Expression and properties of polyvinyl alcohol dehydrogenase from Bacillus cereus

Abstract:

[Background] Via oxydehydrogenation of polyvinyl alcohol (PVA), polyvinyl alcohol dehydrogenase (PVADH) plays an important role in the biodegradation of PVA.[Objective] pvadh gene was extracted from the PVA-degrading Bacillus cereus sp. DG01 for the expression of PVADH in Pichia pastoris and specificity of the enzyme for degrading different PVA species was explored. The findings are expected to guide the application of PVADH in PVA degradation.[Methods] The 1 965 bp pvadh as obtained by reverse-transcription PCR amplification. pPIC9K-cpvadh plasmid was constructed for expression in P. pastoris GS115. Methanol was employed to induce the expression of the protein which was then isolated and purified. The enzymatic properties and degradation specificity were investigated. [Results] The activity of crude PVADH solution yielded under optimal fermentation conditions reached 54.55 U/mL. The purified PVADH had the specific activity of 173.42 U/mg, molecular weight of 67.1 kDa, and isoelectric point of 6.06, and the optimum temperature and pH for PVADH were 41 ℃ and pH 7.5, respectively. The half-life of PVADH was more than 4 h at 27-32 ℃ and pH 7.0-8.0, and 1 mmol/L Ca²⁺ can activate the enzyme. K_mvalues of PVADH for the three substrates PVA1799, PVA1788, and PVA2488, were 1.49 mg/mL, 1.17 mg/mL, and 1.21 mg/mL, separately. [Conclusion] Heterogeneous expression in P. pastoris is a simple method to obtain PVADH and the purification features ease of implementation. The yielded PVADH has stable enzymatic properties and high efficiency in degrading the PVA with low alcoholysis degree.

Key words:polyvinyl alcohol;polyvinyl alcohol dehydrogenase;heterologous expression;enzymatic properties