Cloning And Expression Of Glucose Dehydrogenase Gene And Its Effect On Physicochemical Properties Of Bamboo Shoots Dietary Fiber

Bamboo shoot is a kind of high quality dietary fiber raw material,which can be used as functional additives in the field of food.The food containing bamboo shoot dietary fiber has the functions of lowering blood sugar levels and blood fat,preventing constipation and controlling body weight and so on.The composition,chemical structure and physicochemical properties of bamboo shoot dietary fiber determine its functional characteristics.Therefore,in order to prepare high-quality bamboo shoot dietary fiber and improve the physicochemical properties and efficacy of dietary fiber,it is necessary to adopt appropriate ways to deal with dietary fiber.Enzyme treatment technology is a green and effective method,which can improve the network structure of dietary fiber and form soluble components and small molecular substances.Cyrtotrachelus buqueti as a bamboo shoot exclusive pest,has the ability to degrade lignocellulose of bamboo shoot efficiently.Further studies had shown that the gene encoding glucose dehydrogenase was abundant in its genome.Therefore,in order to improve the physicochemical properties of bamboo shoot dietary fiber and enrich the glucose dehydrogenase resources from insects,a glucose dehydrogenase gene was screened from the genome data of C.buqueti by using bioinformatics method.It was cloned and transformed into Pichia pastoris heterologous expression in vitro,and the enzyme with biological activity was obtained.The enzymatic properties of the recombinant enzyme and its effect on the physicochemical properties of bamboo shoot dietary fiber were studied.The main results are as follows:Bioinformatics analysis showed that the gene contained 1404 bp open reading frame and encoded 467 amino acids.The contents of T,C,A,and G were 26.4%,21.7%,32.3% and 19.6%,respectively.The start codon was ATG,and the stop codon was TAA.The predict the molecular weight of protein was 51.77 kDa and the isoelectric point was 6.03.The sequence had no transmembrane region,no signal peptide.An amino acid homology sequence comparison using NCBI blast revealed a 74.52% homology to Sitophilus oryzae glucose dehydrogenase,followed by 53.98% homology to Anoplophora glabripennis glucose dehydrogenase.17 amino acid sequences with high homology to this gene were selected to construct the NJ phylogenetic tree.It was found that the amino acid sequence was clustered with Sitophilus oryzae,Dendroctonus ponderosae and Anoplophora glabripennis,which was consistent with the results of homologous alignment of NCBI blast.It is suggested that this gene is probably the glucose dehydrogenase gene(CbGDH)from C.buqueti.The total RNA from muscle of C.buqueti was extracted,and then reversed transcription cDNA.The CbGDH gene was cloned and successfully expressed in P.pastoris.The expressed product was subjected to SDS-PAGE after concentration,and there was a clear target protein band at 51.77 kDa,and enzyme was found to exist activity,indicating that the CbGDH gene was successfully expressed in P.pastoris.The results of enzymatic studies showed that the optimum conditions were pH 7.0 and temperature 50 ?.The stability was the best in the range of pH 5-7,the residual enzyme activity could reach more than 70% of the original enzyme activity after being kept in the buffer solution of pH value for one hour.When the enzyme was kept at the optimum temperature for one hour,it can still maintain more than 80% of the enzyme activity.At the optimum pH and temperature,the activity of enzyme was 4.32 ?/mg.Compared with the treatment of bamboo shoot dietary fiber with only complex enzymes(cellulase,xylanase,laccase),the dietary fiber treated with the complex enzyme and 10 mL glucose dehydrogenase CbGDH increased its water holding capacity by 10.39%,and its oil holding capacity Increased by 5.57% and swelling capacity by 13.29%,indicating that glucose dehydrogenase CbGDH might synergistically improved the physicochemical properties of dietary fiber with complex enzymes.In conclusion,we have cloned and expressed the glucose dehydrogenase gene of C.buqueti,and studied the enzymatic properties.In addition,the improvement of the physicochemical properties of bamboo shoot dietary fiber by the combination of glucose dehydrogenase CbGDH and other enzymes was also studied.On the one hand,this paper enriches the resources of glucose dehydrogenase,provides support for further research on the function of glucose dehydrogenase and commercial production in the future,on the other hand,it provides a reference for the application of lignocellulose-degrading enzyme of C.buqueti.