Expression and Characterization of a Small, Xylan/Cellulose-degrading GH43 Protein Derived from Biofertilizer Metagenome
Atcha Oraintara1, 2, *, Pitak Bhunaonin1
Identifiers and Pagination:Year: 2022
E-location ID: e187428582210170
Publisher ID: e187428582210170
Article History:Received Date: 11/5/2022
Revision Received Date: 8/9/2022
Acceptance Date: 15/9/2022
Electronic publication date: 07/12/2022
Collection year: 2022
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
A putative glycosyl hydrolase gene biof1_09 was identified from a metagenomic fosmid library of local biofertilizers in previous report . The gene is renamed as gh43kk in this study.
The gene gh43kk, encoding a putative β-D-xylosidase was amplified by polymerase chain reaction (PCR) and successfully cloned and expressed in Escherichia coli. The expressed recombinant protein was purified by metal affinity chromatography. Its properties were initially verified by enzyme assay and thin layer chromatography (TLC).
The purified recombinant protein showed the highest catalytic activities at acidic pH 4 and 50°C toward beechwood xylan, followed by carboxymethylcellulose (CMC). TLC analysis indicated a release of xylose and glucose when xylan and CMC were treated with Gh43kk protein, respectively, whereas glucose and cellobiose were detected when avicel, cellulose and filter paper were used as substrates, suggesting its dual function as xylanase with cellulase activity. The enzyme indicated great stability in a temperature between 10 to 50 °C and a wide range of pH from 4 to 8. Enzyme activity of Gh43kk was enhanced in the presence of magnesium and manganese ions, while calcium ions, Ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) inhibited the enzyme activity.
These results suggest that Gh43kk could be a potential candidate for application in various bioconversion processes.