis[j]. Gene, 1986, 46(2/3): [11] Sizemore C, Wieland B, Götz F, et al. Regulation of Staphylococcus xylosus xylose utilization genes at the molecular level[j]. Journal of Bacteriology, 1992, 174(9): [12] Kim IC, Cha JH, Kim JR, et al. Catalytic properties of the cloned amylase from Bacillus licheniformis[j]. Journal of Biological Chemistry, 1992, 267(31): [13] Mabrouk SB, Ayadi DZ, Hlima HB, et al. Thermostability improvement of maltogenic amylase MAUS149 by error prone

11 : 273 PCR[J]. Journal of Biotechnology, 2013, 168(4): [14] Ravi-Kumar K, Venkatesh KS, Umesh-Kumar S. The 53-kD proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillusniger has Taka-amylase-like activity[j]. Applied Microbiology and Biotechnology, 2007, 74(5): [15] Mehta DP, Shetters C, Bouldin DW. Meta-algorithms for scheduling a chain of coarse-grained tasks on an array of reconfigurable FPGAs[J]. VLSI Design, 2013: Article ID [16] Vihinen M, Mantsiila P. Microbial amylolytic enzyme[j]. Critical Reviews in Biochemistry and Molecular Biology, 1989, 24(4): [17] Xue B, Wang ZB, Liu ZD, et al. Study on gene cloning, expression and enzymatic characterization of a high maltose-producing alpha-amylase from Thermobifida fusca[j]. Science and Technology of Food Industry, 2013, 34(24): (in Chinese),,,. Thermobifida fusca α- [J]., 2013, 34(24): [18] Wang XB, Li MR, Li X, et al. Gene cloning, expression and enzymatic characterization of a high maltose-producing α-amylase from Saccharomonospora viridis[j]. Chinese Journal of Applied and Environmental Biology, 2013, 19(2): (in Chinese),,,. α- [J]., 2013, 19(2): [19] Nasrollahi S, Golalizadeh L, Sajedi RH, et al. Substrate preference of a Geobacillus maltogenic amylase: a kinetic and thermodynamic analysis[j]. International Journal of Biological Macromolecules, 2013, 60: 1-9 [20] Liu Y, Shen W, Shi GY, et al. Enzymatic properties of mesophilic α-amylase from Bacillus amyloliquefaciens M23[J]. Food Science, 2008, 29(9): (in Chinese),,,. α- [J]., 2008, 29(9): [21] Kim TJ, Kim MJ, Kim BC, et al. Modes of Action of Acarbose Hydrolysis and transglycosylationcatalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain[j]. Applied and Environmental Microbiology, 1999, 65(4): [22] Zi N, Shen W, Shi GY, et al. Gene cloning and characterization of Bacillus licheniformis maltogenic α-amylase[j]. Chinese Journal of Applied and Environmental Biology, 2009, 15(1): (in Chinese),,,. α- [J]., 2009, 15(1): 征订启事欢迎订阅微生物学通报 (F5000) (100101) 1 3 Tel bjb@im.ac.cn tongbao@im.ac.cn M413

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇