Molecular cloning and biochemical characterization of an endo-β-mannanase gene from soybean for soybean meal improvement.

Soybean meal is the most commonly used protein source in animal feeds. Among the undesirable attributes of soybean meal is the high level of β-mannan, which was determined to be detrimental to the growth performance of animals. β-Mannan is a type of hemicellulose in the plant cell wall and can be hydrolyzed by endo-β-mannanase. The goal of this study is to isolate and characterize an endo-β-mannanase gene from soybean that can be used for genetic improvement of soybean meal. From the sequenced soybean genome, 21 putative endo-β-mannanase genes were identified. On the basis of their relatedness to known functional plant endo-β-mannanases, four soybean endo-β-mannanase genes (GmMAN1 to GmMAN4) were chosen for experimental analysis. GmMAN1 and GmMAN4 showed expression in the soybean tissue examined, and their cDNAs without the sequences for signal peptide were cloned and expressed in Escherichia coli to produce recombinant enzymes. Only GmMAN1 showed endo-β-mannanase hydrolase activity. Further gene expression analysis showed that GmMAN1 is specifically expressed in cotyledons of seedlings, suggesting a role of GmMAN1 in degrading mannan-rich food reserves during soybean seedling establishment. Purified recombinant GmMAN1 exhibited an apparent K(m) value of 34.9 mg/mL. The catalytic efficiency (k(cat)/K(m)) of GmMAN1 was determined to be 0.7 mL/(mg·s). GmMAN1 was also shown to be active in hydrolyzing the β-mannan-rich cell wall of soybean seeds.