Mode of action of a β-(1→6)-glucanase from Penicillium multicolor

β-(1→6)-Glucanase from the culture filtrate of Penicillium multicolor LAM7153 was purified by ammonium sulfate precipitation, followed by cation-exchange and affinity chromatography using gentiotetraose (Gen4) as ligand. The hydrolytic mode of action of the purified protein on β-(1→6)-glucan (pustulan) was elucidated in real time during the reaction by HPAEC-PAD analysis. Gentiooligosaccharides (DP 2-9, Gen2-9), methyl β-gentiooligosides (DP 2-6, Gen2-6 β-OMe), and p-nitrophenyl β-gentiooligosides (DP 2-6, Gen2-6 β-p  NP) were used as substrates to provide analytical insight into how the cleavage of pustulan (DP¯ 320) is actually achieved by the enzyme. The enzyme was shown to completely hydrolyze pustulan in three steps as follows. In the initial stage, the enzyme quickly cleaved the glucan with a pattern resembling an endo  -hydrolase to produce a short-chain glucan (DP¯ 45) as an intermediate. In the midterm stage, the resulting short-chain glucan was further cleaved into two fractions corresponding to DP 15-7 and DP 2-4 with great regularity. In the final stage, the lower oligomers corresponding to DP 3 and DP 4 were very slowly hydrolyzed into glucose and gentiobiose (Gen2). As a result, the hydrolytic cooperation of both an endo-type and saccharifying-type reaction by a single enzyme, which plays a bifunctional role, led to complete hydrolysis of the glucan. Thus, β-(1→6)-glucanase varies its mode of action depending on the chain length derived from the glucan.

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► β-(1→6)-Glucanase from Penicillium multicolor was purified by affinity chromatography.
► The hydrolytic mechanism of the purified enzyme on β-(1→6)-glucan was examined.
► Synthetic substrates were used to analyze the hydrolytic mode of action of the enzyme.
► The action of β-(1→6)-glucanase on β-(1→6)-glucan was elucidated.