假交替单胞菌JMUZ2重组κ-卡拉胶酶的异源表达和酶学性质

doi:10.11949/0438-1157.20201827

摘要/Abstract

摘要：

将假交替单胞菌JMUZ2的κ-卡拉胶酶基因在大肠杆菌中异源表达，利用亲和层析对重组κ-卡拉胶酶进行分离纯化，表征重组酶的酶学性质，利用质谱分析κ-卡拉胶的酶解产物，并进行产物的抗氧化活性分析。结果表明，假交替单胞菌JMUZ2的κ-卡拉胶酶属于GH16家族，能专一性降解κ-卡拉胶，重组κ-卡拉胶酶的最适反应温度和pH分别为50℃和8.0，在40℃条件下处理1 h，保持约80%残余活力。重组酶对去垢剂Tween 20、Tween 80和Triton X-100有良好的耐受性。LC-MS分析显示，重组酶水解κ-卡拉胶的终产物为二糖和四糖。酶解产物对·OH自由基、DPPH自由基、ABTS自由基具有一定清除作用，还具有良好的还原能力。重组κ-卡拉胶酶的酶学性质及酶解产物的分析为该酶用于κ-卡拉胶寡糖绿色制备的应用奠定理论基础。

关键词: 假交替单胞菌, κ-卡拉胶酶, 酶学性质, 酶解产物, 抗氧化活性

Abstract:

The κ-carrageenase gene from Pseudoalteromonas sp. JMUZ2 was expressed heterologously in Escherichia coli. After the recombinant κ-carrageenase was purified, the properties of the recombinant enzyme were characterized and the antioxidant activity was explored for the enzymatic hydrolysis products of κ-carrageenan. The results showed that the κ-carrageenase from Pseudoalteromonas sp. JMUZ2 belonged to GH16 family. The recombinant enzyme could specifically degrade κ-carrageenan. The optimal reaction temperature and pH of the recombinant κ-carrageenase were 50℃ and 8.0, respectively. The recombinant κ-carrageenase maintained the residual activity of about 80% after incubation at 40℃ for 1 h. The recombinant enzyme had good tolerance to the detergents of Tween 20, Tween 80 and Triton X-100. LC-MS analysis showed that the final products of κ-carrageenan hydrolyzed by recombinant enzyme were disaccharides and tetrasaccharides. The hydrolysates had scavenging effects on ·OH, DPPH and ABTS radicals, and also had good reducing ability. The enzymatic properties of the recombinant κ-carrageenase and the analysis of the enzymatic hydrolysates lay a theoretical foundation for the application of the enzyme in the green preparation of κ-carrageenan oligosaccharides.

Key words: Pseudoalteromonas, κ-carrageenase, enzymatic properties, enzymatic hydrolysates, antioxidant activity

中图分类号:

Q 814

张成昊, 朱艳冰, 陈艳红, 姜泽东, 倪辉, 李清彪. 假交替单胞菌JMUZ2重组κ-卡拉胶酶的异源表达和酶学性质[J]. 化工学报, 2021, 72(7): 3738-3746.

ZHANG Chenghao, ZHU Yanbing, CHEN Yanhong, JIANG Zedong, NI Hui, LI Qingbiao. Enzymatic properties of the recombinant κ-carrageenase from Pseudoalteromonas sp. JMUZ2[J]. CIESC Journal, 2021, 72(7): 3738-3746.

图/表 10

图1 假交替单胞菌JMUZ2 κ-卡拉胶酶的多序列比对（a）和系统进化树（b）

Fig.1 Multiple sequence alignment (a) and the phylogenetic tree (b) of κ-carrageenase from Pseudoalteromonas sp. JMUZ2

图2 假交替单胞菌JMUZ2 κ-卡拉胶酶的三维建模（黄色:模板5ocq;蓝色:所建模型）

Fig.2 Three-dimensional modeling of κ-carrageenasefrom Pseudoalteromonas sp. JMUZ2

图3 重组κ-卡拉胶酶的SDS-PAGE分析（a）和底物特异性（b）道1—蛋白Mark;道2—含pET-28a空载,IPTG诱导;道3—含重组质粒阳性菌,未诱导;道4—含重组质粒阳性菌,IPTG诱导;道5—纯化的重组蛋白

Fig.3 SDS-PAGE analysis (a) and substrate specificity (b) of the recombinant κ-carrageenase

图4 温度对重组κ-卡拉胶酶活性（a）和稳定性（b）的影响

Fig.4 Effects of temperature on the enzymatic activity (a) and the thermal stability (b) of the recombinant κ-carrageenase

图5 pH对重组κ-卡拉胶酶活性的影响

Fig.5 Effect of pH on the activity of the recombinant κ-carrageenase

图6 金属离子对重组κ-卡拉胶酶活性的影响

Fig.6 Effects of metal ions on the activity of the recombinant κ-carrageenase

表1 添加试剂对重组κ-卡拉胶酶活性的影响

Table 1 Effects of additives on the activity of the recombinant κ-carrageenase

添加试剂	浓度	相对活性/%
对照	—	100.0 ± 0.4
EDTA	1 mmol/L	107.0 ± 3.5
	10 mmol/L	147.2 ± 7.8
β-巯基乙醇	1 mmol/L	82.1 ± 1.3
	10 mmol/L	80.1 ± 3.7
DTT	1 mmol/L	73.9 ± 2.9
	10 mmol/L	47.3 ± 1.7
Tween 20	0.1%（体积分数）	152.4 ± 6.0
	1%（体积分数）	128.8 ± 8.0
Tween 80	0.1%（体积分数）	114.8 ± 1.4
	1%（体积分数）	138.3 ± 6.4
Triton X-100	0.1%（体积分数）	99.2 ± 1.3
	1%（体积分数）	97.5 ± 4.6
CTAB	0.1%（质量分数）	30.3 ± 2.3
	1%（质量分数）	34.7 ± 2.2
SDS	0.1%（质量分数）	5.9 ± 0.5
	1%（质量分数）	6.4 ± 0.6
尿素	2.5 mol/L	71.0 ± 2.4
	5 mol/L	20.9 ± 1.8
盐酸胍	2.5 mol/L	45.3 ± 4.0
	5 mol/L	2.4 ± 0.2

图7 重组κ-卡拉胶酶的双倒数图

Fig.7 The Lineweaver-Burk of the recombinant κ-carrageenase

图8 酶解产物的LC-MS分析

Fig.8 LC-MS analysis of the enzymatic hydrolysis products

图9 酶解产物的抗氧化活性分析

Fig.9 Antioxidant activity analysis of the enzymatic hydrolysis products

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