A carboxymethyl cellulase from a marine yeast (Aureobasidium pullulans 98): Its purification, characterization, gene cloning and carboxymethyl cellulose digestion

Abstract

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase (CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U (mg protein)⁻¹. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40°C, much lower than that of the CMCases from other fungi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity (pH 5.0–6.0). The enzyme was activated by Na⁺, Mg²⁺, Ca²⁺, K⁺, Fe²⁺ and Cu²⁺, however, it was inhibited by Fe³⁺, Ba²⁺, Zn²⁺, Mn²⁺ and Ag⁺. K _m and V _max values of the purified enzyme were 4.7 mg mL⁻¹ and 0.57 µmol L⁻¹ min⁻¹ (mg protein)⁻¹, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose (CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp (EU978473). The protein deduced contained the conserved domain of cellulase superfamily (glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.