Study On Production, Purification, Characterization, Gene Cloning And Expression Of Inulinase From Marine Yeast Pichia Guilliermondii

Inulin is a fructose polymer that occurs as a reserve carbohydrate in Jerusalem artichoke, dahlia tubers and chicory root. This polymer is a recognized source for the production of either ultra-high fructose syrups, with D-fructose content over 95% or for production of oligofructose syrups. Inulinase (β-2,1-D-fructan fructanohydrolase, EC 3.2. 1.7) targets on theβ-2,1 linkage of inulin and hydrolyzes it into fructose. Inulinase is produced by many microorganisms, including fungi, yeasts and bacterias. But little is known about inulinase produced by marine yeasts. Marine yeast strain 1 which was found to secrete a large amount of inulinase into the medium was selected from the collection culture of marine yeasts available in this laboratory. This marine yeast strain was identified to be a strain of Pichia guilliermondii according to the results of routine yeast identification and molecular methods.The optimal medium and cultivation conditions for inulinase production were determined first. Seawater containing 4.0% (w/v) inulin and 0.5% (w/v) yeast extract , cultivated under the optimal condition of pH 8.0, 28 oC and 170 rpm. Over 60 U ml-1 of inulinase activity was produced within 48 h of fermentation at shake flask level. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis, indicating that the crude inulinase had a high exoinulinase activity.The extracellular inulinase in the supernatant of cell culture of the marine yeast Pichia guilliermondii strain 1 was purified to homogeneity with a 7.2-fold increase in 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 mass of the purified enzyme was estimated to be 50.0 kDa. The optimal pH and temperature of the purified enzyme were 6.0 and 60 (C, respectively. The enzyme was activated by Mn2+, Ca2+, K+, Li+, Na+, Fe3+, Fe2+, Cu2+, and Co2+. 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. The Km and Vmax values of the purified enzyme for inulin were 21.1 mg ml-1 and 0.082 mg min-1, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin.The inulinase structural gene was isolated from genomic DNA of P. guilliermondii strain 1. The gene had an open reading frame of 1542 bp long encoding an inulinase (accession number EU195799). The coding region of the gene had no intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids. The protein sequence deduced from the inulinase structural gene contained the consensus sequence of glycosyl hydrolases family 32 N terminal and ten conserved putative N-glycosylation sites. The inulinase encoding DNA was subcloned into pPICZαA expression vector and the resulting recombinant plasmid was named pPICZαAINU. The recombinant plasmid was expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 30.71 U/mL was obtained from supernatant of P. pastoris X-33 harboring pPICZαAINU. The crude recombinant inulinase had hydrolytic activity towards inulin.One mutant (M-30) with enhanced inulinase production was obtained by treating P. guilliermondii strain 1 with UV light and LiCl2. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for maximum inulinase production by mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30 oC and 6.50, respectively. Under the optimized conditions, 455.9 U/gram of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its wild-type only produced 291.0 U/ gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.