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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2019, Vol. 45 Issue (5): 526-532    DOI: 10.3785/j.issn.1008-9209.2018.10.081
生物科学与技术     
克氏原螯虾i-型溶菌酶在巴斯德毕赤酵母中的高效胞外表达及其抑菌活性(英文)
水燕1(),管政兵2,叶俊贤2,史永红3,刘国锋1,徐增洪1
1. 中国水产科学研究院淡水渔业研究中心，农业农村部淡水渔业和种质资源利用重点实验室，江苏 无锡 214081
1. The Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture and Rural Affairs, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, Jiangsu, China
2. 江南大学生物工程学院，工业生物技术教育部重点实验室，江苏 无锡 214122
2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
3. 中国农业科学院上海兽医研究所，上海 200241
3. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China
Efficient extracellular expression and antimicrobial activity of Procambarus clarkii invertebrate-type lysozyme in Pichia pastoris
Yan SHUI1(),Zhengbing GUAN2,Junxian YE2,Yonghong SHI3,Guofeng LIU1,Zenghong XU1
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摘要：

为开发虾类来源的溶菌酶，本研究建立了利用巴斯德毕赤酵母表达系统高效分泌表达重组克氏原螯虾无脊椎动物型溶菌酶1（简称“pc-iLys1”）的方法。经密码子偏好性优化后的pc-iLys1 cDNA在毕赤酵母菌株SMD1168中成功地实现胞外分泌表达，重组蛋白分子质量约为17.1 kDa。在摇瓶中对诱导表达条件进行优化，获得的相对最佳表达条件为：培养基pH 7.0，培养温度28 ℃，甲醇体积分数1.5%，诱导表达时间96 h。采用该优化条件，进一步利用分批补料策略在5 L发酵罐中进行高密度培养诱导，120 h后获得重组pc-iLys1，其酶活性达到 2 052.6 U/mL，最终，酵母干细胞质量浓度达98.1 g/L。抑菌实验表明，重组pc-iLys1对多种革兰氏阳性和阴性细菌均具有明显的抑制活性。总之，本研究实现了克氏原螯虾i-型溶菌酶在毕赤酵母中的重组表达，为其大规模制备打下了基础。
关键词： i-型溶菌酶;  克氏原螯虾;  巴斯德毕赤酵母;  表达条件优化;  抑菌活性;  invertebrate-type lysozyme;  Procambarus clarkii;  Pichia pastoris;  expression condition optimization;  antimicrobial activity    
出版日期: 2019-12-05
CLC:  TS 201.1  
基金资助: orted by the Central Public-interest Scientific Institution Basal Research Fund(2018HY-ZD0402);the Jiangsu Province “Creation of Major New Varieties of Agriculture” development program(PZCZ201746);the National Natural Science Foundation of China(31472003)
通讯作者: 水燕     E-mail: yanshui1201@sina.com
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水燕
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刘国锋
徐增洪

引用本文:

水燕,管政兵,叶俊贤,史永红,刘国锋,徐增洪. 克氏原螯虾i-型溶菌酶在巴斯德毕赤酵母中的高效胞外表达及其抑菌活性(英文)[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(5): 526-532.

Yan SHUI,Zhengbing GUAN,Junxian YE,Yonghong SHI,Guofeng LIU,Zenghong XU. Efficient extracellular expression and antimicrobial activity of Procambarus clarkii invertebrate-type lysozyme in Pichia pastoris. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(5): 526-532.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.10.081    或    http://www.zjujournals.com/agr/CN/Y2019/V45/I5/526

Fig. 1  Alignment of nucleotide sequences between the optimized and the original cDNA of pc-iLys1
Fig. 2  SDS-PAGE and Western blotting analysis of the secretory expression of pc-iLys1 in P. pastorisThe molecular mass of the recombinant pc-iLys1 is about 17.1 kDa. Protein samples loaded into each lane are as follows. M: Marker (20 μL); lane 1: Supernatant (10 μL)of P. pastoris with empty pPIC9K; lanes 2 and 3: Supernatant (5 and 10 μL, respectively) of P. pastoris with pPIC9K-pc-iLys1; lane 4: The purified pc-iLys1 by immobilized metal-affinity chromatography; Lane 5: Western blotting analysis of the expressed pc-iLys1 recognized by an anti-His6-tag mouse monoclonal antibody. The molecular mass marker is shown in the M lane. The arrowhead indicates the band of the recombinant pc-iLys1 protein.
Fig. 3  Effects of induction conditions on the extracellular expression of the recombinant pc-iLys1Each value represents the mean of three independent measurements.
Fig. 4  Time-course profile of three-stage fermentation for the high-yield production of pc-iLys1 by recombinant P. pastoris in a 5 L bioreactor
Fig. 5  Antimicrobial activities of recombinant pc-iLys1 in vitroBSA: Bovine serum albumin; HEWL: Hen egg-white lysozyme. Each value represents the mean of three independent measurements. Single asterisk (*) above the bars indicates a significant difference at the 0.05 probability level.
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