PpGlu5A (PpBglu5A)

Ex-Glu0017

(EC.3.2.1.73) β-1,3/4-glucanase

CAZy Family: GH5

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~43kDa)

Figure 1. Electrophoresis analysis of PpGlu5A. 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, PpGlu5A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

1605 U/mg protein (on β-glucan) at pH6.5 and 50°C

One Unit of β-glucanactivity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute from β-glucan (25 mg/mL) in phosphate buffer(25 mM) pH 6.5.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Substrate specifificity of PpGlu5A on polysaccharides.

¹ One unit refers to the enzyme used for releasing 1 μmol/min reducing sugar from the substrate.

^{2 -}: not detected.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima:6.5

pH Stability: 6.0-8.0

Temperature Optima:50°C

Temperature Stability:<60°C

Fig. 2. Optimal pH (a) and pH stability (b) of PpGlu5A with barley β-glucan as the substrate. The buffffers used were acetate (pH 2.0-6.0), sodium phosphate (pH 6.0-8.0), and glycine-NaOH buffffer (pH 8.0-11.0). The results are shown as means ± SD values.

Fig.3. Optimal temperature (a) and thermostability (b) of recombinant PpGlu5A with barley β-glucan as the substrate. The results are shown as means ± SD values.

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (20 mM) pH6.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1] Ye Yuan, Xinyu Zhang, Han Zhang, et al.Degradative GH5 β-1,3-1,4-glucanase PpBglu5A for glucan in Paenibacillus polymyxa KF-1. Process Biochemistry, 2020, 98:183–192.