title: Combination of site-directed mutagenesis and yeast surface display enhances Rhizomucor miehei lipase esterification activity in organic solvent.
abstract: To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.
authors.affiliation: Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, People's Republic of China.
authors.forename: Shuang-yan
authors.initials: SY
authors.lastname: Han
authors.forename: Jun-hui
authors.initials: JH
authors.lastname: Zhang
authors.forename: Zhen-lin
authors.initials: ZL
authors.lastname: Han
authors.forename: Sui-ping
authors.initials: SP
authors.lastname: Zheng
authors.forename: Ying
authors.initials: Y
authors.lastname: Lin
mesh_terms: D004789:Enzyme Activation; D004795:Enzyme Stability; D004951:Esterification; D024861:Genetic Enhancement / Q000379:methods*; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods; D020103:Rhizomucor / Q000201:enzymology* / Q000235:genetics*; D012997:Solvents / Q000737:chemistry*
chemical_list: D009930:Organic Chemicals; D012997:Solvents; D008049:Lipase