Abstract:
Oligo-1,6-glucosidase (EC 3.2.1.10) shows a good hydrolytic activity for isomaltose existed in the residual sugar of lactic acid fermentation broth. On the basis of previous work and a validation by BRENDA database search, five possible heat-resistant and acid-resistant oligo-1, 6-glucosidase enzyme genes (
Bacillus coagulans ATCC 7050: malL BF29_2011(BC1) and malL BF29_2004(BC2);
Bacillus subtilis 168: yvdL BSU34560(BS1) and yugT BSU31290(BS2);
Bacillus thermoglucosidasius KP 1006: malL(KP)) were selected for recombinant expression in
E. coli. The expressed oligo-1,6-glucosidases were purified and their enzymatic properties were tested. The optimal working pH for BC1, BC2 and KP was determined to be 5.0, and that for BS1 and BS2 was 6.0. The working temperature of the oligo-1,6-glucosidases was in an order of KP (60 ℃), BC1 (56 ℃), BC2 (56 ℃), BS1 (43 ℃), BS2 (43 ℃). In addition, Mg
2+ and NH
4 + could enhance the enzymatic activity of BC1, while Co
2+, K
+, NH
4 + could enhance the enzymatic activity of KP. However, these ions showed minimal effect on the activity of BC2, BS1 and BS2. Zn
2+, Cu
2+ and Ni
+ showed inhibitory effects on the activity of the five oligo-1,6-glucosidases. Using
p-nitrobenzene-
α-
D-glucoside as the substrate, the catalytic efficiency of the enzymes was in an order of KP> BC1> BS1> BS2> BC2. The substrate specificity of the oligo-1,6-glucosidases was tested for maltose, isomaltose, sucrose, lactose, trehalose and the soluble amylose isolated from the residual sugar in lactic acid fermentation broth. The most suitable substrate for BC1, BC2, KP and BS1 was isomaltose, and that for BS2 was maltose.