Biochemical characterization and gene cloning of a novel alkaline endo-1-4-glucanase from Bacillus subtilis DR8806

Abstract

In the pre­sent study, an endo-1-4-glu­canase was iso­lated from Bacil­lus sub­tilis DR8806. The en­zyme was pu­ri­fied to ho­mo­gene­ity via salt pre­cip­i­ta­tion and ion-ex­change chro­matog­ra­phy. SDS-PAGE analy­sis re­vealed a mol­e­c­u­lar mass of 52 kDa. Op­ti­mum pH of en­zyme was 9.5 and the en­zyme was sta­ble at pH range of 8.5–10.5. The op­ti­mum tem­per­a­ture of en­zyme was found to be 55 °C and it showed a re­mark­able sta­bil­ity at tem­per­a­tures be­tween 40 and 60 °C. The en­zyme ac­tiv­ity was stim­u­lated by Co2+, K+, Mg2+, Ca2+ and Na+ ions while Hg2+, Mn2+, Pb2+ and Zn2+ ions were found to in­hibit the en­zyme ac­tiv­ity. The en­zyme ac­tiv­ity was de­creased by in­creas­ing in con­cen­tra­tion of β-mer­cap­toethanol, EDTA, SDS, PMSF and Tri­ton X-100. Or­ganic sol­vents such as hexane, 2-propanol, ace­tone and ethanol (20% v/​v) stim­u­lated en­zyme ac­tiv­ity by 110%, 114%, 119% and 128%, re­spec­tively. Im­i­da­zolium-based ionic liq­uids (ILs) had in­hibitory ef­fects on endo-1-4-glu­canase ac­tiv­ity. Us­ing car­boxymethyl cel­lu­lose as sub­strate, ki­netic pa­ra­me­ters of Km ap­par­ent and Vmax were cal­cu­lated to be 1.49% (W/​V) and 66.66 μM min−1 mg−1, re­spec­tively. Endo-1-4-glu­canase cod­ing gene of B. sub­tilis DR8806 was iden­ti­fied by T/​A cloning. The com­pu­ta­tional mod­el­ing of endo-1-4-glu­canase showed that two Glu residues are at ac­tive site. Our re­sults sug­gest that the en­zyme has great of po­ten­tial ap­pli­ca­tions in hy­drolyz­ing cel­lu­losic sub­strates