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Expression of a thermostable xylanase gene from Bacillus coagulans ST-6 in Lactococcus lactis

Raha AR, Chang LY, Sipat A, Yusoff K, Haryanti T

Lett Appl Microbiol, 2006 Mar;42(3):210-4.
PMID: 16478506

The aim of the study is to evaluate whether xylanase can be used as a potential reporter gene for cloning and expression studies in Lactococcus.

Matched MeSH terms: Endo-1,4-beta Xylanases/genetics
Secretion of recombinant xylanase in Lactococcus lactis using signal peptides Usp45 and Spk1

Roslan AM, Mustafa Kamil A, Chandran C, Song AA, Yusoff K, Abdul Rahim R

Biotechnol Lett, 2020 Sep;42(9):1727-1733.
PMID: 32335791 DOI: 10.1007/s10529-020-02894-1

OBJECTIVE: The effect of two signal peptides, namely Usp45 and Spk1 on the secretion of xylanase in Lactococcus lactis was analysed.
RESULTS: Xylanase was successfully expressed in Lactococcus lactis. Recombinant xylanase fused to either signal peptide Usp45 or Spk1 showed halo zone on Remazol Brilliant Blue-Xylan plates. This indicated that the xylanase was successfully secreted from the cell. The culture supernatants of strains secreting the xylanase with help of the Spk1 and Usp45 signal peptides contained 49.7 U/ml and 34.4 U/ml of xylanase activity, respectively.
CONCLUSION: Although Usp45 is the most commonly used signal peptide when secreting heterologous proteins in Lactococcus lactis, this study shows that Spk1 isolated from Pediococcus pentosaceus was superior to Usp45 in regard to xylanase protein secretion.

Matched MeSH terms: Endo-1,4-beta Xylanases/genetics
Fulltext Characterizing a Halo-Tolerant GH10 Xylanase from Roseithermus sacchariphilus Strain RA and Its CBM-Truncated Variant

Teo SC, Liew KJ, Shamsir MS, Chong CS, Bruce NC, Chan KG, et al.

Int J Mol Sci, 2019 May 09;20(9).
PMID: 31075847 DOI: 10.3390/ijms20092284

A halo-thermophilic bacterium, Roseithermus sacchariphilus strain RA (previously known as Rhodothermaceae bacterium RA), was isolated from a hot spring in Langkawi, Malaysia. A complete genome analysis showed that the bacterium harbors 57 glycoside hydrolases (GHs), including a multi-domain xylanase (XynRA2). The full-length XynRA2 of 813 amino acids comprises a family 4_9 carbohydrate-binding module (CBM4_9), a family 10 glycoside hydrolase catalytic domain (GH10), and a C-terminal domain (CTD) for type IX secretion system (T9SS). This study aims to describe the biochemical properties of XynRA2 and the effects of CBM truncation on this xylanase. XynRA2 and its CBM-truncated variant (XynRA2ΔCBM) was expressed, purified, and characterized. The purified XynRA2 and XynRA2ΔCBM had an identical optimum temperature at 70 °C, but different optimum pHs of 8.5 and 6.0 respectively. Furthermore, XynRA2 retained 94% and 71% of activity at 4.0 M and 5.0 M NaCl respectively, whereas XynRA2ΔCBM showed a lower activity (79% and 54%). XynRA2 exhibited a turnover rate (kcat) of 24.8 s-1, but this was reduced by 40% for XynRA2ΔCBM. Both the xylanases hydrolyzed beechwood xylan predominantly into xylobiose, and oat-spelt xylan into a mixture of xylo-oligosaccharides (XOs). Collectively, this work suggested CBM4_9 of XynRA2 has a role in enzyme performance.

Matched MeSH terms: Endo-1,4-beta Xylanases/genetics
Biodegradation of fibrillated oil palm trunk fiber by a novel thermophilic, anaerobic, xylanolytic bacterium Caldicoprobacter sp. CL-2 isolated from compost

Widyasti E, Shikata A, Hashim R, Sulaiman O, Sudesh K, Wahjono E, et al.

Enzyme Microb Technol, 2018 Apr;111:21-28.
PMID: 29421033 DOI: 10.1016/j.enzmictec.2017.12.009

Oil palm trunk (OPT) is one of the most promising lignocellulosic bioresources. To develop effective biodegradation, thermophilic, anaerobic microorganisms were screened from bovine manure compost using fibrillated OPT (f-OPT) pretreated by wet disk milling as the substrate. One thermophilic, anaerobic bacterium, strain CL-2, whose 16S rDNA gene has 98.6% sequence identity with that of Caldicoprobacter faecale DSM 20678T, exhibited high degradation activity (32.7% reduction in total dry solids of f-OPT). Strain CL-2 did not use cellulose as a carbon source, but used hemicelluloses such as xylan, arabinoxylan, starch and pectin at 70 °C. Phylogenetic and morphologic analyses and the polysaccharide use suggest that CL-2 may be classified as a novel species of Caldicoprobacter, named Caldicoprobacter sp. CL-2. To characterize enzymatic activities of CL-2, extracellular enzymes were prepared from culture broth using beechwood xylan as the carbon source. The extracellular enzymes showed high xylanase activity, but low cellulase activity, suggesting that f-OPT degradation may depend on xylanase activity. To understand the xylanase system of CL-2, a major xylanase was cloned and characterized. The xylanase (CalXyn11A) had a modular structure consisting of a glycoside hydrolase (GH) family-11 domain and a family 36 carbohydrate-binding module. CalXyn11A did not show f-OPT degradation activity, but a strong synergistic effect was observed when CalXyn11A was added to the extracellular enzyme preparation. These results indicate that, rather than working alone, CalXyn11A has an important role in enhancing total lignocellulose degradation activity by cooperation with other GHs.

Matched MeSH terms: Endo-1,4-beta Xylanases/genetics
Heterologous expression, purification and biochemical characterization of a new endo-1,4-β-xylanase from Rhodothermaceae bacterium RA

Liew KJ, Ngooi CY, Shamsir MS, Sani RK, Chong CS, Goh KM

Protein Expr Purif, 2019 12;164:105464.
PMID: 31376486 DOI: 10.1016/j.pep.2019.105464

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.

Matched MeSH terms: Endo-1,4-beta Xylanases/genetics*