A PUTATIVE β-GLUCOSIDASE AND AN ENDO-1,4-β-GLUCANASE FROM POME METAGENOMIC DNA
| Main Authors: | Benbelgacem, Parman, Bellag, Akhyar, Alam, Salleh |
|---|---|
| Format: | Article Journal |
| Bahasa: | eng |
| Terbitan: |
, 2022
|
| Subjects: | |
| Online Access: |
https://zenodo.org/record/5826148 |
Daftar Isi:
- Functional metagenomic approach with high-throughput screening can be used to identify tapped and untapped biocatalysts. Metagenomic DNA libraries of 4.49 Gbase were constructed from microbes in Malaysian palm oil mill effluent (POME). After culture experiment based on natural selection metagenomic DNA was extracted and cloned to pCC1FOS vector and transformed into EPI300T1R. Cellulose-degrading enzyme activity was screened with microtiter assay using methylumbelliferyl-β-D-glucopyranoside (MUGlc) and methylumbelliferyl-β-D-cellobioside (MUC) as fluorogenic substrates. Reads were normalized using robust z-score and 100 highly rated clones were selected. Fosmids of these clones were isolated and sequenced with Hiseq strategy. Using Solexa, Velvet, SSPACE, Prodigal and Blastp, genes IDs of 96 putative cellulose-degrading enzymes were identified. Two putative metagenomic cellulose-degrading enzymes, MCDE1 with β-glucosidase activity and MCDE3 with endo-1,4-β-glucanase activity were produced, purified, and partially biochemically characterized.