Purification and characterisation of recombinant xylanase from Roseithermus Sacchariphilus strain RA
Hemicellulases are important enzymes for applications in pulp and paper, animal feed, food and beverages, and biofuel industry. In the search for hemicellulases with promising properties, Roseithermus sacchariphilus strain RA could be a candidate source of enzymes. Strain RA is a Gram-negative halo-...
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| Format: | Thesis |
| Language: | English |
| Published: |
2019
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| Subjects: | |
| Online Access: | http://eprints.utm.my/id/eprint/101704/1/TeoSengChongMFS2019.pdf http://eprints.utm.my/id/eprint/101704/ http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:146168 |
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| Summary: | Hemicellulases are important enzymes for applications in pulp and paper, animal feed, food and beverages, and biofuel industry. In the search for hemicellulases with promising properties, Roseithermus sacchariphilus strain RA could be a candidate source of enzymes. Strain RA is a Gram-negative halo-thermophilic bacteria isolated from a hot spring in Langkawi, Malaysia. Complete genome analysis revealed the bacterium encodes 57 glycoside hydrolases (GHs) that are affiliated to 30 GH families. Hemicellulases from GH 3, 10, and 43 are present in this bacterium. The objective of this project is to analyse an endo-xylanase (EC 3.2.1.8, GH10), designated as XynRA2. The XynRA2 gene consisted of 2,439 nucleotides encoding a protein with 813 amino acids. The protein sequence has the highest identity (98.6%) to the xylanase from R. sacchariphilus MEBiC09517T. Three domains are present in XynRA2, namely (i) carbohydrate-binding module (CBM4_9), (ii) glycoside hydrolase family 10 (GH10) domain, and (iii) a C-terminal domain (CTD). In this study, two genes were cloned; native XynRA2 and its mutant, XynRA2ΔCBM, which lacked the CBM. The genes were cloned into the pET-28a(+) vector and expressed intracellularly in E. coli BL21 (DE3). The recombinant proteins carry a 6X-His-tag at both the termini were purified using Ni-NTA columns yielding proteins with weights of 89.5 kDa and 68.5 kDa, respectively. The enzyme activities were assessed using 3,5-dinitrosalicylic acid (DNS) assay against 1% (w/v) purified beechwood xylan. XynRA2 exhibited an optimum activity at 70°C and pH 8.5 in Tris-HCl buffer, with excellent activity (71%) in 5.0 M NaCl. On the other hand, XynRA2ΔCBM was most active at 70°C and pH 6.0 in acetate buffer although with a lower activity (54%) in 5.0 M NaCl. XynRA2 has a half-life of 45 min at 70°C but XynRA2ΔCBM has a shorter half-life (37 min). The specific activity and kcat of XynRA2 were 300 U·mg-1 and 24.8 s-1, whereas XynRA2ΔCBM were 160 U·mg-1 and 15.7 s-1, respectively. Metal ions such as Na+, K+, and Ca2+ enhanced XynRA2 activity. Both XynRA2 and XynRA2ΔCBM hydrolysed exclusively xylose-based substrate including beechwood xylan and oat-spelt xylan. However, the product yield of XynRA2 on oat-spelt xylan was higher than XynRA2ΔCBM. The major end-products of hydrolysis by the enzymes were xylobiose (X2) and xylotriose (X3). Altogether, the results suggested removal of CBM affected the stability and activity of XynRA2. In summary, XynRA2 is an alkaline xylanase capable of withstanding high temperature and high NaCl concentration. These properties implied XynRA2 favours applications that require a combination of alkaline pH, high temperature, and the saline environment. |
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