CcGlu1C

Ex-Glu0013

(EC.3.2.1.192)exo-β-Glucosidase

CAZy Family: GH1

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~56 kDa)

Figure 1. Electrophoresis analysis of CcGlu1C. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before IPTG induction; lane 2, culture lysate after IPTG induction; lane 3, CcGlu1C purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

8.4 U/mg protein (on pNP-β-glu) at pH 6.0 and 30^oC

One Unit of pNP-β-glu activity is defined as the amount of enzyme required to release 1 μmol of glucose per minute from pNP-β-glu (5 mM) in phosphate buffer (50 mM) pH 6.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of CcGlu1C on different substrates^a.

^aReactions were performed with 1 mM (p-nitrophenyl glycosides and disaccharides) or 5 mg/ml (polysaccharides) substrate, pH 6.0, at 30^oC for 10 min.

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

Fig. 2. Analysis of ginsenosidase activity of CcBgl1C by TLC. S1, standards of Rh2, Rg3, Rd, Rc and Rb1; S2, standards of CK, Gyp LXXV, Gyp XVII and Rb2; 1, biotransformation substrate Rb1; 2, transformed products of Rb1；3, biotransformation substrate Rb2; 4, transformed products of Rb2; 5, biotransformation substrate Rb3; 6, transformed products of Rb3; 7, biotransformation substrate Rc; 8, transformed products of Rc; 9, biotransformation substrate Rd ; 10, transformed products of Rd; 11, biotransformation substrate Re ; 12, transformed products of Re; 13, biotransformation substrate Rg1; 14, transformed products of Rg1; S3, standards of Rh1, Rg1, Re and Rb3.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 6.0

pH Stability: 6.0-10.0

Temperature Optima: 45°C

Temperature Stability:<35°C

Fig. 3. Effect of pH on activity (solid symbols) and stability (hollow symbols) of CcGlu1C. The optimal pH of CcBgl1C was determined by ranging pH from 2.0 to 11.0 using following buffers: sodium acetate buffer, pH 2.0–6.0; Na₂HPO₄–NaH₂PO₄buffer, pH 6.0–8.0; Glycine–NaOH buffer, pH 8.0–11.0. The maximum activity obtained was defined as 100%. The pH stability of CcGlu1C was determined by pre-incubating CcGlu1C in different pH for 24 h at 4^oC and then determining the percentage of residual activity under standard assay conditions. The activity of CcGlu1C without pre-incubating was defined as 100%. Results are presented as means ± standard deviations (n = 3).

Fig. 4. Effect of temperature on activity (a) and stability (b) ofCcGlu1Cusing pNP-Glu as substrate. The optimal temperature (a) was determined at different temperatures from 20 to 80^oC. The maximum activity obtained was defined as 100% activity. Thermal stability was determined by incubating the enzyme for 1 h at different temperatures. The activity of the enzyme before incubation was defined as 100%. Results are presented as means ± standard deviations(n = 3).

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (50 mM) pH 6.0. Swirl to mix the enzyme immediately prior to use.