前导肽区域N-糖基化影响骆驼凝乳酶原在毕赤酵母中的分泌表达和热稳定性研究

doi:10.13304/j.nykjdb.2022.0213

摘要/Abstract

摘要：

骆驼凝乳酶原在毕赤酵母中的表达量较低，不能满足产业化需求。为提高骆驼凝乳酶原的表达量，采用定点突变的方法，分别在其前导肽的第11、26、34、35、38、39位氨基酸处引入1个N-糖基化位点，构建成6种骆驼凝乳酶原的N-糖基化突变体：chy11、chy26、chy34、chy35、chy38和chy39，并分别导入到毕赤酵母中进行分泌表达。测定和比较了野生型（wild）和各突变体骆驼凝乳酶原的表达量。Chy34、chy35、chy38和chy39的表达量显著提高，其中chy34的表达量最高。6种突变体均保持前导肽自切割活性。在50 ℃保温8 h，wild酶活完全丧失，而突变体仍有20%~80%的相对酶活，表明6种突变体的热稳定性均显著增强。研究结果为提高骆驼凝乳酶原在毕赤酵母中的表达量和增强热稳定性提供了新的研究思路。

关键词: 骆驼凝乳酶原, N-糖基化, 毕赤酵母, 分泌表达, 热稳定性

Abstract:

The expression level of camel prochymosin in Pichia pastoris is low and cann’t meet the needs of industrialization. In order to improve the expression of camel prochymosin， N-glycosylation sites were introduced into the 11^th，26^th，34^th，35^th，38^th，39^th amino acids of propeptide region， respectively，to constructe 6 N-glycosylation mutants： chy11，chy26，chy34，chy35，chy38 and chy39. These mutants were introduced into P. pastoris for secretory expression and the expression levels of wild type and mutants were measured and compared. The secretory expression of chy34， chy35， chy38 and chy39 increased significantly， and the expression level of chy34 was the highest. The leading peptide of all 6 mutants maintained self cleavage activity. At 50 ℃ for 8 h， the enzyme activity of wild was completely lost， while the mutants still had 20%～80% relative enzyme activity， indicating that the thermotability of the 6 mutants was significantly enhanced. The results provided new research idea for improving the expression of camel prochymosin in P. pastoris and enhancing thermostability.

Key words: camel prochymosin, N-glycosylation, P. pastoris, secreted expression, thermostability

中图分类号:

王楠, 杨彩峰, 彭华康, 郭文芳, 王梦琪, 李刚强, 刘德虎. 前导肽区域N-糖基化影响骆驼凝乳酶原在毕赤酵母中的分泌表达和热稳定性研究[J]. 中国农业科技导报, 2022, 24(10): 71-78.

Nan WANG, Caifeng YANG, Huakang PENG, Wenfang GUO, Mengqi WANG, Gangqiang LI, Dehu LIU. N-glycosylation in the Propeptide Improved Thermostability of Camel Prochymosin and Enhanced Its Secretion in Pichia pastoris[J]. Journal of Agricultural Science and Technology, 2022, 24(10): 71-78.

图/表 7

表1 本研究所用引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Sequence （5’-3’）
chy-F	GGGTATCTCTCGAGAAAAGACACCATCACCATCACCACTCCGGTATCACCAGAATCC
chy11-F	CTCCGGTATCACCAGAATCCCATTGCACAAGAACAAGACCTTGAGAAAGGCTTTGAAGG
chy26-1F	TGAGAAAGGCTTTGAAGGAGAGAGGTTTGTTGGAGAACTTCTCCCAAAGACAACAATAC
chy26-2F	CTCCGGTATCACCAGAATCCCATTGCACAAGGGTAAGACCTTGAGAAAGGCTTTGAAGG
chy34-F	CTTGCAAAGACAACAATACAACGTCTCCTCCAAGTACTCCTCCTTGGGTAAGGTCGC
chy34-R	GCGACCTTACCCAAGGAGGAGTACTTGGAGGAGACGTTGTATTGTTGTCTTTGCAAG
chy35-F	CTTGCAAAGACAACAATACGCTAACTCCTCCAAGTACTCCTCCTTGGGTAAGGTCGC
chy35-R	GCGACCTTACCCAAGGAGGAGTACTTGGAGGAGTTAGCGTATTGTTGTCTTTGCAAG
chy38-F	CAACAATACGCTGTCTCCTCCAACTACTCCTCCTTGGGTAAGGTCGCCAGAGAG
chy38-R	CTCTCTGGCGACCTTACCCAAGGAGGAGTAGTTGGAGGAGACAGCGTATTGTTG
chy39-F	CAACAATACGCTGTCTCCTCCAAGAACTCCTCCTTGGGTAAGGTCGCCAGAGAG
chy39-R	CTCTCTGGCGACCTTACCCAAGGAGGAGTTCTTGGAGGAGACAGCGTATTGTTG
chy-R	GTCTAAGGCGAATTAATTCGCGGCCGCTTAGATGGCCTTGGCCAAACCGAC
qPCR-F	TTGAGATCCATGCCAACCGT
qPCR-R	TTGAGATCCATGCCAACCGT
actin-F	AGTGTTCCCATCGGTCGTAG
actin-R	GGTGTGGTGCCAGATCTTTT

图1 骆驼凝乳酶前导肽的N-糖基化突变位点注：下划线所示为N-糖基化保守序列。

Fig. 1 Mutant sites of N-glycosylation in the propeptide of prochymosin.Note： Mutant conserved N-glycosylated sequences are indicated by underlines.

图2 野生型和突变体凝乳酶原Western-blot分析注：A-发酵液上清Western-blot鉴定；B-发酵液上清去糖基化处理后Western-blot鉴定。白色箭头表示无糖基化形式，黑色箭头表示低糖基化形式。

Fig. 2 Western-blot analysis of wild and mutant prochymosinsNote： A-Western-blot identification of supernatant of fermentation broth； B-Western blot identification of the supernatant of fermentation broth after deglycosylation. The white arrows indicate non-glycosylated forms of prochymosin， black arrows indicate low glycosylated forms.

图3 发酵液上清中野生型和突变体凝乳酶原的相对分泌表达量比较注：***表示在P < 0.001水平差异显著。

Fig. 3 Secretion level comparison of wild and mutant prochymosins by western-blotNote： *** indicates significant difference at P < 0.001 level.

图4 荧光定量PCR比较凝乳酶原基因的相对转录水平注：*表示与野生型相比在P < 0.05水平差异显著。

Fig. 4 qPCR analysis of the relative mRNA levels of the prochymosin genesNote： * indicates significant difference compared with wild at P< 0.05 level.

图5 凝乳酶原前导肽的自切割活性分析

Fig. 5 Analysis of autocatalytic cleavage of the propeptide by confirming the milk-clotting acitity

图6 野生型和突变体凝乳酶原的热稳定性比较

Fig. 6 Comparison of thermostability of wild and mutant prochymosins

参考文献 26

1	KUMAR S, SHARMA G J, BATISH V K. Chymosin and other milk coagulants: sources and biotechnological interventions [J]. Crit. Rev. Biotechnol., 2010, 30(4): 243-258.
2	JOHNSON M E. A 100-Year Review: Cheese production and quality [J]. J. Dairy Sci., 2017, 100(12):9952-9965.
3	STEFAN R K, HANS J M VAN DEN BRINK, HENRIK R N, et al.. Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk [J]. Biochem. Biophys. Res. Commun., 2006, 342(2):647-654.
4	JESPER L J, ANNE M, JENS-CHRISTIAN N P, et al.. Camel and bovine chymosin: the relationship between their structures and cheese-making properties [J]. Acta Crystallogr. D. Biol. Crystallogr., 2013, 69(5):901-913.
5	PORRO D, SAUER M, BRANDUARDI P, et al.. Recombinant protein production in yeasts [J]. Mol. Biotechnol., 2005, 31(3):245-259.
6	WANG N, WANG K Y, LI G Q, et al.. Expression and characterization of camel chymosin in Pichia pastoris [J]. Protein Expres. Purif., 2015, 111:75-81.
7	APWEILER R, HERMJAKOB H, SHARON N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database [J]. Biochim. Biophys. Acta, 1999, 1473(1):4-8.
8	李剑凤,阎岩,王庆民,等. N-糖基化对毕赤酵母表达的DSPAα1分泌和活性的影响 [J].生物工程学报,2010, 26(09):1287-1292.
	Li J F, Yan Y, Wang Q M, et al.. Effect of N-linked glycosylation on secretion and activity of recombinant DSPAα1 expressed in Pichia pastoris [J]. Chin. J. Biotechnol., 2010, 26(9):1287-1292.
9	TIAN B, CHEN Y, DING S J. A combined approach for improving alkaline acetyl xylan esterase production in Pichia pastoris, and effects of glycosylation on enzyme secretion, activity and stability [J]. Protein Expres. Purif., 2012, 85(1): 44-50.
10	HAN M H, WANG W X, JIANG G C, et al.. Enhanced expression of recombinant elastase in Pichia pastoris through addition of N-glycosylation sites to the propeptide [J]. Biotechnol. Lett., 2014, 36(12):2467-2471.
11	WANG P, ZHANG J, SUN Z Y, et al.. Glycosylation of prourokinase produced by Pichia pastoris impairs enzymatic activity but not secretion [J]. Protein Expres. Purif., 2000, 20(2):179-185.
12	WANG Z Y, GUO C, LIU L, et al.. Eﬀects of N-glycosylation on the biochemical properties of recombinant bEK L expressed in Pichia pastoris [J]. Enzyme Microb. Technol., 2018, 114: 40-47.
13	WILLIAM S A, TINA J, GREGG T B, et al.. Probing the role of N-linked glycans in the stability and activity of fungal cellobiohydrolases by mutational analysis [J]. Cellulose, 2009, 16: 699-709.
14	YANG M, YU X W, ZHENG H Y, et al.. Role of N-linked glycosylation in the secretion and enzymatic properties of Rhizopus chinensis lipase expressed in Pichia pastoris [J]. Microb. Cell Fact., 2015 (14) :40.
15	JUAN A V, JOSE M A, MARGARITA P, et al.. Cloning and expression of buffalo active chymosin in Pichia pastoris [J]. J. Agric. Food Chem., 2008, 56(22):10606-10610.
16	MARIA C V H, ALICIA G C, JESÚS V . et al .. Molecular cloning and expression in yeast of caprine prochymosin [J]. J. Biotechnol., 2004, 114(1-2): 69-79.
17	ROGELJ I, PERKO B, FRANCKY A, et al.. Recombinant lamb chymosin as an alternative coagulating enzyme in cheese production [J]. J. Dairy Sci., 2001, 84(5):1020-1026.
18	Tyagi A, KUMAR A, MOHANTY A K, et al.. Expression of buffalo chymosin in Pichia pastoris for application in mozzarella cheese [J]. LWT-Food Sci. Technol., 2017, 84:733-739.
19	WANG N, WANG K Y, XU F F, et al.. The eﬀect of N-glycosylation on the expression of the tetanus toxin fragment C in Pichia pastoris [J/OL]. Protein Expres. Purif., 2020, 166:105503 [2022-03-02]. .
20	NAIRN A V, YORK W S, HARRIS K., et al.. Regulation of glycan structures in animal tissues: transcript proﬁling of glycan-related genes [J]. J. Biol. Chem., 2008, 283:17298-17313.
21	DALIT S B, YAAKOV L. Folding of glycoproteins: toward understanding the biophysics of the glycosylation code [J]. Curr. Opin. Struct. Biol., 2009, 19 (5): 524-533.
22	DURCHSCHLAG H, CHRISTL R, JAENICKE R. Comparative Determination of the Particle Weight of Glycoproteins by SDS-PAGE and Analytical Ultracentrifugation [M]. Steinkopff, Germany: Progress in Analytical Ultracentrifugation, 1991: 41-56.
23	HAN C, WANG Q Q, SUN Y X, et al.. Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites [J/OL]. Biotechnol. Biofuels, 2020, 13(1): 30 [2022-03-20]. .
24	ZHAI Z Y, NUYLERT A, KIMIYASU I, et al.. Effects of codon optimization and glycosylation on the hig‑level production of hydroxynitrile lyase from Chamberlinius hualienensis in Pichia pastoris [J]. J. Ind. Microbiol. Biotechnol., 2019, 46(7): 887-898.
25	PETRESCU A J, MILAC A L, PETRESCU S M, et al.. Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding [J]. Glycobiology, 2004, 14(2):103-114.
26	DALIT S B, YAAKOV L. Effect of glycosylation on protein folding: a close look at thermodynamic stabilization [J]. Proc. Natl. Acad. Sci. USA, 2008,105(24):8256-8261.

[1]	张艳丽1,2,任柳3,张宇宏1*,张伟1. 棉铃虫葡萄糖氧化酶HaGOX在毕赤酵母中的重组表达和性质研究[J]. 中国农业科技导报, 2020, 22(8): 56-63.
[2]	任雅馨1,2,罗会颖1,姚斌 1,王国增2,涂涛1. Cordyceps fumosorosea和Beauveria bassiana来源的碱性蛋白酶在毕赤酵母中的异源表达与性质测定[J]. 中国农业科技导报, 2020, 22(11): 69-78.
[3]	张丽洁1,2,徐欣欣2,田健2,初晓宇2,朱宝成1,伍宁丰2. 特异腐质霉来源漆酶基因的克隆及其在毕赤酵母中的表达[J]. 中国农业科技导报, 2019, 21(2): 46-53.
[4]	罗艳1,涂涛2,姚斌2,罗会颖2,徐波1. 嗜热子囊菌JCM12803来源的阿魏酸酯酶FAE-2515酶学性质研究[J]. 中国农业科技导报, 2018, 20(9): 57-64.
[5]	张祺珮1,李雅楠2,孟昆1,柏映国1,黄火清1*,罗会颖1. 新型高产β-胡萝卜素巴斯德毕赤酵母表达宿主菌的构建[J]. 中国农业科技导报, 2018, 20(2): 48-55.
[6]	李烨青1,涂涛2,王苑 2,马锐2,姚斌2,罗会颖2,徐波1*. Bispora sp. MEY-1来源的新型嗜热多聚半乳糖醛酸酶的酶学性质研究及其应用评估[J]. 中国农业科技导报, 2017, 19(10): 36-44.
[7]	郭超1,2,赵军旗2,齐西珍2,孙文良2,刘浩1,田朝光2. 粗糙脉孢菌GH45家族内切纤维素酶基因ncGH45*在毕赤酵母中表达及重组酶的性质表征[J]. 中国农业科技导报, 2016, 18(4): 64-72.
[8]	杨文霞,李可,潘霞,苏小运,杨培龙*. Neosartorya fischeri P1来源的外切聚半乳糖醛酸酶异源表达及性质研究[J]. 中国农业科技导报, 2016, 18(1): 73-80.
[9]	龚攀1,2,范家佑1,田健2,伍宁丰2,初晓宇2*. 热稳定性对甲基对硫磷水解酶在毕赤酵母中分泌的影响[J]. , 2015, 17(3): 42-48.
[10]	刘赟1,2,卢海强2,石鹏君2,谢响明1*. Achaetomium sp.Xz8来源的甘露聚糖酶Man5Xz8 热稳定性的研究[J]. , 2015, 17(3): 49-55.
[11]	罗晓亮1§,顾杏露2§,田健1,初晓宇1*,伍宁丰1. 组合突变提高甲基对硫磷水解酶MPH热稳定性的研究[J]. , 2015, 17(1): 167-172.
[12]	孙乔乔1,谭笑2,吕依2,黄火清2,张会图1*,路福平1. 嗜热真菌Neosartorya fischeri P1脂肪酶基因的克隆、表达及酶学性质分析[J]. , 2014, 16(5): 53-59.
[13]	刘小芸1,2,姚冬生1,2,3*. 饲用酶蛋白的理性设计及其改良技术[J]. , 2013, 15(5): 46-52.
[14]	杨雯涵,郭晓晶,陈轶群,吕俊楠,谢飞,曹云鹤*. 一种酸性木聚糖酶在毕赤酵母中的分泌表达及体外活性评价研究[J]. , 2013, 15(5): 59-66.
[15]	聂春明1,2§,宁晓彦2§,张宇宏2,樊晓虎1,2,赵国芬1,张伟2. 应用融合标签技术提高乳糖酶在毕赤酵母中的分泌表达[J]. , 2012, 14(5): 71-77.