Abstract:
The aims of this study are to produce and characterize mannanase from the selected Thai Aureobasidium strain and use the enzyme for hydrolysis of spent coffee ground extract (SCGE) to prepare mannooligosaccharide (MOs). The optimum condition for SCGE extraction by hot water was 1:30 solid to liquid ratio with 60 min incubation time at 121°C 15 lbs per in2. The maximum yield was 9.5 g/100 g substrate. The second dominant composition of SCGE was hemicellulose at 28 %. Aureobasidium pullulans NRRL 58524 was selected as the best mannanase producer with 8.07 U/mL. The mannanase production medium was optimized by using SCGE and ammonium sulphate without L-asparagine at a C/N ratio of 1.15. The highest enzyme yield was obtained after 72 hours. The optimal condition for mannanase was at 55 °C in 50 mM citrate buffer (pH 4.0). The enzyme was highly thermostable at 50 and 55 °C, retaining most of its activity after 6 h incubation. It was also stable in 50 mM citrate buffer (pH 4.0) with more than 80 % of initial activity remained after 6 h. The mannanase was highly specific to glucomannan and galactomannan, and its activity was enhanced by Cu2+, Ca2+ and Mg2+. The enzyme was inhibited by mannose and galactose at 40 mM and 30 mM, respectively. The optimal MOs preparation was by using the enzyme at 66.7 U/g substrate and incubating for 5 days 9 h and 36 min which yielded the maximal reducing sugars at 0.66 g/g substrate. The produced SCG MOs failed to enhance the growth of all probiotic bacteria tested.
