A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. 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 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. 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＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol 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 flame of 954bp （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.

Screening and performance of L-14, a novel, highly efficient and low temperature-resistant cellulose-degrading strain

In view of the low bioconversion efficiency of agricultural biomass waste in low-temperature environments in winter,a low-temperature-resistant cellulose-degrading strain,L-14,was successfully screened by restrictive cultures from humus-rich soil in the Daqing Zhalong wetland region.According to morphological observations and 18S rDNA sequence analysis,the cellulose-degrading strain L-14 was identified as a Neurospora sp,belonging to fungus.Different parameters,such as temperature,initial pH,carbon,nitrogen and lecithin,were optimized using a single-factor experiment and a response surface methodology(RSM).When the temperature was 16°C,the optimal conditions for enzyme production were an initial pH 8.20,10.45 g/L of bran,5.28 g/L of yeast powder,and 4.25 g/L of lecithin.The carboxymethyl cellulase(CMCase)activity of strain L-14 was 63.598 IU/mL.Strain L-14 had a high level of cellulose degradation activity,excellent resistance to low temperatures and environmental adaptability,indicating its good application prospects in substrates pretreatment of biogas engineering.