Production Of Isomaltooligosaccharides By Recombinant α-glucosidase From Potato Wastes

Using potato waste to produce isomaltooligosaccharides(IMOs) is a new method for utilization of potato waste. Because of its unique physiological fuctions, IMOs have extensive applications in the pharmaceutical, food and feed industries.In the present paper, the production of IMOs by potato waste was investigated; The gene ofα-glucosidase was expressed in Pichia pastoris; The production of IMOs transglucosylated by recombinantα-glucosidase was also studied. The following researches had been done:1. The components of potato waste from starch was analyzed. The waste contained 38.96 % starch after dehydration. The optimal condition was as follow: Firstly, potato waste was sacchrified by fungalα-amylase,β-amylase and pullulanase(0.5 mL/kg waste added)for 2 hours; Secondly,α- glucosidase was used to transglucosylated for 24 hours. Final product contained isomaltose, panose and isomaltotriose about 28.1 %, 13.0 %, 11.2 % respectively, and total amount of them reached up to 52.3 %. It suggested that potato waste coulded be produced to isomaltooligosaccharides.2. Total DNA was extracted from Aspergillus niger SG136. According to the nucleotide sequence ofα- glucosidase from Aspergillus niger CBS 513.88 in NCBI database, eight PCR primers were designed. The completed gene ofα- glucosidase was amplified through each exons ligated by over-lap PCR and the aglu was sequenced.3. The sequencing result showed that the gene is 2880 bp and encoded a protein of 960 amino acids. The gene was as long as that of A. niger CBS 513.88(Gene ID: 4991096). However, there was one amino acid different in 898 sites. It was serine in A. niger SG136 while proline in A. niger CBS 513.88.4. The expression vector pPIC9K-aglu was constructed by subcloning the gene into plasmid pPIC9K, and then transformed into P. pastoris through electroporation after linearized by BglⅡdigestion. The recombinant P. pastoris KM71/pPIC9K-aglu were screened in YPD/G418 plates, and identified by PCR. In shaking culture condition, methanol was added to a final concentration of 1% to induce the secretion ofα-glucosidase. Electrophoresis analysis of KM71/pPIC9K-aglu culture supernatant showed that there were two major protein bands corresponding to 98 kDa and 33 kDa respectively in SDS-PAGE, and there was only one band in Native PAGE; while in the control experiment of KM71/pPIC9K, there were no visible bands. The activity of recombinantα-glucosidase was 0.549 U/mL at 120 h after induction incubation.5. Under the optimal conditions of pH 5 and 60℃, after transglucosidae for 24 h, the contents of isomaltooligosaccharides were up to 26.0 %. Potato waste was liquefied by thermostableα-amylase, sacchrified by fungalα-amylase,β-amylase and pullulanase and transglucosylated by recombinantα-glucosidase, the products contained 9.5 % isomaltose, 7.3 % panose and 4.0 % isomaltotriose, and the total amount was 20.8 %.