Production, purification, and characterization of a thermo-alkali stable and metal-tolerant carboxymethylcellulase from newly isolated Bacillus methylotrophicus Y37

Abstract

A carboxymethylcellulose (CMC)-degrading bacterium was isolated from soil, identified as Bacillus methylotrophicus according to the physiological properties and analyses of 16S rRNA and a partial sequence of the gyrase A (gyrA) gene, and named as B. methylotrophicus Y37. The CMCase enzyme was purified to homogeneity by 20.4-fold with 21.73% recovery using single-step hydrophobic interaction chromatography and biochemically characterized. CMCase showed a molecular weight of approximately 50 kDa as determined by SDS-PAGE. The activity profile of the CMCase enzyme exhibited optimum activity at 45 $^{\circ}$C and pH 5.0. The activity was highly stable at alkaline pH levels. More than 90% of the original CMCase activity was maintained at relatively high temperatures ranging from 55 to 65 $^{\circ}$C. The enzyme activity was induced by Ca$^{2+}$, Cd$^{2+}$, Co$^{2+}$, K$^{+}$, Mg$^{2+}$, and Na$^{1+}$, whereas it was strongly inhibited by phenylmethanesulfonyl fluoride and iodoacetic acid. The enzyme tolerated Hg$^{2+}$ up to 10 mM and presented hydrolytic activity towards glucan, filter paper, laminarin, and CMC but not $o$-nitrophenyl $\beta $-D-galactopyranoside. Kinetic analysis of the purified enzyme showed K$_{m}$ and V$_{max}$ values of 0.19 mg mL$^{-1}$ and 7.46 U mL$^{-1}$, respectively. The biochemical properties of this CMCase make the enzyme a good candidate for many industrial applications.