Cloning of the endomannanase from Scopulariopsis candida LMK008 and evaluation of its effect on the digestibility on animal feed.
Present within the biodiverse hypersaline environment are a wide variety of halotolerant filamentous fungi. Many of these phytopathogens are capable of hydrolysing plant cell wall polysaccharides such as hemicellulose which are comprised of mannans and heteromannans which are polymers of the mannose sugars. Endoacting hydrolytic enzymes such as endo-β-1,4-mannanases are secreted into the extracellular environment and are involved in the catalysis of the random hydrolysis of β-1,4-mannosidic linkages within the backbone of mannan, galactomannan, glucomannan, and galactoglucomannan. Poultry are monogastric animals that are unable to efficiently digest high-fibre and mannan rich feeds such as soybean meals and this results in decreased or depressed animal performance. The use of feeds supplemented with β-mannanases has been shown to enhance the feeding value of mannan-based meals. In the current study, the degradation of β-mannan polysaccharides present in poultry feed by halotolerant Scopulariopsis candida LMK008 β-mannanase was investigated. SDS-PAGE, Native-PAGE in conjunction with zymogram analysis was used to assess the molecular weight of the endomannanases. At least three isozymes were detected: two of 56 kDa (pI 3.5 and 6.7) and one of 28 kDa. Anion exchange chromatography was used to purify the 28 kDa isozyme. Three mannan-based substrates, viz., locust bean gum, guar gum and soybean flour, were used to evaluate the hydrolysis capability of the crude as well as the purified β-mannanase via the release of reducing sugars and was detected using the DNS assay. The β-mannanase exhibited low activity with pure guar gum but high activity with locust bean gum galactomannan and soybean flour mannan. The hydrolysis activities of the crude and purified enzyme were then tested further on mannan-based soybean meals. In general it was found that more reducing sugars were released from the grower feed than the starter and layer feeds. Another common hydrolysis pattern observed in all feed types was that after prolonged incubation of 24 h there was a decrease in the amount of reducing sugars released which could be attributed to the presence of naturally-occurring microorganisms in the feed sample which metabolised the simple sugars resulting from the enzymatic hydrolysis of the mannan components in the feed samples. This was confirmed by standard plate count assays. The results obtained are encouraging and the purified β-mannanase could be applied as an industrial feed additive within the animal feed industry, however, further testing of the enzyme in situ is needed in order to prove its applicability. The cloning of the endomannanase has to date proven unsuccessful despite numerous techniques being employed and further research is also needed to accomplish this task.