期刊
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
卷 69, 期 18, 页码 5344-5352出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.1c00913
关键词
mono- and diacylglycerol lipase; crystal structure; site-directed mutagenesis; molecular dynamic simulations; hydrolysis
资金
- National Science Fund for Distinguished Young Scholars [31725022]
- Key Program of National Natural Science Foundation of China [31930084]
- Science and Technology Planning Project of Guangdong Province [2019A050503002]
- Chinese Academy of Sciences
- Shenzhen Institutes of Advanced Technology, CAS, Shenzhen government
- Guangdong province [2019QN01Y306]
Enzyme engineering based on protein structures is a rational and efficient approach to acquire biocatalysts with desired performances. In this study, improved variants of a special MDGL were discovered by replacing specific residues, with V269D showing a six-fold increase in hydrolysis activity compared to the wild type. The substitution of Asp enhanced the substrate affinity of AOL, shedding light on the catalytic process and helping tailor MDGLs for biotechnological applications.
Engineering of enzymes on the basis of protein structures are rational and efficient approaches to acquire biocatalysts of desired performances. In this study, we focused on a special mono- and diacylglycerol lipase (MDGL) isolated from the lipolytic enzyme-enriched fungus Aspergillus oryzae and discovered improved variants based on its crystal structure. We first solved the crystal structure of Aspergillus oryzae lipase (AOL) at 1.7 angstrom resolution. Structure analysis and sequence alignment of AOL and other MDGLs revealed that the residue V269 is of vital importance for catalysis. Replacement of the V269 in AOL with the corresponding residues in other MDGLs has led to noticeable changes in hydrolysis without sacrificing the thermostability and substrate specificity. Among the investigated variants, V269D exhibited about a six-fold higher hydrolysis activity compared to the wild type. Molecular dynamics simulations and protein-ligand interaction frequency analyses revealed that the Asp substitution enhanced the substrate affinity of AOL. Our work sheds light on understanding the catalytic process of AOL and helps tailoring MDGLs with desired catalytic performance to fulfill the demand for biotechnological applications.
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