title: Modification of recombinant elastase expressed in Pichia pastoris by introduction of N-glycosylation sites.
abstract: A novel N-glycosylation site was introduced into recombinant elastase (rPAE) at N36, N67, or N264 through the site-directed mutagenesis of I38T, A69T, or N266T, respectively. The A69T mutation completely inhibited the expression of rPAE. As expected, the I38T and N266T mutant proteins exhibited higher degrees of N-glycosylation compared with the wild type rPAE. The I38T mutant was more efficient in the hydrolysis of casein in aqueous medium and exhibited higher specific activity and k(cat) values and a lower K(m) value. In contrast, the N266T mutant and the wild type displayed similar values. Importantly, the I38T mutant achieved in higher rates and yields of peptide synthesis in 50% (v/v) dimethylsulfoxide, whereas the N266T mutant was similar to the wild type rPAE. Furthermore, the maximum yield of Z-Ala-Phe-NH2 synthesis catalyzed by the I38T mutant protein (87%) was higher than those achieved by the wild type (78%) and N266T mutant (78%) proteins. Neither the I38T nor the N266T mutation exerted significant effects on the rPAE solvent stability. In aqueous medium, the I38T mutation decreased the rPAE thermostability, and the N266T mutation slightly improved that. In conclusion, the I38T mutation improved the potential of rPAE in industrial applications.
authors: Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Jiangsu Key Laboratory for Eco-Agricultural Biotechnology Around Hongze Lake, Huaiyin Normal University, Huaian 223300, China. Electronic address: minghaihan@gmail.com.
authors: Minghai
authors: M
authors: Han
authors: Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Jiangsu Key Laboratory for Eco-Agricultural Biotechnology Around Hongze Lake, Huaiyin Normal University, Huaian 223300, China.
authors: Xinfeng
authors: X
authors: Wang
authors: Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Jiangsu Key Laboratory for Eco-Agricultural Biotechnology Around Hongze Lake, Huaiyin Normal University, Huaian 223300, China.
authors: Guilong
authors: G
authors: Yan
authors: Huaian Institute of Supervision & Inspection on Product Quality, Huaian 223300, China.
authors: Weixian
authors: W
authors: Wang
authors: Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Jiangsu Key Laboratory for Eco-Agricultural Biotechnology Around Hongze Lake, Huaiyin Normal University, Huaian 223300, China.
authors: Yuan
authors: Y
authors: Tao
authors: Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Jiangsu Key Laboratory for Eco-Agricultural Biotechnology Around Hongze Lake, Huaiyin Normal University, Huaian 223300, China.
authors: Xin
authors: X
authors: Liu
authors: Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Jiangsu Key Laboratory for Eco-Agricultural Biotechnology Around Hongze Lake, Huaiyin Normal University, Huaian 223300, China.
authors: Hui
authors: H
authors: Cao
authors: Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors: Xiaobin
authors: X
authors: Yu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D010196:Pancreatic Elastase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010196:Pancreatic Elastase
keywords: Enzyme activity; Enzyme stability; N-glycosylation; Peptide synthesis; Pseudomonas aeruginosa elastase