The production, purification and characterization of endo-1,4-ß-mannanase from newly isolated strains from Scopulariopsis candida

Abstract

English: Mannan polysaccharides occur in hemicellulose fraction of the plant cell walls. The hydrolysis of these polymers involves the action of enzymes such as β-mannanase, β-mannosidase and α- galactosidase which are produced by both fungi and bacteria. The current study reports on the production of β-mannanase, β-mannosidase and α-galactosidase by newly isolated Scopulariopsis candida strains LMK004 and LMK008. The effect of medium composition and carbon source on growth and enzyme production was evaluated in a liquid culture. A combination of Vogel’s medium and locust bean gum was found to stimulate growth and increase β-mannanase production. Optimal β-mannanase production of 7800 nkat/g biomass for LMK004 and 13300 nkat/g biomass for LMK008 was achieved in media containing 10% NaCl, 1X Vogel’s medium, 1% yeast extract and 1% locust bean gum. Both strains secreted trace amounts (less than 1 nkat/ml) of β-mannosidase and α-galactosidase indicating that these enzymes may be retained intracellularly. Native-PAGE and SDS-PAGE were used together with the zymogram to assess purity and to estimate the molecular weight of the proteins. The molecular weight of LMK004 β-mannanase was estimated to be ≈41 kDa whereas that of LMK008 β-mannanase could not be determined due to excessive loss of protein material during dialysis. The β-mannanase from LMK004 was most active at pH 5 and 50 °C, and retained ≥ 80% of its activity at pH 5 – 6.5 after 24 hrs of incubation at 4 °C. In contrast, the LMK008 β- mannanase retained ≥ 60% activity between pH 6 – 7. Both enzymes remained stable for 3 hrs at temperature between 30 °C and 40 °C, and showed loss of activity at higher temperatures. The two enzymes displayed different degrees of halotolerance. The LMK008 β-mannanase tolerated high NaCl concentrations with 60% activity remaining after incubation for 2 hrs at 20% NaCl, whereas the LMK004 β-mannanase was only active between 0% - 10% NaCl. It is clear from the current study that the two strains of S. candida produce distinct β-mannanases which may be useful candidates in low water activity reactions.

Afrikaans: Mannaanpolisakkariede kom voor in die hemisellulose fraksie van plantselwande. Ensieme soos β-mannanase, β-mannosidase en α-galaktosidase, geproduseer deur beide fungi en bakterië, is betrokke by die hidrolise van hierdie polimere. Hierdie studie handel oor die produksie van β- mannanase, β-mannosidase en α-galaktosidase deur nuwe isolate van Scopulariopsis candida, stamme LMK004 en LMK008. Die invloed van mediumsamestelling en koolstofbron op groei en ensiemproduksie is geëvalueer in n vloeibare kultuur. Daar is gevind dat ‘n kombinasie van Vogel se medium en locust bean gum groei stimuleer en produksie van β-mannanase verhoog. Optimale b-mannanase produksie van 7800 nkat/g biomassa vir LMK004 en 13300 nkat/g biomassa vir LMK008 is verkry in media bestaande uit 10% NaCl, 1X Vogel se medium, 1% gisekstrak en 1% locust bean gum. Beide stamme het spoorhoeveelhede (minder as 1 nkat/ml) β- mannosidase en α-galaktosidase uitgeskei, wat daarop mag dui dat hierdie ensieme intrasellulêr behou word. “Native”-PAGE en SDS-PAGE is saam met zymogram gebruik om die suiwerheid en molekulêre massas van die proteïene te bepaal. Die molekulêre massa van die LMK004 β- mannanase is ≈41 kDa terwyl dié van die LMK008 β-mannanase nie bepaal kon word nie van weë n uitermatige verlies van proteïen gedurende dialise. Die LMK004 β-mannanase was mees aktief by pH 5 en 50 oC en het ≥ 80% aktiwiteit behou by pH 5 – 6.5 na 24 uur inkubasie by 4 oC. Inteenstelling daarmee, het die LMK008 β-mannanase ≥ 60% aktiwiteit behou by pH 6 - 7. Beide ensieme was stabiel vir 3 ure by temperature tussen 30 oC en 40 oC en het n verlies aan aktiwiteit getoon by hoer temperature. Die twee ensieme het verskilende grade van halotoleransie getoon. Die LMK008 β-mannanase kon hoer NaCl konsentrasies verduur met 60% aktiwiteit na inkubasie vir 2 ure by 20% NaCl, waa die LMK004 β-mannanase slegs aktief was tussen 0% - 10% NaCl. Uit hierdie studie blyk dit duidelik dat die twee stamme van S. candida erskillende β-mannanases produseer wat nuttig mag wees in reaksies by lae wateraktiwiteite.