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| Biosynthesis of Crucian Carp (Carassius auratus) c-type Lysozyme Based on Recombinant Pichia pastoris and Its Characteristics |
| SONG Yu-Fang1, TAO Yan1,2,*, XIE Jing1,2, QIAN Yun-Fang1,2 |
1 College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China;
2 Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China;
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Abstract As key immune protein in innate immune mechanism of crucian carp (Carassius auratus), c-type lysozyme is a good candidate for the development of green feed additives or natural antimicrobial agents. In present study, the cDNA encoding crucian carp c-type lysozyme was cloned by reverse transcription PCR (RT-PCR). Subsequently, 3 times of PCR were performed to add the XhoⅠrestriction endonuclease site, Kex2 signal peptidase cleavage site and a 6×His tag to its 5' end, and the XbaⅠrestriction endonuclease site and stop codon to its 3' end; the obtained target gene was named CrCLm (crucian carp c-type lysozyme mature peptide). Using pPICZɑA as expression vector and Pichia pastoris X-33 as engineering strain, the recombinant P. pastoris strain X-33/pPICZɑA-CrCLm was constructed. After the recombinant strain was induced at initial pH 6.0, 28 ℃ and 250 r/min for 96 h with 1.0% methanol, the obtained recombinant protein was purified by a nickel ion affinity chromatography column. The purified protein was used for Western blot and LC-MS/MS analysis. On the other hand, the antibacterial activity and stability for the recombinant protein were determined. The results indicated that under above fermentation conditions, the recombinant protein with a yield of 18.3 mg/L was obtained, and it was identified by LC-MS/MS analysis as expected recombinant CrCLm with a molecular weight of 14.5 kD. The results of antibacterial test showed that the recombinant CrCLm had obvious activity against Gram-positive Staphylococcus aureus, Bacillus subtilis, B. cereus and Listeria monocytogenes, and Gram-negative Escherichia coli, Salmonella, Vibrio parahaemolyticus and Pseudomonas aeruginosa. In addition, different acidic environments, high temperature treatment and hydrolysis of proteinase (such as trypsin, pepsin and proteinase K) did not affect its antibacterial activity. The recombinant P. pastoris strain constructed in the present study can effectively synthesize CrCLm with obvious antibacterial activity and good stability, which could provide technical support for its large-scale preparation and application.
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Received: 20 January 2021
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Corresponding Authors:
* ytao@shou.edu.cn
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[1] 冯亚东, 陶妍, 李雯, 等. 2017. 斑点叉尾鮰C型溶菌酶在毕赤酵母中的表达及其抑菌活性[J]. 生物技术通报, 33(7): 195-202.
(Feng Y D, Tao Y, Li W, et al. 2017. Expression of channel catfish c-type lysozyme in Pichia pastoris and its bacteriostatic activity[J]. Biotechnology Bulletin, 33(7): 195-202.)
[2] 何春征. 2019. 翼手目动物C型溶菌酶的功能及分子进化研究[D]. 硕士学位论文, 沈阳农业大学, 导师: 刘洋, pp. 26-40.
(He C Z. 2019. Functional analyses and molecular evolution on C-type lysozyme in Bats[D]. Thesis for M.S., Shenyang Agricultural University, Supervisor: Liu Y, pp. 26-40.)
[3] 李锋涛, 孙亚东, 柳公卿. 2009. 溶菌酶的性质及在畜牧生产中的应用[J]. 养殖与饲料, 9: 49-51.
(Li F T, Sun Y D, Liu G Q. 2009. Properties of lysozyme and its application in animal husbandry[J]. Animal Breeding and Feed, 9: 49-51.)
[4] 李雯, 陶妍. 2017. 鲤鱼g型溶菌酶基因的cDNA克隆及其在毕赤酵母中的表达[J]. 福建农林大学学报, 46(1): 81-88.
(Li W, Tao Y. 2017. cDNA cloning of g-type lysozyme gene from common carp and its expression in Pichia pastoris[J]. Journal of Fujian Agriculture and Forestry University, 46(1): 81-88.)
[5] 刘纪红, 马美湖. 2018. 蛋清溶菌酶改性研究进展[J]. 中国家禽, 40(15): 44-48.
(Liu J H, Ma M H. 2018. Research advances on modification of egg white lysozyme[J]. China Poultry, 40(15): 44-48.)
[6] 刘真英, 李文利. 2015. 密码子优化后的柞蚕溶菌酶在酵母中的表达及活性测定[J]. 微生物学通报, 43(02): 292-300.
(Liu Z Y, Li W L. 2015. Expression and activity determination of codon optimized Antheraea pernyi lysozyme in Pichia pastori[J]. Microbiology China, 43(02): 292-300.)
[7] 王强厚, 陶妍, 崔旭, 等. 2018. 三疣梭子蟹C型溶菌酶在毕赤酵母中的表达及其抑菌活性[J]. 生物技术通报, 34(10): 141-148.
(Wang Q H, Tao Y, Cui X, et al. 2018. Expression of swimming crab C-type lysozyme in Pichia pastoris and its bacteriostatic activity [J]. Biotechnology Bulletin, 34(10): 141-148.)
[8] 徐永平, 汪婷婷, 孙永欣, 等. 2011. 水产动物溶菌酶研究的最新进展[J]. 水产科学, 30(5): 63-66.
(Xu Y P, Wang T T, Su Y X, et al. 2011. Current research on function and application of aquatic animalm lysozyme[J]. Fisheries Science, 30(5): 63-66.)
[9] 颜倩倩, 陶妍, 李雯, 等. 2019. 鲤鱼c型溶菌酶在毕赤酵母中的重组表达及其抑菌活性[J]. 农业生物技术学报, 27(11): 1912-1922.
(Yan Q Q, Tao Y, Li W, et al. 2019. Recombinant expression of common carp, 27(11): 1912-1922.)
[10] 杨杰, 丛丽娜, 夏涛, 等. 2012. 日本对虾c型溶菌酶的高效重组表达及产物分析[J]. 生物技术通报, 6: 99-105.
(Yang J, Cong L N, Xia T, et al. 2012. High level recombinant expression of c-type lysozyme from Marsupenaeus japonicus and product analysis[J]. Biotechnology Bulletin, 6: 99-105.)
[11] 张江凡, 齐甜甜, 董传举, 等. 2108. 中国不同鲫鱼品系系统发育关系研究进展[J]. 河南水产, 3: 25-27.
(Zhang J F, Qi T T, Dong C J, et al. 2018. Development of phylogenetic relationship of different crucian carp, 3: 25-27.)
[12] 张赛南, 施柳佳, 廖志银, 等. 2020. 蛋清溶菌酶基因的密码子优化及其在毕赤酵母中的分泌表达[J]. 浙江大学学报(理学版), 47(4): 476-483.
(Zhang S N, Shi L J, Liao Z Y, et al. 2020. Codon optimization of egg white lysozyme gene and its secretory expression in Pichia pastoris[J]. Journal of Zhe Jiang University (Engineering Science), 47(4): 476-483.)
[13] 赵震. 2020. 青蛤Mytimacin抗菌肽和C型溶菌酶在毕赤酵母中的表达及其抑菌活性[D]. 硕士学位论文, 上海海洋大学, 导师: 陶妍, pp. 41-51.
(Zhao Z. 2020. Expression of Cyclina sinensis mytimacin antibacterial peptide and C-type lysozyme in Pichia pastoris and their antibacterial activity[D]. Thesis for M.S., Shanghai Ocean University, Supervisor: Tao Y, pp. 41-51.)
[14] Aisenbrey C, Marquette A, Bechinger B.2019. The Mechanisms of Action of Cationic Antimicrobial Peptides Refined by Novel Concepts from Biophysical Investigations[M]. Antimicrobial Peptides, Singapore, pp. 33-64.
[15] Chung C R, Jhong J H, Wang Z, et al.2020. Characterization and identification of natural antimicrobial peptides on different organisms[J]. International Journal of Molecular Sciences, 21(3): 986-1012.
[16] Daly R, Hearn M T.2005. Expression of heterologous protion in Pichia pastoris: A useful experimental tool in protein enigneering and production[J]. Journal of Molecular Recognition, 18(2): 119-138.
[17] Dautigny A, Prager E M, Pham-Doinh D, et al.1991. cDNA and amino acid sequences of rainbow trout (Oncorhynchus mykiss) lysozymes and their implications for the evolution of lysozyme and lactalbumin[J]. Journal of Molecular Evolution, 32(2): 187-198.
[18] Eom G T, Seung H L, Bong K S, et al.2013. High-level extracellular production and characterization of Candida antarctica lipase B in Pichia pastoris[J]. Journal of Bioscience and Bioengineering, 116(2): 165-170.
[19] Huang P, Li W S, Xie J, et al.2011. Characterization and expression of HLysG2, a basic goose-type lysozyme from the human eye and testis[J]. Molecular Immunology, 48(4): 524-531.
[20] Huang P, Sun Q, Shi W, et al.2018. Efficient production of human goose-type lysozyme 2 in the methylotrophic yeast, Pichia pastoris[J]. Journal of Biotechnology, 275: 44-52.
[21] Marilaine M M, Gisèle L, Christophe L, et al.2000. MICs of mutacin B-Ny266, nisin A, vancomycin, and oxacillin against bacterial pathogens[J]. Antimicrobial Agents and Chemotherapy, 44(1): 24-29.
[22] Pan C, Huang T, Wang Y, et al.2012. Oral administration of recombinant epinecidin-1 protected grouper (Epinephelus coioodes) and zebrafish (Danio rerio) from Vibro vulnificus infection and enhanced immune-related gene expression[J]. Fish & Shellfish Immunology, 32(6): 947-957.
[23] Ting C, Chen Y, Chen J, et al.2018. Nile tilapia fry fed on antimicrobial peptide epinecidin-1-expression artemiacyst exhibit enhanced immunity against acute bacterial infection[J]. Fish & Shellfish Immunology, 81: 37-48.
[24] Wang M J, Zhao X L, Kong X H, et al.2016. Molecular characterization and expressing analysis of the c-type lysozyme in Qihe crucian carp Carassius auratus[J]. Fish & Shellfish Immunology, 52: 210-220.
[25] Wang R J, Feng J B, Li C.2013. Four lysozymes (one c-type and three g-type) in catfish are drastically but differentially induced after bacterial infection[J]. Fish & Shellfish Immunology, 35(1): 136-145.
[26] Wei S, Huang Y, Cai J, et al.2012. Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides[J]. Fish & Shellfish Immunology, 33(2): 186-196.
[27] Wiegand I, Hilpert K, Hancock R E W.2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances[J]. Nature Protocols, 3(2): 163-175.
[28] Yang D L, Wang Q, Cao R W, et al.2017. Molecular characterization, expression and antimicrobial activities of two c-type lysozymes from manila clam Venerupis philippinarum[J]. Developmental and Comparative Immunology, 73: 109-118.
[29] Yu Y, Zhou X Y, Wu S, et al.2014. High-yield production of the human lysozyme by Pichia pastoris SMD1168 using response surface methodology and high-cell-density fermentation[J]. Electronic Journal of Biotechnology, 17(6): 311-316.
[30] Zhou X Y, Yu Y, Tao J J, et al.2014. Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation[J]. Journal of Bioscience and Bioengineering, 118(4): 420-425. |
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