Cloning and Expression of Protease SpP1 Gene and Characterization of Enzymatic Properties

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

Abstract

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

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.

Figures/Tables 7

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

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）

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

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

Fig. 5 Enzyme kinetic curves of SpP1

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

Fig. 7 Effects of organic solvent on SpP1

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