BcGlu13A（BcGlu6A）

Ex-Glu0005

(EC.3.2.1.10) oligo-α-1,6-Glucosidase

CAZy Family: GH13

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~66kDa)

Figure 1. Electrophoresis analysis of BcGlu13A. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before IPTG induction; lane 2, culture lysate of after IPTG induction; lane 3, BcGlu13A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

147 U/mg protein (on pNP-α-glu) at pH7.0 and 30°C

One Unit of pNP-α-glu activity is defined as the amount of enzyme required to release 1 μmol of glucose per minute from pNP-α-glu (5 mM) in phosphate buffer(10 mM) pH7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of BcGlu13A on different substrates^a.

^aReactions were performed with 5mM substrate, pH7.0, at 30°C for 10 min.

^bAbsorption caused by releasedp-nitrophenol was measured at 405 nm. The relative activity onpNPGlu (147 mol/min/mg) was taken as 100%.

^cThe data are reported as means±standard errors from the mean for three independent experiments.

4.PHYSICOCHEMICAL PROPERTIES

Temperature Optima:40°C

Temperature Stability: <40°C

Figure2.Effect of temperature on the activities (A) and stabilities (B) of oligo-1,6-glucosidasesBcGlu13A(○) and from B. cereus ATCC7064 (●). The activity was measured as in Materials and Methods, except that the reaction was carried out at the temperatures ranging over 2-60°C. The reaction mixture contained 0.5pg (0.15 U) of each enzyme. The activity observed at 35°C was expressed as 100%. The enzyme (0.85 pg, 0.27 U) was incubated in 50 μl buffer A for 10 min at the indicated temperatures, mixed with 450 μl buffer A at the end of the incubation, then assayed for remaining activity. The activity observed after the treatment at 35°C was defined as 100%.

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (10 mM) pH7.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1] KWatanabe, K Kitamura, H Iha, et al.Primary structure of the oligo-l,6-glucosidase of BaciZZus cereus ATCC7064 deduced from the nucleotide sequence of the cloned gene. Eur. J. Biochem. 1990,192:609-620.