The Study On Production, Purification, Characterization Of Extracellular Inulinase And Molecular Cloning Of Extracellular Inulinase Gene From A Marine Yeast Cryptococcus Aureus

Inulin is the main carbohydrate stored in Jerusalem artichoke, dahlia tubers and chicory root plants. Inulinase can hydrolyze inulin into fructose and fructooligosaccharides. Fructoses possess many merits such as high sweet, low caloric, pretects from decayed teeth and diabetes and so on. Fructooligosaccharides have a variety of physiological functions such as raising immunity, improving lipid metabolism, reducing blood fatty cholesterol, promoting the absorbing of minerals. So inulinase has many applications.Many hydrolytic enzymes such as lipase, inulinase, protease and phytase have been commercially produced by yeasts. Among the enzymes, inulinase has received much attention. Although there are many studies about inulinase production by terrestrial yeasts, little is known about inulinase and inulinase production from marine yeasts.After we screened over 500 marine yeast strains from different marine environments, the marine yeast strain G7a isolated from sediment of China South Sea was found to secrete a large amount of inulinase into the medium. This marine yeast strain was identified to be a strain of Cryptococcus aureus according to the results of routine yeast identification and molecular methods. To our knowledge, this is the first report that Cryptococcus aureus from the marine environment could produce extracellular inulinase. The crude inulinase produced by this marine yeast showed the higher activity at pH 5.0 and 50 oC. The optimal medium for inulinase production was artificial seawater containing inulin 4.0% (w/v), K2HP04 0.3% (w/v), yeast extract 0.5% (w/v),KCl 0.5% (w/v), CaCl2 0.12% (w/v), NaCl 4.0% (w/v) and MgCl2·6H2O 0.6% (w/v). Under the optimal conditions, over 85.0 U/mL of inulinase activity was produced within 42 h of fermentation at shake flask level. This is very high level of inulinase activity produced by yeasts. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.In order to explore the potential application of this inulinase we purified this enzyme and carried out a comprehensive study about the properties of enzyme. After the supernatant was obtained from the cell culture of the marine yeast Cryptococcus aureus G7a, the extracellular inulinase in the supernatant was purified to homogeneity with a 2.44-fold increase in specific inulinase activity as compared to that in the supernatant by ultrafiltration, concentration, gel filtration chromatography (SephadexTM G-75) and anion exchange chromatography (DEAE Sepharose Fast Flow Anion Exchange). The molecular weight of the purified enzyme was estimated to be 60.0 kDa. The optimal pH and temperature of the purified enzyme were 5.0 and 50°C, respectively. The enzyme was activated by Ca2+, K+, Na+, Fe2+ and Zn2+. However, Mg2+, Hg2+ and Ag+ acted as inhibitors in decreasing activity of the purified inulinase. The enzyme was strongly inhibited by Phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid and EDTA. The Km and Vmax values of the purified enzyme for inulin were 0.1114 mol/L and 0.0085 mg/min, respectively. A large amount of monosaccharides were detected after the hydrolysis, indicating the purified inulinase had a high exoinulinase activity.Crude enzyme with high concentration of inulinase is needed for hydrolysis of large amount of inulin. The optimization of process parameters for high inulinase production by the marine yeast strain Cryptcoccus aureus G7a in solid-state fermentation (SSF) was carried out using response surface methodology (RSM) based on Plackett–Burman design. Then, five levels of the five factors were further optimized using a Plackett–Burman design. Finally, the optimal parameters obtained with RSM were the initial moisture 61.5%, inoculum 2.75%, the amount ratio of wheat bran to rice husk 0.42, temperature 29oC, pH 5.5. 420.9 U/gram of dry substrate (gds) of inulinase activity was reached in the solid fermentation culture of strain G7a within 120h under the optimized conditions whereas the predicted maximum inulinase activity of 436.2 U/gds was derived from RSM regression. This is the highest inulinase activity produced by the yeast strain reported so far.The extracellular inulinase structural gene was isolated from cDNA of Cryptococcus aureus G7a by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1557 bp long encoding an inulinase. There is not intron in the coding region of the gene. The inulinase encodes 518 amino acid residues of a protein with a putative signal peptide of 21 amino acids. The protein sequence deduced from the extracellular inulinase structural gene contained six conserved putative N-glycosylation sites. The protein sequence deduced from the inulinase gene exhibited 71% and 58% identity with that of inulinase from Cryptococcus neoformans and Aspergillus fumigatus.In addition, the marine yeast strain G7a can not only secret high extracellular inulinase but also is a good single cell protein source. So it has a highly potential application in industry.