Characterization and overexpression of esterases-encoding Lip900 and Lip3954 through metagenomic sequencing of paddy soil

Abstract

Abstract A lot of industrial genes can be explored from the metagenomic sequences. In this study, metagenome sequencing of paddy soil was carried out, and several putative open reading frames (ORFs) involved in the lipolytic activity can be identified. Lipolytic enzymes are widely used in different industrial applications, such as biodiesel production, bioremediation, and waste treatment. To verify the lipolytic enzymes of assembly ORFs, two putative genes encoding esterase, namely Lip900 and Lip3594, which shared 47.6% and 43.7% identities with the uncharacterized esterase proteins, were synthesized and constructed with pET-30a for Escherichia coli overexpression. Lip900 and Lip3594 belonging to VI and XII families were successfully obtained and characterized. The results of recombinant proteins indicated that Lip900 and Lip3594 preferred to hydrolyze short-length p-nitrophenyl (p-NP) esters such as p-NP butyrate (C4). The optimal temperature and pH for both Lip900 and Lip3594 were 30°C and pH 7, respectively. Nevertheless, Lip3594 had a higher relative activity than Lip900 when the temperature was over 40°C. The effect of various reagents on Lip900 and Lip3594 activities was determined. The inhibition of Lip900 and Lip3594 was observed in the presence of MgSO4, MnSO4, NiSO4, and sodium dodecyl sulfate (SDS). However, the addition of ethylenediaminetetraacetic acid (EDTA) can improve the lipolytic activity, indicating these esterases without metal ions as the cofactor. Moreover, Lip900 and Lip3594 were resistant to methanol, ethanol, and butanol. Over 81.6% of the relative activity of Lip900 can be attained when these organic solvents of alcohol were added to 10%. These results revealed that Lip900 and Lip3594 have potential applications in various industries.