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1 微生物学通报 Microbiology China May 20, 2017, 44(5): DOI: /j.microbiol.china 研究报告褐藻胶裂解酶基因的克隆表达及重组酶酶学性质 * 韩伟林娟谢勇徐凡叶秀云 ( ) 摘要 : 目的克隆交替假单胞菌(Pseudoalteromonas sp.) BYS-2 的褐藻胶裂解酶基因, 实现其在大肠杆菌细胞中异源表达, 对分离纯化的重组酶进行酶学性质研究方法以交替假单胞菌 BYS-2 菌株基因组 DNA 为模板, 克隆得到褐藻胶裂解酶基因 alg738, 构建重组基因工程菌 BL21(DE3)/pET22b-alg738, 诱导表达, 表达产物通过 Ni-NTA 树脂纯化后进行酶学性质研究结果重组酶的最适反应 ph 为 8.0, 在 ph 范围内 37 C 保温 1 h 仍能保持 84% 以上的相对酶活力, 具有较好的 ph 稳定性 ; 最适反应温度为 45 C, 热稳定性实验显示在 37 C 下保温 60 min 其残余酶活力仍达 66.6%; 在 5 mmol/l 浓度下,Na + Mg 2+ Mn 2+ 对该酶具有明显的促进作用,Ni 2+ Co 2+ Cu 2+ Hg 2+ Zn 2+ EDTA β- 巯基乙醇 SDS 具有明显的抑制作用动力学参数 K m V max 分别为 1.11 g/l 和 g/(l min), 底物特异性分析表明该重组酶为偏好聚甘露糖醛酸钠 (Poly M) 裂解作用的双功能酶结论重组褐藻胶裂解酶具有良好的酶学特性, 为褐藻胶裂解酶的开发应用打下基础关键词 : 褐藻胶裂解酶, 交替假单胞菌, 克隆表达, 重组酶, 酶学性质 Expression and characterization of a recombinant alginate lyase HAN Wei LIN Juan * XIE Yong XU Fan YE Xiu-Yun (Fujian Provincial Key Laboratory of Marine Enzyme Engineering, Fuzhou University, Fuzhou, Fujian , China) Abstract: [Objective] The alginate lyase gene of Pseudoalteromonas sp. BYS-2 was cloned and expressed in Escherichia coli, and the properties of recombinase were characterized. [Methods] The alginate lyase gene alg738 was cloned from the genomic DNA of Pseudoalteromonas sp. BYS-2 and the recombinant strain BL21(DE3)/pET22b-alg738 was constructed. The recombinase was purified with Ni-NTA resin and the enzymatic properties were studied. [Results] The optimum ph of recombinase was 8.0. It was stable in the ph range of 6.0 to 9.0 and could maintain more than 84% of its relative enzyme activity after incubation at 37 C for 1 hour. The optimum temperature of recombinase was 45 C and 66.6% of enzyme activity was remained after incubation at 37 C for Foundation item: State Oceanic Administration of Marine Public Welfare Industry Research (No ); Enterprise Technology Innovation Project of Fujian Province; Fuzhou Science and Technology Project (No. 2016X0005) *Corresponding author: ljuan@fzu.edu.cn Received: July 17, 2016; Accepted: September 22, 2016; Published online ( September 28, 2016 基金项目 (No ) (No. 2016X0005) * 通讯作者 : ljuan@fzu.edu.cn 收稿日期 : ; 接受日期 : ; 优先数字出版日期 (

2 : minutes. At the concentration of 5 mmol/l, Na +, Mg 2+ and Mn 2+ had significant stimulation on the enzyme activity, while Ni 2+, Co 2+, Cu 2+, Hg 2+, Zn 2+, EDTA, β-mercaptoethanol and SDS had inhibitory effects on the enzyme. The kinetic parameters K m and V max of ralg738 were 1.11 g/l and g/(l min), respectively. Moreover, this recombinase was a bifunctional enzyme which prefers sodium polymannuronate (Poly M). [Conclusion] The properties of the recombinase ralg738 has laid a good foundation for its future development and application. Keywords: Alginate lyase, Pseudoalteromonas sp., Cloning and expression, Recombinant enzyme, Characterization (Alginate) [1-5] 3 (Macrocystis) (Laminaria) (Ascophyllum) C5 [β-d- (M) α-l- (G)] [6] 3 G M G/M [7-8] [9] [10] ph 1 材料与方法 1.1 材料菌株和质粒 : BYS-2 Top10 BL21(DE3) TransGen pet-22b(+) 主要试剂和仪器 :Pfu DNA Polymerase TransGen OMEGA T4 DNA Marker Thermo Fisher Scientific IPTG ( ) PCR Veriti 96-Well Thermal Cycler Applied Biosystems JS-680 DYY-8C DYCP-31DY YXQ-LS-100SII SW-CJ-2FI B SHP N CF16RXⅡ HITACHI ZWY-2101C

3 1076 微生物学通报 Microbiol. China 2017, Vol.44, No 实验方法引物设计与基因克隆 : alg738-f 5 -AACTGAATTCAGCGCATCCAAATTTGGTAATA ACC-3 alg738-r 5 -AAGTCTCGAGCTCCTGATTA TTCTTCATCGCATAAAC-3 BYS-2 DNA 50 µl alg738 2 Pfu PCR master 25 µl 20 pmol/l alg738-f 1.5 µl 20 pmol/l alg738-r 1.5 µl DNA 3 µl ddh 2 O 19 µl 94 C 5 min 94 C 30 s 60 C 30 s 72 C 2.5 min C 10 min 10 C [11] PCR 表达载体构建 : alg738 pet-22b(+) EcoR I Xho I T4 DNA pet-22b-alg738 Top10 Invitrogen BL21(DE3) 序列分析 : Expasy ProtParam ( alg738 SignalP 4.1 ( services/signalp/) SWISS- MODEL ( Alg 重组酶 ralg738 诱导表达 : LB [12] ( Amp 100 mg/l) 37 C 200 r/min OD IPTG ( 1 mmol/l) IPTG 4 C r/min 5 min 重组酶 ralg738 活力检测 : DNS (3,5- ) [13] 0.1 ml 0.9 ml 0.3% 40 C 15 min 1 ml DNS 5 min 5 ml 540 nm 1 μmol (U) 重组酶 ralg738 分离纯化 : [14] 2 L Tris-HCl (ph 7.0) Ni-NTA ralg738 SDS-PAGE 重组酶 ralg738 比活力测定 : (Bradford) [15-16] ( / ) 重组酶 ralg738 纯酶性质研究 :(1) ph ph 40 C ph 100% ph ph 37 C 1 h ph 100% ph (2) ph ( C) 100% ( min) ph 100% (3) 1 mmol/l 5 mmol/l 14 (Na + Ca 2+ K + Co 2+ Cr 3+ Li + Cu 2+ Mg 2+ Ni 2+ Fe 2+ Fe 3+ Mn 2+ Hg 2+ Zn 2+ ) 2 (EDTA SDS) (4) K m V max ( g/l)

: 1077 3 K m V max [17] (5) 0.3% ( ) 2 结果与分析 2.1 褐藻胶裂解酶全长基因调取及序列分析 BYS-2 DNA alg738-f/r PCR 2 200 bp ( 1) Vector NCBI alg738 alg738 2 217 bp ProtParam 738 79.

2 重组酶 ralg738 的表达与纯化 BL21(DE3)/pET22b-alg738 IPTG ralg738 SDS-PAGE ( 3) IPTG 80 kd 图 1 褐藻胶裂解酶基因 alg738 的 PCR 扩增电泳图 Figure 1 PCR product of alginate lyase gene

4 : K m V max [17] (5) 0.3% ( ) 2 结果与分析 2.1 褐藻胶裂解酶全长基因调取及序列分析 BYS-2 DNA alg738-f/r PCR bp ( 1) Vector NCBI alg738 alg bp ProtParam kd 6.42 Asp Glu 79 Arg Lys 72 SignalP 4.1 N 28 NCBI BLASTp Alginate lyase Hepar_ ⅡⅡ _ SWISS-MODEL Alg738 4nei.1.A Alg ( 2) 50.07% N 370 α- β- 2.2 重组酶 ralg738 的表达与纯化 BL21(DE3)/pET22b-alg738 IPTG ralg738 SDS-PAGE ( 3) IPTG 80 kd 图 1 褐藻胶裂解酶基因 alg738 的 PCR 扩增电泳图 Figure 1 PCR product of alginate lyase gene alg738 Note: M: bp DNA marker; 1: PCR product. 图 2 三维结构模拟示意图 Figure 2 Three-dimensional simulated structure of Alg738 ( kd) alg738 ( 3) ralg U/L 0.34 U/mg ralg738

5 1078 微生物学通报 Microbiol. China 2017, Vol.44, No.5 图 3 重组酶 ralg738 的 SDS-PAGE 分析 Figure 3 SDS-PAGE analysis of ralg738 M Protein marker 1 IPTG 2 IPTG 3. Note: M: Protein marker; 1: Fermentation without IPTG; 2: Fermentation with IPTG; 3: The purified recombinant enzyme. 2.3 重组酶 ralg738 性质分析最适反应 ph 和 ph 稳定性 : ralg738 ph ph 8.0 ph ( 4A) ph 37 C 1 h ph % ph ( 4B) 最适反应温度和热稳定性 : ralg C ( 5A) 图 4 重组酶 ralg738 最适反应 ph (A) 和 ph 稳定性 (B) Figure 4 Effect of ph on activity (A) and stability (B) of ralg738 图 5 重组酶 ralg738 最适反应温度 (A) 和热稳定性 (B) Figure 5 Effect of temperature on activity (A) and stability (B) of ralg738

6 : C 37 C 60 min 66.6% 50 C 15 min 0 ( 5B) 金属离子及化学试剂对酶活性影响 : (1 mmol/l) Na + K + Ca 2+ Co 2+ Hg 2+ Zn 2+ Ni 2+ EDTA Cu 2+ SDS 5 mmol/l Na + Mg 2+ Mn 2+ Mn % K + Ca 2+ Fe 2+ Ni 2+ Co 2+ EDTA β- Cu 2+ Hg 2+ Zn 2+ SDS 酶促反应动力学参数测定 : Linewaeaver-Burk ralg738 V max g/(l min) K m 1.11 g/l ( 6) 表 1 不同添加剂对重组酶 ralg738 活力的影响 Table 1 Effects of different factors on ralg738 activity 1 mmol/l 5 mmol/l Additives 1 mmol/l Relative activity (%) 5 mmol/l Relative activity (%) Blank Li ± ±1.00 Na ± ±6.43 K ± ±4.04 Ca ± ±2.42 Mg ± ±4.98 Ni ± ±1.36 Co ± ±0.63 Cu Fe ± ±2.46 Fe ± ±3.87 Mn ± ±7.86 Hg ± Gr ± ±1.32 Zn ± SDS 0 0 EDTA 67.91± ±2.34 β ± ±2.79 β-mercaptoethanol 图 6 重组酶 ralg738 的 Linewaeaver-Burk 作图 Figure 6 Linewaeaver-Burk plot of ralg 重组酶 ralg738 的底物特异性 : -D- (M) -L- (G) 1,4- -D- 1,4- -L- (Poly M) (Poly G) Poly M ( 2)

7 1080 微生物学通报 Microbiol. China 2017, Vol.44, No.5 表 2 重组酶 ralg738 的底物特异性 Table 2 Substrate specificity of ralg738 Substrate Relative activity (%) Sodium alginate ±1.55 Poly M ±1.86 Poly G 44.54± 结论 BYS-2 DNA alg738 BL21(DE3)/pET22b(+)-alg738 ralg738 ph C ph C (1 mmol/l) Na + K + Ca 2+ (5 mmol/l) Na + Mg 2+ Mn 2+ 1 mmol/l 5 mmol/l Cu 2+ Co 2+ Hg 2+ Zn 2+ Ni 2+ EDTA SDS K m V max 1.11 g/l g/(l min) (Poly M) 参考文献 [1] Raybaudi-Massilia RM, Mosqueda-Melgar J, Martín-Belloso O. Edible alginate-based coating as carrier of antimicrobials to improve shelf-life and safety of fresh-cut melon[j]. International Journal of Food Microbiology, 2008, 121(3): [2] Li CX, Sun Y, Guan HS. Progress of marine drug propylene glycol alginate sodium sulfate (PSS) and inspiration[j]. Chinese Bulletin of Life Sciences, 2012, 24(9): (in Chinese),,. [J]., 2012, 24(9): [3] Suzuki Y, Tanihara M, Ohinishi K, et al. Cat peripheral nerve regeneration across 50 mm gap repaired with a novel nerve guide composed of freeze-dried alginate gel[j]. Neuroscience Letters, 1999, 259(2): [4] Wang Q, Zhang N, Hu XW, et al. Alginate/polyethylene glycol blend fibers and their properties for drug controlled release[j]. Journal of Biomedical Materials Research Part A, 2007, 82(1): [5] Ashton RS, Banerjee A, Punyani S, et al. Scaffolds based on degradable alginate hydrogels and poly(lactide-co-glycolide) microspheres for stem cell culture[j]. Biomaterials, 2007, 28(36): [6] Ji MH. Seaweed Chemistry[M]. Beijing: Science Press, 1997: (in Chinese). [M]. :, 1997: [7] Zhang YJ, Luo FW, Yao ZA, et al. Research progress about biological activity of sodium alginate oligosaccharides[j]. China Brewing, 2014, 33(1): 5-8 (in Chinese),,,. [J]., 2014, 33(1): 5-8 [8] Zhu L, Cheng L, Cai JP. Progress in algin oligosaccharide potential medicinal value[j]. Chinese Herbal Medicine, 2006, 29(9): (in Chinese),,. [J]., 2006, 29(9): [9] Li LY, Guan HS, Jiang XL, et al. Advances in algae toolenzymes: alginate lyases[j]. Chinese Journal of Biotechnology, 2011, 27(6): (in Chinese),,,. [J]., 2011, 27(6): [10] Liu Y, Jiang XL, Guan HS. Research advances on alginate lyases[j]. Journal of Fishery Sciences of China, 2001, 7(4): (in Chinese),,. [J]., 2001, 7(4): [11] Wang LP, Liu YP, Sun XH, et al. Expression of alginate lyase gene in E. coli and characterization of the enzyme[j]. Journal of Food Science and Biotechnology, 2012, 31(2): (in Chinese),,,. [J]., 2012, 31(2): [12] Shen P, Chen XD. Experiment of Microbiology[M]. Beijing: Higher Education Press, 2010: (in Chinese),. [M]. :, 2010: [13] Hou BB, Liu SL, Zhang JY, et al. Optimization of alginate-degrading bacterial fermentation process for alginate lyaseproduction[j]. Fisheries Science, 2009, 28(11): (in Chinese),,,. [J]., 2009, 28(11): [14] Dou WF, Wei D, Li H, et al. Purification and characterisation of a bifunctional alginate lyase from novel Isoptericola halotolerans CGMCC 5336[J]. Carbohydrate Polymers, 2013, 98(2): [15] Wang XP, Xing SL. Determination of protein guantitation using the method of coomassie brilliant blue[j]. Tianjin: Chemical Industry, 2009, 23(3): (in Chinese),. [J]., 2009, 23(3): [16] Li J, Zhang YT, Zeng W, et al. Determination of total protein content applications Coomassie brilliant blue[j]. Chinese Journal of Biologicals, 2000, 13(2): (in Chinese),,,. [J]., 2000, 13(2): [17] Li JW. Study on the alginate lyases secreted by the marine bacterium Pseudoalteromonas sp. SM0524[D]. Jinan: Doctoral Dissertation of Shandong University, 2011 (in Chinese). Pseudoalteromonas sp. SM0524 [D]. :, 2011

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