title: Cloning, Expression, and Purification of Xylanase Gene from Bacillus licheniformis for Use in Saccharification of Plant Biomass.
abstract: The xylanase gene (xynA) of Bacillus licheniformis 9945A was cloned and expressed in Escherichia coli BL21(DE3) using pET-22b(+) as an expression vector. The recombinant xylanase enzyme was purified by ammonium sulfate precipitation, followed by single-step immobilized metal ion affinity chromatography with a 57.58-fold purification having 138.2 U/mg specific activity and recovery of 70.08 %. Molecular weight of the purified xylanase, 23 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable for up to 70 °C with a broad pH range of 4-9 pH units. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA, indicating that the xylanase was a metalloenzyme. However, an addition of 1-4 % Tween 80, β-mercaptoethanol, and DTT resulted in the increase of enzyme activity by 51, 52, and 5 %, respectively. Organic solvents with a concentration of 10-40 % slightly decreased the enzyme activity. The xylanase enzyme possesses the ability of bioconversion of plant biomasses like wheat straw, rice straw, and sugarcane bagasse. Among the different tested biomasses, the highest saccharification percentage was observed with 1 % sugarcane bagasse after 72 h of incubation at 50 °C with 20 units of enzyme. The results suggest that recombinant xylanase can be used in the bioconversion of natural biomasses into simple sugars which could be further used for the production of biofuel.
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
authors.forename: Asma
authors.initials: A
authors.lastname: Zafar
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan. nauman535@yahoo.com.
authors.forename: Muhammad Nauman
authors.initials: MN
authors.lastname: Aftab
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
authors.forename: Zia Ud
authors.initials: ZU
authors.lastname: Din
authors.affiliation: Department of Environmental Science, Lahore College for Women University, Lahore, Pakistan.
authors.forename: Saima
authors.initials: S
authors.lastname: Aftab
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
authors.forename: Irfana
authors.initials: I
authors.lastname: Iqbal
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
authors.forename: Anam
authors.initials: A
authors.lastname: Shahid
authors.affiliation: Department of Environmental Science, Lahore College for Women University, Lahore, Pakistan.
authors.forename: Arifa
authors.initials: A
authors.lastname: Tahir
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
authors.forename: Ikram Ul
authors.initials: IU
authors.lastname: Haq
mesh_terms: D001407:Bacillus / Q000235:genetics*; D018533:Biomass; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D031786:Saccharum / Q000378:metabolism*; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases
keywords: Biomass; Cloning; Fermentation; Saccharification; Xylanase