Cloning, Expression And Characterization Of α-glucosidase From Aspergillus Niger

a-Glucosidases (EC3.2.1.20) are a group of typical exo-type carbohydrases, which catalyze the liberation of a-glucose from non-reducing terminals of substrates. A. niger a-glucosidase not only hydrolyze glycosides but also can transfer a glucosyl residue to the 6-OH of the accepting unit and yield isomaltose, panose, isomaltotriose and tetrasaccharides from maltose. In the industry of functional oligosaccharides, the transglycosylation activity of A. niger a-glucosidase has been applied to produce isomaltooligosaccharides (IMOs).In our previous work, the cDNA of A. niger SG136 a-glucosidase was cloned by overlap-PCR and expressed in P. pastoris. However, the biological activity of the recombinant enzyme was low. In the present study, a-glucosidase cDNA from A. niger CICIM F0620 was amplified through RT-PCR and expressed in P. pastoris. In addition, the optimized condition of fermentation and the characterization of the recombinant enzyme were also investigated. The main results were listed as follows:(1) The cDNA of a-glucosidase was amplified from the total RNA of A. niger by RT-PCR and cloned into P. pastoris expression vector pPIC9K then transformed into P. pastoris stain KM71. The recombinants were screened in MD and YPD/G418 plates. After 120 h of induction on methanol in the shake flask, the pNPG hydrolase activity of recombinant KM71/pPIC9K-aglu culture supernatant was 0.44 U/mL.(2) In the shake flask, the optimized ferment condition of the recombinant P. pastoris has been determined:the optimal initial pH, initial cell density, methanol supplementation quantity and induction temperature were 5.0,50 (OD600),1.5%(in every 24 h) and 28℃, respectively. After 120 h of induction, the maximum yield of a-glucosidase was achieved to 1.15 U/mL, which was 18.2-fold higher than that of native a-glucosidase extracted from A. niger.(3) In order to enhance the production of a-glucosidase, fed-batch cultivation of the recombinant P. pastoris KM71/pPIC9k-aglu was carried out in a 3 L fermentor. When the initial cell density (OD600) and the initial methanol concentration were 100 and 0.5%(v/v), the pNPG hydrolase activity and protein concentration of the culture supernatant reached to 3.51 U/mL and 1759μg/mL after 96 h of induction, which were 3.0-fold and 3.4-fold higher than those in shake condition, respectively. The fermentation was also performed on a 30 L fermentor, and the activity of a-glucosidase was up to 5.12 U/mL.(4) The molecular weight of recombinant enzyme was estimated to be about 145 kDa by SDS-PAGE, and it reduced to 115 kDa after treated with deglycosylase (Endo Hf). The optimal temperature of the recombinant a-glucosidase was 60℃and it was stable below 50℃. In addition, the recombinant a-glucosidase had maximal activity at pH 4.5 and had great stability on the range of pH 3.0-7.0. The Km and Vmax values for pNPG at 50℃and pH 5.5 were 0.45 mM and 43.48 U/mg respectively. The enzymatic activity of recombinant a-glucosidase was not significantly affected by a range of ions.(5) The recombinant a-glucosidase could produce IMOs from maltose. The transglycosylation activity of recombinant a-glucosidase was similar to a commercial a-glucosidase. The optimized condition of transglycosylation was 0.05 U of a-glucosidase per gram of maltose in 100 mM citric acid/disodium hydrogen phosphate buffer at pH 5.0 and 60℃for 24 h. In this condition,183.12 g/L IMOs could be obtained when using 300 g/L maltose. These studies provide the basis for the application of recombinant a-glucosidase in the industry of functional oligosaccharides.