A novel multifunctional starch debranching enzyme from Myxococcus sp. V11
摘要
MDBE, a novel multifunctional isoamylase derived from Myxococcus sp. V11, was heterologously expressed in the Escherichia coli BL21 (DE3). Phylogenetic analysis indicated that MDBE is a multifunctional starch debranching enzyme belonging to the glycoside hydrolase GH13_11 family. Functional identification revealed that MDBE exhibits bifunctional hydrolytic activity, enabling it to hydrolyze both α-1,4 and α-1,6 glycosidic bonds, thereby allowing the degradation of both amylose and amylopectin. Moreover, MDBE can transfer α-1,4-glucan oligosaccharides between molecules through a newly discovered 4-α-D-glucanotransferase activity, resulting in the production of serial oligosaccharides. Under conditions devoid of any added ions, the specific enzyme activity of MDBE was determined to be 250.5 μmol·min⁻1·mg⁻1 and 209. μmol·min⁻1·mg⁻1 when using corn starch and amylose as substrates, respectively. Application of the Michaelis–Menten equation allowed the calculation of the Vmax, Km, and kcat of MDBE for amylose, yielding values of 3171.8 μmol·min⁻1·mg⁻1, 17.7 mM, and 18.9 min⁻1, respectively. The hydrolysis rates of MDBE towards corn starch, amylose, and amylopectin were 50%, 48%, and 44%, respectively. In combined enzyme hydrolysis of amylopectin, the yields of G1 and G2 increased by 1.1-fold and 1.8-fold, respectively, compared to the application of maltogenic amylase (BMAL) alone. Our results suggest that MDBE has potential for future applications in starch hydrolysis and bioconversion, pending further optimization of its catalytic efficiency and stability under industrial conditions.