High level expression of β-mannanase (RmMan5A) in Pichia pastoris for partially hydrolyzed guar gum production.

Abstract

Partially hydrolyzed guar gum (PHGG), an important supplemental dietary fiber, has been used as food ingredient in many industries. In this study, a novel β-mannanase gene (RmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris and subjected for PHGG production. Enzyme activity of fermentation supernatant reached 85,200UmL-1 after 168h high cell density fermentation. The purified RmMan5A exhibited the highest enzyme activity at pH 7.0 and 65°C. RmMan5A was then employed for guar gum hydrolysis and PHGG obtained demonstrated a weight-average molecular weight (Mw) of 2.5×104Da. Total dietary fiber accounted 90.6% of PHGG and 24.9% (w/w) of PHGG were identified as manno-oligosaccharides with degree of polymerization<7. PHGG was further fractionated (F1-F4) by gradual ethanol precipitation. PHGG F1 with an Mw value of 3.6×104Da and a mannose/galactose (M/G) ratio of 1.47 was precipitated initially, followed by PHGG F2 and F3 which showed lower Mw and higher M/G ratio. According to the structure analysis, the distribution of α-d-galactose of PHGG F1 was compact and regular, and that of other fractions was more random. A suitable β-mannanase for PHGG production and some useful information of PHGG are provided in this paper.