Purification and Characterization of Alkaline Xylanase Secreted from <i>Paenibacillus macquariensis</i>

Abstract

An alkaline xylanase secreted by Paenibacillus macquariensis RC 1819 has been purified using ammonium sulfate fractionation, ion exchange chromatography using DEAE-cellulose and gel filtration chromatography over Sephadex G-200 and Sephadex G-100. The purified enzyme had the specific activity, 25.2 units/mg protein with birchwood xylan as a substrate. The purified enzyme showed a single protein band over sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme has been found to be 31,000 ± 2000 as determined by using Sephadex G-200 gel filtration chromatography. The subunit molecular weight has also been found to be ~31,000 as determined using SDS-PAGE indicating monomeric enzyme. The enzyme showed optimum activity at pH 8.6 and temperature, 50˚C. The Michaelis constant (Km) of the enzyme for birchwood xylan was 2.2 mg/ml as determined using velocity saturation plot. The metal ions viz. Co +2 and Mn +2 stimulated xylanase enzyme activity whereas Hg +2 inhibited the enzyme activity. AiM 35 Xylanase-Metal Ions Binding StudiesXylanase aliquots (0.5 ml) were incubated at the room temperature (25˚C) with 1 mM metal ions viz. Ca +2 , Mg +2 , Hg +2 , Fe +2 , Cu +2 , Mn +2 , Co +2 , Zn +2 , As +3 , Mo +2 for 2 hours. Thereafter, enzyme activity was determined using 0.1 ml of the incubated enzyme with 0.9 ml of 1% birchwood xylan dissolved in 50 mM glycine-NaOH buffer, pH 8.6 and incubated at 50˚C for 15 min. Km DeterminationBirchwood xylan hydrolysis rates were determined at all substrate concentrations ranging from 0.5% to 8% in 50 mM glycine-NaOH buffer at pH 8.6.[6] C.

Purification, characterization and thermostability improvement of xylanase from Bacillus amyloliquefaciens and its application in pre-bleaching of kraft pulp

“…There is wide variation in the specific activity of xylanase from different bacterial species. The specific activity of purified xylanase from B. amyloliquefaciens strain SK-3 in the present study was higher than the previous reports (Sharma et al 2013 ; Bajaj and Manhas 2012 ; Mishra and Thakur 2010 ). Kamble and Jadhav ( 2012 ) reported xylanase from Bacillus arseniciselenatis strain DSM-15340 having high specific activity of 299.25 IU/mg protein.…”

Response surface optimization of xylanase production by indigenous thermoalkalophillic Bacillus sp.

“…The supernatant obtained was used as a source of crude xylanase enzyme. The quantitative estimation of xylanase activity was done with some modifications according to the procedure of Sharma et al (2013). A reaction mixture was prepared containing 0.5 ml supernatant and 0.5 ml of 1% Birchwood xylan (HiMedia, India) solution prepared in 50 mM Glycine-NaOH buffer (pH 9.2).…”

Production of thermostable multiple enzymes from Bacillus amyloliquefaciens KUB29

“…Resultado similar foi reportado por Sanjivkumar M e colaboradores (2017) para uma xilanase produzida por Streptomyces, cuja presença de Ca 2+ e Fe 2+ , bem como de Co 2+ , Mn 2+ e Hg 2+ aumentaram sua atividade hidrolítica. Todavia, a presença de íons pode não influenciar a atividade de xilanases, como observado para Paenibacillus macquariensis RC 1819, com os íons Ca 2+ e Fe 2+(Sharma M, Mehta S, Kumar A, 2013); ou pode ainda inibir sua atividade, como observado para Bacillus sp., com Hg 2+ , Cu 2+ , Fe 2+…”