微生物学通报 Microbiology China tongbao@im.ac.cn May 20, 2017, 44(5): 1074 1080 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.160518 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 ( 350116) 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 6.0 9.0 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 0.011 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 350116, 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. 201305015); Enterprise Technology Innovation Project of Fujian Province; Fuzhou Science and Technology Project (No. 2016X0005) *Corresponding author: E-mail: ljuan@fzu.edu.cn Received: July 17, 2016; Accepted: September 22, 2016; Published online (www.cnki.net): September 28, 2016 基金项目 (No. 201305015) (No. 2016X0005) * 通讯作者 :E-mail ljuan@fzu.edu.cn 收稿日期 :2016-07-17; 接受日期 :2016-09-22; 优先数字出版日期 (www.cnki.net):2016-09-28
: 1075 60 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 0.011 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 1.1.1 菌株和质粒 : BYS-2 Top10 BL21(DE3) TransGen pet-22b(+) 1.1.2 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-250 752N CF16RXⅡ HITACHI ZWY-2101C
1076 微生物学通报 Microbiol. China 2017, Vol.44, No.5 1.2 实验方法 1.2.1 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 30 72 C 10 min 10 C [11] PCR 1.2.2 表达载体构建 : alg738 pet-22b(+) EcoR I Xho I T4 DNA pet-22b-alg738 Top10 Invitrogen BL21(DE3) 1.2.3 序列分析 : Expasy ProtParam (http://web.expasy.org/cgi-bin/protparam/protparam) alg738 SignalP 4.1 (http://www.cbs.dtu.dk/ services/signalp/) SWISS- MODEL (http://swissmodel.expasy.org/interactive) Alg738 1.2.4 重组酶 ralg738 诱导表达 : LB [12] ( Amp 100 mg/l) 37 C 200 r/min OD 600 0.5 0.8 IPTG ( 1 mmol/l) IPTG 4 C 13 000 r/min 5 min 1.2.5 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) 1.2.6 重组酶 ralg738 分离纯化 : [14] 2 L Tris-HCl (ph 7.0) Ni-NTA ralg738 SDS-PAGE 1.2.7 重组酶 ralg738 比活力测定 : (Bradford) [15-16] ( / ) 1.2.8 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 (20 30 40 50 60 70 C) 100% (2 5 8 15 30 45 60 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 (0.8 6.0 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 79.512 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 Alg738 28 734 ( 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: 5 000 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 (79.512 kd) alg738 ( 3) ralg738 560 U/L 0.34 U/mg ralg738
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 2.3.1 最适反应 ph 和 ph 稳定性 : ralg738 ph ph 8.0 ph 5.0 10.0 0 ( 4A) ph 37 C 1 h ph 6.0 9.0 84% ph 4.0 11.0 ( 4B) 2.3.2 最适反应温度和热稳定性 : ralg738 45 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
: 1079 43 C 37 C 60 min 66.6% 50 C 15 min 0 ( 5B) 2.3.3 金属离子及化学试剂对酶活性影响 :14 2 1 (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 2+ 50.7% K + Ca 2+ Fe 2+ Ni 2+ Co 2+ EDTA β- Cu 2+ Hg 2+ Zn 2+ SDS 2.3.4 酶促反应动力学参数测定 : Linewaeaver-Burk ralg738 V max 0.011 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 100 100 Li + 87.35±5.71 94.82±1.00 Na + 103.04±2.54 122.81±6.43 K + 102.38±3.28 114.54±4.04 Ca 2+ 110.18±1.43 107.41±2.42 Mg 2+ 93.44±6.57 130.50±4.98 Ni 2+ 70.42±1.55 56.00±1.36 Co 2+ 47.96±2.58 38.96±0.63 Cu 2+ 0 0 Fe 2+ 98.30±2.46 117.01±2.46 Fe 3+ 83.00±6.70 80.45±3.87 Mn 2+ 89.16±1.08 150.70±7.86 Hg 2+ 47.42±1.10 0 Gr 3+ 83.56±1.08 89.53±1.32 Zn 2+ 38.99±1.97 0 SDS 0 0 EDTA 67.91±1.28 56.93±2.34 β- 89.45±3.77 77.89±2.79 β-mercaptoethanol 图 6 重组酶 ralg738 的 Linewaeaver-Burk 作图 Figure 6 Linewaeaver-Burk plot of ralg738 2.3.5 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)
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