A highly active endo-ß-1,4-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of Eisenia fetida.

A xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum, guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManK, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50°C and pH 7.0, ManK exhibited extraordinary high specific activities of 7109 IU/mg and 5158 IU/mg toward locust bean gum and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. Thin layer chromatography revealed that the enzyme degraded locust bean gum to mannobiose and mannotetraose. No detectable amount of mannose was produced from hydrolytic reactions with the substrates. ManK strongly attached to Avicel, ß-cyclodextrin, lignin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManK suggest that it is a unique endo-ß-1,4-mannanase without additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.

Cloning and characterization of a modular GH5 ß-1,4-mannanase with high specific activity from the fibrolytic bacterium Cellulosimicrobium sp. strain HY-13.

The gene (1272-bp) encoding a ß-1,4-mannanase from a gut bacterium of Eisenia fetida, Cellulosimicrobium sp. strain HY-13 was cloned and expressed in Escherichia coli. The recombinant ß-1,4-mannanase (rManH) was approximately 44.0 kDa and has a catalytic GH5 domain that is 65% identical to that of the Micromonospora sp. ß-1,4-mannosidase. The enzyme exhibited the highest catalytic activity toward mannans at 50 °C and pH 6.0. rManH displayed a high specific activity of 14,711 and 8498 IU mg⁻¹ towards ivory nut mannan and locust bean gum, respectively; however it could not degrade the structurally unrelated polysaccharides, mannobiose, or p-nitrophenyl sugar derivatives. rManH was strongly bound to ivory nut mannan, Avicel, chitosan, and chitin but did not attach to curdlan, insoluble oat spelt xylan, lignin, or poly(3-hydroxybutyrate). The superior biocatalytic properties of rManH suggest that the enzyme can be exploited as an effective additive in the animal feed industry.

Kinetic study on the pretreatment and enzymatic saccharification of rice hull for the production of fermentable sugars.

The production of fermentable sugars from rice hull was studied by dilute acid pretreatment and enzymatic saccharification. Rice hull (15%, w/v) was pretreated by 1% (v/v) sulfuric acid at high temperature (120 approximately 160 degrees C) for 15, 30, 45, and 60 min, respectively. The maximum sugar concentration from rice hull in the prehydrolysate was obtained at 140 degrees C for 30 min, but the enzymatic saccharification yield from the corresponding pretreated rice hull is not high. To another aspect, the maximum enzymatic saccharification yield was achieved at 160 degrees C for 60 min, while the recovery of fermentable sugars was the poorest. To take account of fermentable sugars from pretreatment and enzymatic saccharification, the maximum yield of sugars was obtained only when rice hull was treated at 140 degrees C for 30 min. Under this condition, 72.5% (w/w) of all sugars generated from the raw material can be recovered. The kinetic study on the enzymatic saccharification of dilute acid pretreated rice hull was also performed in this work by a modified Michaelis-Menten model and a diffusion-limited model. After calculation by a linear and a non-linear regression analysis, both models showed good relation with the experimental results.