Enhanced Expression of Protease K in Pichia pastoris through Molecular Chaperones and Analysis of Its Effect on Wool Scale Layer

doi:10.13560/j.cnki.biotech.bull.1985.2024-0136

Abstract

Abstract:

【Objective】 To augment the expression level of protease K and pave the way for the implementation of efficient enzymatic scaling technology, the co-expression of molecular chaperones was employed to enhance the secretion of the heterologous protease K in Pichia pastoris, and the mechanism underpinning its chlorine-free wool scale stripping efficacy has been meticulously investigated. 【Method】 We used the Pichia pastoris expression system to have the tprK gene heterologously expressed. Then at the first time we analyzed the effects of overexpressions of molecular chaperone Ssa1, Erj5, Sil1, Hac1, Kar2, Lhs1, and Ydj1, which affect protein folding and quality control, on the expressions and enzymatic activities of TPRK. Also we analyzed the effect of TPRK treatment on wool fibers. 【Result】 TPRK is expressed in P. pastoris GS115, and its optimal reaction conditions are 65℃ and pH 9.0, with good thermal stability and pH stability. The enzyme activities of recombinant strains overexpressing the ssa1, hac1, erj5, and sil1 genes increased by 36.8%, 20.0%, 17.7%, and 14.8%, respectively. High density fermentation was carried out in a 5 L fermentation tank, and after 72 h of induction, the TPRK enzyme activity reached 77 471.99 U/mL. In the application of wool hydrolysis, TPRK hydrolyzed the inner layer of wool scales to gradually peel them off, achieving a peeling effect. The optimal hydrolysis conditions are: TPRK addition of 300 U/mL, reaction temperature of 55℃, pH 9.0, and reaction time of 2 h. 【Conclusion】 Overexpression of molecular chaperone Ssa1 can effectively increase the expression of TPRK. Using this TPRK to process wool fibers can effectively remove the wool scale layer, while causing less damage to the core cortex layer of wool. This lays the foundation for the promotion and application of protease K in wool scale removal technology.

Key words: proteinase K, molecular chaperone, Pichia pastoris, biological enzymatic treatment, anti-felting shrinkage

CAI Yi-an, ZHANG Yi-qun, YANG Zi-xuan, LIU Ye-xue, LIU Wen-long, LU Fu-ping, LI Yu. Enhanced Expression of Protease K in Pichia pastoris through Molecular Chaperones and Analysis of Its Effect on Wool Scale Layer[J]. Biotechnology Bulletin, 2024, 40(7): 307-313.

Figures/Tables 6

Fig. 1 Expression of TPRK in P. pastoris A: High copies screening of recombinant strains; B: SDS-PAGE analysis of TPRK in culture supernatant

Fig. 2 Enzymatic properties of TPRK A: Optimal temperature of TPRK; B: thermal stability of TPRK; C: optimal pH of TPRK; D: pH stability of TPRK

Fig. 3 Effects of co-expressing molecular chaperones on the secretory expression of TPRK

Fig. 4 Fermentation results of TPRK 5 L fermentation tank

Fig. 5 Electron microscopy of wool scales hydrolyzed by different proteases A: Untreated wool sample; B: TPRK treatment; C: alkaline protease treatment; D: keratinase treatment

Fig. 6 Electron microscopy of TPRK hydrolyzed wool scales A: 100 U/mL TPRK hydrolyzed wool for 0.5 h, 1 h, 1.5 h, and 2 h; B: 300 U/mL TPRK hydrolyzed wool for 0.5 h, 1 h, 1.5 h, and 2 h; C: 500 U/mL TPRK hydrolyzed wool for 0.5 h, 1 h, 1.5 h, and 2 h

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