分子伴侣增强蛋白酶K在毕赤酵母中的表达及对羊毛鳞片层的作用分析

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

摘要/Abstract

摘要：

【目的】 通过分子伴侣共表达增强毕赤酵母中异源蛋白酶K的分泌水平，并对其的羊毛无氯剥鳞作用机制进行解析，旨在提高蛋白酶K的表达量并为高效酶法剥鳞技术的应用奠定基础。【方法】 利用毕赤酵母表达系统对tprK基因进行异源表达，首次分析了影响蛋白质折叠和质量控制的分子伴侣Ssa1、Erj5、Sil1、Hac1、Kar2、Lhs1和Ydj1分别过表达对TPRK的表达量和酶活力的作用，并对TPRK处理的羊毛纤维效果进行分析。【结果】 TPRK在毕赤酵母GS115中表达，其最适反应条件为65℃、pH 9.0，且具有良好的热稳定性和pH稳定性。过表达ssa1、hac1、erj5和sil1基因的重组菌株酶活分别提升了36.8%、20.0%、17.7%和14.8%。5 L发酵罐进行高密度发酵，诱导72 h后，TPRK的酶活达到77 471.99 U/mL。在羊毛水解应用中TPRK可水解羊毛鳞片内层使鳞片逐渐剥落，起到剥鳞效果，并且最适水解条件为：TPRK添加量为300 U/mL、反应温度55℃、pH 9.0和反应时间2 h。【结论】 过表达分子伴侣Ssa1能够有效提升TPRK的表达量，利用该TPRK处理羊毛纤维，可有效去除羊毛鳞片层，而对羊毛核心皮质层造成的损伤较小。

关键词: 蛋白酶K, 分子伴侣, 毕赤酵母, 生物酶处理, 防毡缩

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

蔡逸安, 张轶群, 杨子璇, 刘业学, 刘文龙, 路福平, 李玉. 分子伴侣增强蛋白酶K在毕赤酵母中的表达及对羊毛鳞片层的作用分析[J]. 生物技术通报, 2024, 40(7): 307-313.

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.

图/表 6

图1 TPRK在毕赤酵母中的表达 A：高拷贝重组转化子的筛选；B：培养物上清液中的TPRK的SDS-PAGE分析

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

图2 TPRK的酶学性质 A：TPRK的最适作用温度；B：TPRK的热稳定性；C：TPRK的最适pH；D：TPRK的pH稳定性

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

图3 共表达分子伴侣对TPRK分泌表达的影响

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

图4 TPRK 5 L发酵罐发酵结果

Fig. 4 Fermentation results of TPRK 5 L fermentation tank

图5 不同蛋白酶水解羊毛鳞片的电镜图 A：未处理羊毛样品；B：TPRK处理；C：碱性蛋白酶处理；D：角蛋白酶处理

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

图6 TPRK水解羊毛鳞片的电镜图 A：100 U/mL TPRK依次水解羊毛0.5 h、1 h、1.5 h、2 h电镜图；B：300 U/mL TPRK依次水解羊毛0.5 h、1 h、1.5 h、2 h电镜图；C：500 U/mL TPRK依次水解羊毛0.5 h、1 h、1.5 h、2 h电镜图

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

参考文献 33

[1]	Rani S, Kadam V, Rose NM, et al. Wheat starch, gum arabic and chitosan biopolymer treatment of wool fabric for improved shrink resistance finishing[J]. Int J Biol Macromol, 2020, 163: 1044-1052. doi: S0141-8130(20)33811-3 pmid: 32673714
[2]	张腾飞. 基于蛋白酶K的羊毛剥鳞技术研究[D]. 上海: 东华大学, 2023.
	Zhang TF. Reaserch on wool scale-peeling technology based on proteinase K[D]. Shanghai: Donghua University, 2023.
[3]	Li B, Li JY, Shen YQ, et al. Development of environmentally friendly wool shrink-proof finishing technology based on L-cysteine/protease treatment solution system[J]. Int J Mol Sci, 2022, 23(21): 13553.
[4]	Bhari R, Kaur M, Sarup Singh R. Chicken feather waste hydrolysate as a superior biofertilizer in agroindustry[J]. Curr Microbiol, 2021, 78(6): 2212-2230. doi: 10.1007/s00284-021-02491-z pmid: 33903939
[5]	An FF, Fang KJ, Liu XM, et al. Protease and sodium alginate combined treatment of wool fabric for enhancing inkjet printing performance of reactive dyes[J]. Int J Biol Macromol, 2020, 146: 959-964. doi: S0141-8130(19)34207-2 pmid: 31726143
[6]	Wang L, Yao JB, Niu JR, et al. Eco-friendly and highly efficient enzyme-based wool shrinkproofing finishing by multiple padding techniques[J]. Polymers, 2018, 10(11): 1213.
[7]	Ren YX, Luo HY, Huang HQ, et al. Improving the catalytic performance of proteinase K from Parengyodontium album for use in feather degradation[J]. Int J Biol Macromol, 2020, 154: 1586-1595.
[8]	Li R, Liu Z, Jiang F, et al. Enhancement of thermal stability of proteinase K by biocompatible cholinium-based ionic liquids[J]. Phys Chem Chem Phys, 2022, 24(21): 13057-13065.
[9]	Yadollahi E, Shareghi B, Eslami farsani R. Molecular aspects of the interaction of organic solvents and proteinase K: kinetics and docking studies[J]. Iran J Sci Technol Trans A Sci, 2019, 43(1): 57-62.
[10]	Skowron PM, Krefft D, Brodzik R, et al. An alternative for proteinase K-heat-sensitive protease from fungus Onygena corvina for biotechnology: cloning, engineering, expression, characterization and special application for protein sequencing[J]. Microb Cell Fact, 2020, 19(1): 135.
[11]	Yang JX, Chu N, Chen XW. Preparation of polyoxometalate-based composite by solidification of highly active cobalt-containing polytungstate on polymeric ionic liquid for the efficient isolation of proteinase K[J]. Molecules, 2023, 28(8): 3307.
[12]	Yang H, Zhai C, Yu XH, et al. High-level expression of proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains[J]. Protein Expr Purif, 2016, 122: 38-44.
[13]	Cai P, Duan XP, Wu XY, et al. Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris[J]. Nucleic Acids Res, 2021, 49(13): 7791-7805.
[14]	李登科, 王欣驰, 陈雪佳, 等. 不同β-葡萄糖苷酶性能分析及对罗汉果苷的转化[J]. 食品科学技术学报, 2019, 37(3): 48-54.
	Li DK, Wang XC, Chen XJ, et al. Properties analysis and transformation ability to mogroside of β-glucosidase[J]. J Food Sci Technol, 2019, 37(3): 48-54.
[15]	Raschmanová H, Weninger A, Knejzlík Z, et al. Engineering of the unfolded protein response pathway in Pichia pastoris: enhancing production of secreted recombinant proteins[J]. Appl Microbiol Biotechnol, 2021, 105(11): 4397-4414. doi: 10.1007/s00253-021-11336-5 pmid: 34037840
[16]	Fauzee YNBM, Taniguchi N, Ishiwata-Kimata Y, et al. The unfolded protein response in Pichia pastoris without external stressing stimuli[J]. FEMS Yeast Res, 2020, 20(7): foaa053.
[17]	Herrera-Estala AL, Fuentes-Garibay JA, Guerrero-Olazarán M, et al. Low specific growth rate and temperature in fed-batch cultures of a beta-propeller phytase producing Pichia pastoris strain under GAP promoter trigger increased KAR2 and PSA1-1 gene expression yielding enhanced extracellular productivity[J]. J Biotechnol, 2022, 352: 59-67. doi: 10.1016/j.jbiotec.2022.05.010 pmid: 35618082
[18]	Tyson JR, Stirling CJ. LHS1 and SIL1 provide a lumenal function that is essential for protein translocation into the endoplasmic reticulum[J]. EMBO J, 2000, 19(23): 6440-6452. doi: 10.1093/emboj/19.23.6440 pmid: 11101517
[19]	Wang YX, Luo X, Zhao YQ, et al. Integrated strategies for enhancing the expression of the AqCoA chitosanase in Pichia pastoris by combined optimization of molecular chaperones combinations and copy numbers via a novel plasmid pMC-GAP[J]. Appl Biochem Biotechnol, 2021, 193(12): 4035-4051.
[20]	Li Y, Hu XY, Sang JC, et al. An acid-stable β-glucosidase from Aspergillus aculeatus: gene expression, biochemical characterization and molecular dynamics simulation[J]. Int J Biol Macromol, 2018, 119: 462-469.
[21]	罗漫杰, 徐灿, 王梁, 等. 一种蛋白酶K高表达工程菌株的构建及应用: CN113481225A>[P]. 2021-10-08.
	Luo MJ, Xu C, Wang L, et al. Construction and application of an engineering strain with high expression of protease K: CN113481225A[P]. 2021-10-08.
[22]	杨琥, 陈莹, 谭华菊. 一种蛋白酶K多拷贝菌种构建方法: CN112359035A[P]. 2021-02-12.
	Yang H, Chen Y, Tan HJ. A method for constructing multi copy strains of proteinase K: CN112359035A[P]. 2021-02-12.
[23]	王华明, 林艳梅. 一种高产蛋白酶K的黑曲霉菌株及其应用: CN104480027A[P]. 2015-04-01.
	Wang HM, Lin YM. A high-yield proteinase K strain of Aspergillus niger and its application: CN104480027B[P]. 2016-09-21.
[24]	Li WH, Singer RH. Detecting the non-conventional mRNA splicing and translational activation of HAC1 in budding yeast[J]. Methods Mol Biol, 2022, 2378: 113-120. doi: 10.1007/978-1-0716-1732-8_8 pmid: 34985697
[25]	Zahrl RJ, Prielhofer R, Ata Ö, et al. Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion[J]. Metab Eng, 2022, 74: 36-48. doi: 10.1016/j.ymben.2022.08.010 pmid: 36057427
[26]	Li C, Lin Y, Zheng XY, et al. Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris[J]. BMC Biotechnol, 2015, 15: 88.
[27]	Killian AN, Miller SC, Hines JK. Impact of amyloid polymorphism on prion-chaperone interactions in yeast[J]. Viruses, 2019, 11(4): 349.
[28]	Fürsch J, Voormann C, Kammer KM, et al. Structural probing of Hsp26 activation and client binding by quantitative cross-linking mass spectrometry[J]. Anal Chem, 2021, 93(39): 13226-13234. doi: 10.1021/acs.analchem.1c02282 pmid: 34542282
[29]	Deng JB, Li JQ, Ma MP, et al. Co-expressing GroEL-GroES, Ssa1-Sis1 and Bip-PDI chaperones for enhanced intracellular production and partial-wall breaking improved stability of porcine growth hormone[J]. Microb Cell Fact, 2020, 19(1): 35.
[30]	de Jesus JR, Aragão AZB, Arruda MAZ, et al. Optimization of a methodology for quantification and removal of zinc gives insights into the effect of this metal on the stability and function of the zinc-binding co-chaperone Ydj1[J]. Front Chem, 2019, 7: 416. doi: 10.3389/fchem.2019.00416 pmid: 31263692
[31]	Gaur D, Singh P, Guleria J, et al. The yeast Hsp70 cochaperone Ydj1 regulates functional distinction of Ssa Hsp70s in the Hsp90 chaperoning pathway[J]. Genetics, 2020, 215(3): 683-698. doi: 10.1534/genetics.120.303190 pmid: 32299842
[32]	Gaur D, Kumar N, Ghosh A, et al. Ydj1 interaction at nucleotide-binding-domain of yeast Ssa1 impacts Hsp90 collaboration and client maturation[J]. PLoS Genet, 2022, 18(11): e1010442.
[33]	Li WJ, Zhang N, Wang Q, et al. A sustainable and effective bioprocessing approach for improving anti-felting, anti-pilling and dyeing properties of wool fabric[J]. Fibres Polym, 2021, 22(11): 3045-3054.