Engineered variants of a lipase from Yarrowia lipolytica with improved trypsin resistance for enzyme replacement therapy
| Autor: | Jing Zhang, Huan Liu, Xiaonong Wu, Yujie Dai, Haikuan Wang, Cuixia Zhou, Rong Yue, Huitu Zhang, Tongwei Sun, Fuping Lu, Ying Zhang, Guoguo Wu, Fufeng Liu |
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| Rok vydání: | 2019 |
| Předmět: |
Mutant
Yarrowia Bioengineering Protein Engineering Biochemistry Pichia Pichia pastoris Fungal Proteins 03 medical and health sciences Protein Domains Enzyme Stability medicine Humans Enzyme Replacement Therapy Trypsin Amino Acid Sequence Lipase Site-directed mutagenesis Molecular Biology 030304 developmental biology chemistry.chemical_classification 0303 health sciences Binding Sites biology 030302 biochemistry & molecular biology Temperature Hydrogen-Ion Concentration biology.organism_classification Recombinant Proteins Amino acid Enzyme chemistry Mutagenesis Site-Directed biology.protein Biotechnology medicine.drug |
| Zdroj: | Protein Engineering, Design and Selection. 32:375-383 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/gzaa001 |
| Popis: | To improve the proteolytic stability of the lipase LIP2 from Yarrowia lipolytica, the peptide bonds susceptible to trypsin in LIP2 were analyzed by tandem mass spectrometry and redesigned by site-directed mutagenesis. Different variants of the enzyme were expressed in Pichia pastoris GS115 and their biochemical properties were subsequently investigated. Although most of the variants were still cleaved by trypsin, some of them did show an evident increase of resistance against proteolytic degradation. The most stable mutant was LIP2-C5, in which five trypsin-cleavage sites were replaced by non-preferred amino acids. Upon incubation with human trypsin for 80 min at 37°C, the mutant LIP2-C5 was found to retain >70% of its initial activity, compared to only 10% for the wild-type. |
| Databáze: | OpenAIRE |
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