Cloning-expression And Application Of ?-Glucosidase From Aspergillus Nidulans

?-Glucosidase?EC.3.1.20?,also known as?-D-glucoside hydrolase,can cleave?-1,4glycosidic bonds from the non-reducing end of oligosaccharide substrate,and release glucose.The glucose unit is transferred to molecule such as maltotriose,maltose or glucose by an?-1,6 bond to obtain an oligosaccharide product such as isomaltotriose,panose or isomaltose.It exists in nature,with a wide variety and different property.At present,?-glucosidase has been widely used in various fields such as oligomeric isomaltose production,metabolic physiology research,and disease prevention and treatment.In this study,we obtained the?-glucosidase from Aspergillus nidulans,which has strong transglucosidic ability and weak hydrolysis ability.The A.nidulans?-glucosidase gene AgdB was ligated into the expression vector pPIC9K,expressed in Pichia pastoris KM71,and co-expressed with N-acetyltransferase to optimize the fermentation of the recombinant strain in a 3.6 L fermentor.The enzymatic properties of recombinant?-glucosidase and its application in synthesizing oligomeric isomaltose and increasing the resistance content of pyrodextrin were studiedThe main findings are as follows:?1?To compare the genetic evolutionary classification characteristics and three-dimensional structural characteristics of different?-glucosidases in the database,the?-glucosidase derived from A.nidulans?Amino acid sequence GenBank:BAB39856.1?may have strong transglucosidic ability and weak hydrolysis ability.The target gene AgdB was codon-optimized and synthesized,the recombinant plasmid pPIC9K-AgdB was constructed and integrated into P.pastoris KM71 for expression.?2?In order to increase the expression level and enzyme activity of the target protein,the N-acetyltransferase?Mpr1?was co-expressed,and the recombinant strain P.pastoris KM71/pPIC9K-AgdB/pPICZ A-Mpr1 was obtained.The recombinant strain was subjected to shake flask induction fermentation,and the enzyme activity reached 22.56 U?mL^-1,which is 1.92times that of the Mpr1 strain not co-expressed.The 3.6 L fermentor fermentation conditions were optimized.The results showed that when the induction temperature was 25°C,and the methanol concentration was 1%in the induction phase,the enzyme activity reached 128.12U?mL^-1,Which is 5.68 times of the shake flask enzyme activity.?3?Recombinant?-glucosidase was purified,the enzymatic properties analysis showed that the optimum pH was 5.5,and the optimum reaction temperature was 45°C.The recombinant enzyme was stable at pH 3.5-8.0 and had a half-life of 24 h at 50°C.The K_m values of the transglucoside kinetic parameters of the recombinant A.nidulans?-glucosidase to maltose and maltotriose were 1.78 mM and 2.09 mM,respectively.The transglucoside affinity was 10.42 and 1.56 times that of A.niger?-glucosidase,respectively.The hydrolysis kinetic parameters of recombinant A.nidulan?-glucosidase to maltose and maltotriose are2.91 mM and 5.39 mM.The affinity of A.niger?-glucosidase for maltose and maltotriose was 5.49 and 7.19 times that of A.nidulans?-glucosidase.It is proved that A.nidulans?-glucosidase has strong transglucoside ability and weak hydrolysis ability,which is beneficial to the accumulation of transglucoside products.?4?The process for the preparation of isomaltooligosaccharide by?-glucosidase was optimized.The optimum conditions for preparing isomaltooligosaccharide with maltose as substrate was as follows:pH 5.5,45°C,the concentration of?-glucosidase is 5.1 U·g^-1maltose,and the substrate concentration is 30%.The yield of isomaltooligosaccharide reached a maximum of 216.36 g·L^-1,and the conversion rate was 72.12%.It is the highest level of isomaltooligosaccharide synthesized with maltose as a substrate.To improve the resistance content from pyrodextrin,the condition was optimized as follows:pH 5.5,45°C,enzyme amout 2.75 U·g^-1 pyrodextrin,and the reaction time 6 h.The resistance content increased from 44.10%to 63.10%.The TfSBE,CGTase and?-glucosidase was compound.The optimized conditions were:20%(w·v^-1)pyrodextrin concentration,first react at 40°C,pH 6.5,the amount of TfSBE were 1500 U·g^-1 pyrodextrin,after 12 h of reaction,adjust the pH to 5.5,and add 5 U·g^-1 pyrodextrin?-glucosidase and CGTase at 45°C for 12 h,the resistance content increased from 44.10%to 70.44%.