Characterisation of Klebsiella pneumoniae Xylanase and Increment of Its Activity in Heterologous Expression System

Abstract

A xylanase DNA sequence with a total length of 642 bp was previously isolated from a xylanolytic Klebsiellapneumoniae. Xylanase gene primers were designed with the addition of BamH1 and EcoR1 restriction enzymesites in order get a full xylanase gene that is in-frame with pSTAG expression vector. The isolated xylanasegene was amplified using the designed primers through PCR, then cloned and expressed in E. coli BL21 (DE3).In-silico characterization showed that the recombinant xylanase has a molecular weight of 23.9 kDa and a pI of9.32. The signal peptide cleavage site for the recombinant xylanase was predicted to be between residues 61and 62. The activity of the crude recombinant xylanase was 2.015 U/mL, which was higher than the crudenative xylanase activity, with maximum at 0.642 U/mL. Staining of the birchwood xylan agar plate with Congored showed a clearing zone around E. coli BL21 (DE3) colonies with recombinant pSTAG plasmid evenwithout being induced with IPTG. This implied leaky expression of the E. coli BL21 (DE3) secretion system,which recognized the signal sequence of the recombinant xylanase, and proceeded to cleave and secreted outthe mature protein into the culture medium. MALDI-TOF analysis of a 20 kDa protein present in the culturemedium confirmed that the recombinant xylanase had been secreted into the culture medium.

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“…As described in Hussain et al (2011), insilico characterization of the native xylanase from K. pneumoniae showed 99.5% identity to the isolated xylanase from B. subtilis (Jalal et al, 2009). Jalal et al (2011) reported that in heterologous expression, the E. coli secretion system had recognized the signal sequence of the native xylanase and proceeded to cleave it, then secreted out the mature protein into the culture medium.…”

“…With the S-tag removed, the recombinant xylanase is predicted to have 219 amino acids with a molecular weight of 23.9 kDa and a pI of 9.32. The recombinant xylanase was predicted to have a signal peptide cleavage site between amino acid residues 61 and 62 (sequence not shown) due to presence of the S-tag peptides, while the native xylanase signal peptide cleavage site was predicted to be between residues 28 and 29 (Hussain et al, 2011). The signal peptide was left intact to direct the xylanase into the secretory pathway of E. coli.…”

“…Plasmids used in this experiment were pGEM-T Easy Vector (Promega) and pSTAG expression vector (Edwards et al, 1999). The xylanase gene had been cloned into the pGEM-T Easy Vector in previous work by Hussain et al (2011).…”

“…The primers were designed based on the isolated xylanase gene from K. pneumoniae by Hussain et al (2011). The designed primers were 5´-GCG GAT CCA TGT TTA AGT TTA AAA AGA ATT TC-3´as a forward primer and 5´-GCG AAT TCT ATT ACC ACA CTG TTA CGT TAG-3´ as a reverse primer.…”

“…Heterologous expression of a gene encoding β-1,4-endoxylanase in E. coli BL21 (DE3) was performed in this study. The xylanase gene was isolated from xylanolytic Klebsiella pneumoniae, a bacterium that had been locally isolated from soil from a sago plantation by Hussain et al (2011). Here, we report on the expression of a xylanase gene from K. pneumoniae in E. coli BL21 (DE3) and to determine the characteristics of the recombinant xylanase expressed.…”