TmGal36A

Ex-Gal0028

(EC.3.2.1)exo-α-1,3/6-Galactosidase

CAZy Family: GH36

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~64kDa)

Figure 1. Electrophoresis analysis ofTmGal36A. 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, TmGal36A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

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

One Unit of pNP-α-gal activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenyl per minute from pNP-α-gal (5 mM) in phosphate buffer buffer(20 mM) pH7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of TmGal36A on different substrates^a.

^aReactions were performed with 5 mM substrate, pH 8.0, at 37°C for 5 min.

^bAbsorption caused by releasedp-nitrophenol was measured at 405 nm. The relative activity on pNPαGal was taken as 100%.

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

Figure 2.¹H NMR analysis of the stereoselectivity for the TmGal36A catalyzed reaction.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima:5.0-5.5

pH Stability:5.0-8.0

Temperature Optima:90-95°C

Temperature Stability:<90°C

Figure 3. pH dependence of kinetic parameters. log[(kcat)/(kcat)max] of TmGal36A, D327G, and D387G enzymes versus pH.

5.STORAGE CONDITIONS

The enzyme should be stored at -20 °C. For assay, this enzyme should be diluted in pH5.5