LpGal42A

Endo-Gal0067

(EC.3.2.1.164)endo-β-1,6-Galactanase

CAZy Family: GH42

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~55kDa)

Figure 1. Electrophoresis analysis of LpGal42A. 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, LpGal42A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

0.46 U/mg protein (on pNP-β-gal) at pH 7.0 and 37°C.

One Unit of pNP-β-gal activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenyl per minute from pNP-β-gal (5 mM) in phosphate buffer (20 mM) pH 7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of LpGal42A on different substrates^a.

^aReactions were performed with 5mM substrate, pH 7.0, at 37°C for 10 min.

^bAbsorption caused by released p-nitrophenol was measured at 405 nm. The relative activity on pNP-β-Glu was taken as 100%.

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

Figure 2.HPAEC was used to detect the hydrolysis of LpGal42A on galactise and galactan.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima:6.0

pH Stability:5.5-9.5

Temperature Optima: 40°C

Temperature Stability:< 40°C

Figure 3.Effect of pH on the activity and stability of recombinantLpGal42A. (a) Optimum pH. Assays were carried out at 37°C for30 min in buffers ranging in pH from 2.0 to 11.0. The activity at optimum pH was defifined as 100%. (b) pH stability. Assays were carried out after the incubation of the enzyme in buffers ranging from pH 2.0-11.0 at 4°Cfor24 h.

Figure 3.Effect of temperature on the activity and stability of recombinantLpGal42A. (a) Optimum temperature. Activity was measured between 20°C and 90°Cat pH 6.0 for 30 min. The activity at optimum temperature was defifined as 100%. (b)Thermostability. Assays were carried out in 50 mM phosphate buffer (pH6.0) at 35°C-55°C for 12 h. The activity without heat treatment was defifined as 100%.

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (20 mM) pH 7.0. Swirl to mix the enzyme immediately prior to use.

6.  REFERENCES

[1] 张涵. 植物乳杆菌中β-1,6-半乳糖苷酶的重组表达及酶学性质研究[D].东北师范大学, 2020.