Screening, cloning and expression of a xylanase from palm oil mill effluent (pome) by functional metagenomics approach

Abstract

Metagenomics approach is an alternative method to study the novel enzyme. Therefore, a metagenomic fosmid library of approximately 50,000 clones was screened to identify the novel xylanase enzymes. The metagenomics deoxyribonucleic acid (DNA) used in this study is from the samples of palm oil mill effluent (POME) from Felda Palm Industry Sdn. Bhd. in Mempaga, Pahang. Clones of the metagenomics were screened using fluorescence substrate of chlorocoumarin xylobioside. The high score reads obtained from screening were sent for next-generation sequencing (NGS) of Illumina HiSeq2000. The sequences were further analysed to identify the predicted xylanase genes using automated and manual bioinformatic tools. A total of 34 predicted xylanases were identified, and five predicted xylanase genes #11, #15, #16, #17, and #18 of various microbial origins were chosen to be further analysed. The translated sequences of these five genes later were analysed to determine the primary, secondary, and tertiary protein structures in predicting feature and function of predicted xylanases. Next, based on the integrity checking in agarose gel electrophoresis (GE), Gene #15 with approximately 1.2 kb was chosen to be cloned into the pBAD-TOPO vector. Gene #15 has the percentage identity of 99.3% to the Ochrobactrum intermedium with glycoside hydrolase (GH) 10 as the conserved domain. After cloning, pBAD-TOPO-Xyl was used to transform E. coli cells and expressed using the inducer, L-arabinose. Protein #15 (P15) was later purified using immobilised metal affinity chromatography (IMAC), and the molecular mass of SDS-PAGE of approximately 46 kDa was confirmed. The P15 also fluoresced when checked with chlorocoumarin xylobioside substrate, which suggests the protein is a xylanase enzyme.