Enhanced Activity And Thermostability Of Rhizomucor Miehei Lipase By Directed Evolution

Lipases (EC3.1.1.3, systematic name:triacylglycerol hydrolases) are a versatile group of biocatalysts. They are wildly used in oil production, food industry, chemical industry and agriculture industry. Rhizomucor miehei Lipase also known as RML (Genebank accession no.A34959) specifically hydrolyzes the1,3-position C (O)-O bonds of triglyceride esters. The products (diesters and monoesters) are of special interest in the food industry and other commercial branches.Natural RML includes a signal peptide (24amino acids), a propeptide (70amino acids) and a mature region (269amino acids). The molecular weight of mature protein is31.6kD. The object of the present work is the gene containing the propeptide and the mature region. It was the first report on the overexpression of RML in E. coli. The present work improved the amount of the soluble protein through three methods:1. To induce the RML at16℃.2. To fuse a new chaperone (Skp) and RML at N terminal;3. To fuse the Trx.tag in pET32a and RML at N terminal. In particular, the expression result by low-temperature inducing was best and the amount of target protein was5.4-fold compared to the37℃inducing. It was shown that the temperature was the key factor of controling the amount of soluble protein.Propeptides are short sequences that facilitate the folding of their associated proteins. The present study found that the propeptide of RMLwas not proteolytically removed in E. coli. Moreover, RML was not expressed if the propeptide was removed artificially during the cloning process in E. coli. It was demonstrated that the propeptide was essential for the correct folding of RML. This behavior in E. coli permitted the application of directed evolution to full length RML.The high activity of enzyme is one effective way of reducing the enzyme cost. The work selected the adaptive mutants by error-prone PCR firstly. Then it was concluded that the propeptide and the mature protein folded separately rather than fused. Therefore, the3D-structure of the mature protein (PDB:3TGL) could be the basis to select the adative mutations obtained from error-prone PCR for site-directed mutagenesis and saturation mutagenesis. There were4rounds of error-prone PCR,2rounds of site-directed mutagenesis and2rounds of saturation mutagenesis totally. Not only the specific activity was improved obviously, but also the thermostability of RML was enhanced.15mutants was obtained including M13(D48V/V67A/S168P/S240V/S311C/D313H) with the activity of3609±100U/mg (14.3-fold of wild type) and M15(D48V/V67A/S168P/S240A/S311C/D313H) with the activity of1316±50U/mg (5.2-fold of wild type without heat treatment) after the heat treatment at70℃for two hours. The high activity and high thermostability of enzyme facilitated the application in industry. It was shown that the semi-rational was important for the better results in directed evolution.It was the first report on the role of the propeptide of RML in the directed evolution. It was concluded that the change of RML improved the protein activity even the function of the propeptide was mainly the assistance of its connected protein and the propeptide of RML was folded separately. It was found that the N terminal and the C terminal of the propeptides mainly contained the hydrophobic amino acids through the analysis of23propeptides from the lipase family. It was also found that the mutations in the propeptide of RML were mainly located in the N terminal and the C terminal of the propeptide and tend to hydrophobic amino acids. It was shown that the change of the propeptide towarded to the formation of more stable nucleation which was essential for propeptide folding and may be beneficial for the "perfect folding" of mature region. Therefore, the directed evolution of full length proteins including their propeptides is a strategy and simultaneous process. The evolution of the propeptide was essential and the attention should not be limited to the mature region. Such conclusion proposed a new strategy for the directed evolution of other proteins including the propeptides.