Figure 3. Biochemical properties of LHyal.

(a) Purification of the H6LHyal protein using Ni-NTA agarose. M = Marker proteins, C = fermentation supernatant of the control strain P. pastoris GS115, FS = fermentation supernatant of H6LHyal, PE = purified H6LHyal. (b) Effect of temperature on LHyal activity. The enzymatic activity of LHyal was measured at various temperatures in 50 mM citrate buffer (pH 5.5) using HA (1.6 mg ml⁻¹) as the substrate. The logarithm (Ln) of the specific activity (U mg⁻¹) was plotted against the reciprocal of the absolute temperature (T). The activation energy (E_a, kJ mol⁻¹) was calculated from the value of the slope (S) and the general gas constant (R, 8.3 J mol⁻¹ K) according to the Arrhenius equation: E_a = −S × 2.306 × R. (c) After pre-incubating the purified enzyme (0.45 mg ml⁻¹) at 38°C (red ), 50°C (purple ), 55°C (blue ) or 60°C (green ) for up to 180 min, the remaining enzymatic activity was determined at 38°C in 50 mM Na₂HPO₄-citrate buffer (pH 5.5). (d) Effect of pH on LHyal activity and stability. The enzyme activity was determined at various pH values at 38°C. The stability was assayed at 38°C in 50 mM Na₂HPO₄-citrate buffer (pH 5.5) after pre-incubating the purified enzyme (0.45 mg ml⁻¹) for 30 min at 25°C in buffers of pH values from 3.0–8.0. The relative activity is represented as a percentage of the maximum activity. () 50 mM Na₂HPO₄-citrate buffer (pH 3.0–6.0); () 50 mM Na₂HPO₄–NaH₂PO₄ buffer (pH 6.0–8.0). Analysis of the degradation of chitin (e), heparin (f) and chondroitin sulphate (g) by HAases using the DNS method. In the control group, the corresponding substrates (2 mg ml⁻¹) were incubated without enzyme in 50 mM citrate buffer (pH 5.5) at 38°C for 30 min. In the LHyal and BTH groups, substrate was incubated with LHyal (1,000 U) or bovine testicular hyaluronidase (BTH, 1,000 U) under the same treatment conditions.