蛋白酶SpP1基因克隆、表达及酶学性质的表征

doi:10.13560/j.cnki.biotech.bull.1985.2023-0813

摘要/Abstract

摘要：

【目的】对螺旋藻来源的一个蛋白酶基因进行克隆、表达，并探究重组酶酶学性质，为藻类蛋白酶的深入研究奠定基础。【方法】从钝顶螺旋藻（Spirulina platensis）基因组扩增获得了蛋白酶基因SpP1，进而构建 pET28a-SpP1重组质粒，将其转入Escherichia coli BL21（DE3）实现了异源表达，利用镍柱分离纯化重组蛋白酶并研究其酶学性质。【结果】螺旋藻蛋白酶SpP1属于丝氨酸蛋白酶家族成员，分子量为47.04 kD，最适温度和pH值分别为50℃和8.0。其热稳定性较差，在pH=8.0-9.0的范围内具有良好的酸碱稳定性。以酪蛋白为底物时，最大反应速度 V_max=8.237 U/mL,米氏常数K_m=16.369 μg/mL。Mn²⁺对其具有较强的激活作用，添加0.1 mol/L Mn²⁺后其酶活提高了18倍，同时0.1 mol/L 的Fe³⁺、Zn²⁺、Ca²⁺、乙二胺四乙酸（ethylenediaminetetraacetic acid，EDTA）等对酶活也具有明显的促进作用。【结论】螺旋藻来源的蛋白酶SpP1具有丝氨酸蛋白酶家族成员的典型结构和性质特征，具有较好的酸碱稳定性，添加金属锰离子可有效提升其催化活性。

关键词: 钝顶螺旋藻, 蛋白酶, 异源表达, 酶学性质

Abstract:

【Objective】The protease gene of Spirulina was cloned and expressed, and the enzymatic properties of recombinant enzyme were investigated, which lays a foundation for the further research of algal protease.【Method】The protease gene SpP1 was amplified from the genome of Spirulina platensis, and the pET28a-SpP1 recombinant plasmid was constructed, which was transfected into Escherichia coli BL21（DE3）to achieve heterologous expression, and the recombinant protease was isolated and purified by using a nickel column to study its enzymatic properties.【Result】The protease SpP1 was a member of the serine protease family, with a molecular weight of 47.04 kD, and its optimal temperature and pH values were 50℃ and 8.0, respectively; its thermal stability was poor, and it had a good acid-base stability in the pH=8.0-9.0 range. When casein was used as the substrate, the maximum reaction velocity V_max=8.237 U/mL, and the Michaelis constant K_m=16.369 μg/mL. The activity increased by 18-fold with the addition of 0.1 mol/L Mn ²⁺. Also 0.1 mol/L Fe³⁺, Zn²⁺, Ca²⁺, ethylenediaminetetraacetic acid（EDTA）had a significant promotion effect on the enzyme activity.【Conclusion】The protease SpP1 has the typical structure and property characteristics of serine protease family members, with good acid-base stability, and the addition of manganese ions can effectively enhance its catalytic activity.

Key words: Spirulina platensis, protease, heterologous expression, enzymatic property

殷亮, 王代玮, 刘悦莹, 刘海燕, 罗光宏. 蛋白酶SpP1基因克隆、表达及酶学性质的表征[J]. 生物技术通报, 2024, 40(4): 278-286.

YIN Liang, WANG Dai-wei, LIU Yue-ying, LIU Hai-yan, LUO Guang-hong. Cloning and Expression of Protease SpP1 Gene and Characterization of Enzymatic Properties[J]. Biotechnology Bulletin, 2024, 40(4): 278-286.

图/表 7

图1 蛋白酶SpP1的三维结构 Swiss-Model预测的SpP1三级结构：粉色表示 β-sheet；青色表示 α-helix；橙色为coil。黑色圆圈内为催化残基D165H203S380及底物结合口袋

Fig. 1 3-D structure of peptidase SpP1 The 3D structure of SpP1 predicted by Swiss-Model. Pink indicates β-sheet; cyan indicates α-helix; orange indicates coil. The black circle shows the catalytic residues D165H203S380 and the substrate binding pocket

图2 SpP1基因的凝胶电泳（A）和SpP1的SDS-PAGE（B） A: SpP1基因凝胶电泳图（M：DL10000 DNA marker；1：PCR扩增的SpP1基因目的条带）；B: SpP1蛋白的SDS-PAGE（M：蛋白marker；1：沉淀；2：上清；3：纯化的SpP1）

Fig. 2 SpP1 gel electrophoresis（A）and SDS - PAGE of SpP1（B） A: Gel electrophoresis map of SpP1 gene（M: DL10000 DNA marker. 1: PCR-amplified band of SpP1 gene）. B: SDS-PAGE of SpP1protein（M: protein marker. 1: Precipitation. 2: Supernatant. 3: Purified SpP1 protein band）

图3 SpP1最适pH（A）及pH稳定性（B）

Fig. 3 Optimal pH（A）and pH stability（B）of the SpP1

图4 SpP1最适温度（A）及温度稳定性（B）

Fig. 4 Optimal temperature（A）and temperature stability（B）of the SpP1

图5 SpP1的动力学曲线

Fig. 5 Enzyme kinetic curves of SpP1

图6 金属离子和化学试剂对SpP1的影响 ***: P<0.001

Fig. 6 Effects of metal ions and chemical reagents on SpP1

图7 有机溶剂对SpP1的影响 **: P<0.01

Fig. 7 Effects of organic solvent on SpP1

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