Kinetics and thermodynamics of a novel endoglucanase (CMCase) from Gymnoascella citrina produced under solid-state condition

Abstract

Gymnoascella citrina produced two isoforms of endoglucanases (CMCase-I and -ІІ) under solid-state condition. Purified CMCase-I was novel because it was apparently holoenzyme in nature. The enzyme was monomeric as its native and subunit mass were almost the same, i.e., 43 and 42 kDa, respectively. E _a for carboxymethylcellulose (CMC) hydrolysis was 36.2 kJ mol⁻¹. The enzyme was stable over a pH range of 3.5–6.5, while temperature optimum was 55 °C. V _max, K _m and k _cat for CMC hydrolysis were 39 U mg⁻¹ protein, 6.25 mg CMC mL⁻¹ and 27.5 s⁻¹, respectively. The pKa₁ and pKa₂ of ionizable groups of active site were 2.8 and 7.4, respectively. Thermodynamic parameters for CMC hydrolysis were as follows: ΔH^* = 33.5 kJ mol⁻¹, ΔG^* = 70.42 kJ mol⁻¹ and ΔS^* = −114.37 J mol⁻¹ K⁻¹. The removal of metals resulted into complete loss of enzymatic activity and was completely recovered in the presence of 1 mM Mn²⁺, whereas inhibition initiated at 5 mM. The other metals like Ca²⁺, Zn²⁺ and K¹⁺ showed no inhibition up to 7 mM, Co²⁺ completely inhibited the activity, while Mg²⁺ could not recover the initial activity up to 7 mM. So we are reporting for the first time, kinetics and thermodynamics of CMCase-Ι from G. citrina.