Overexpression Of The ?-glucosidase Gene From Filamentous Fungi And Its Characteristics Research

Beta-Glucosidases(EC3.2.1.21)remove the nonreducing terminal ?-D-glucosyl residue to free aglycones and glucose.The aglycones are always biological active intermediates,which widely used in medicine,food industry,sweeteners,etc.The activity and functions of P-glucosidases had become the focus of scientific research nowadays.The ?-glucosidases derived from microorganisms,especially filamentous fungi,have the higher enzymes activity,which have great industrial application prospect.At the same time,it is more complex of the fermentation enzymes from filamentous fungi and low content of ?-glucosidases were derived.This study aims to screen ?-glucosidase gene from the filamentous fungi microorganisms and overexpress the coding genes in Pichia pastoris to study its characters,functions and the hydrolyzed products.The main research results were showed as follows.We queried the database of beta-D-gucosidase gene from NCBI to choose different relationship?-glucosidase genes and get different attributes ?-glucosidase,by reconstruction of a phylogenetic tree to analyze these enzymes and available researchs.We choosed the enzymes from Aspergillus niger TCCC 41064,Aspergillus oryzae TCCC 41672,Penicillium decumhens TCCC 41604,Trichoderma reesei TCCC 41022 as the research objects.The ?-glucosidase genes bglA,hglA(o)-1 and bglA(o)-2,bglP,bglT were respectively amplified by PCR.The ?-glucosidase genes from Aspergillus aculeatus and Neurospora crassa were optimized according to the usage of P.pastoris and synthesized.The target genes subsequently cloned into pPIC9K.The recombinant plasmid was linearized and transformed into P.pastoris GS115 by electroporation.Through shaking flask fermentation,the BglA enzyme activity of culture supernatant was the best,reached to 90.85±3.5 U/mL.And BglA(o)-1,BglA(o)-2,BglT have no enzyme activity.The purified BglA enzyme activity reached to 324.4 U/mg,which was the best of all the recombinat enzymes.The optimal pH and optimum temperature of the recombinant enzymes was 5.0-6.5 and 55-70?.The BglA,BglA(a)moreover 80%of the original enzymatic activity remained after the incubation of recombinant enzyme at 50? for 10 hours,showing the better temperature stability.The BglA,BglA(a)and the BglP,BgIN respectively showed stability in pH 5.0-7.0 and pH 3.0-8.0.It showed that Ca2+ and Mg2+strongly activated the recombinant enzymes activity.Due to different sources of the ?-glucosidases hydrolysis ability have significant difference for one substrate.Meanwhile,one ?-glucosidase hydrolysis ability have some difference for different substrates.Two model protein 4IIB and 3ZYZ were finded in PDB.The BgIA,BglP,BglA(a)structure similar degree with 4IIB was respectively 90%,82%and 100%.The BgIN structure similar degree with 3ZYZ was 75%.The hydrolysis ability for substrates of model protein 4IIB from top to bottom was arbutin,salicin,geniposide,polydatin,cellobiose,while model protein 3ZYZ was geniposide,arbutin,polydatin,salicin,cellobiose.There were some differences in hydrolysis ability for substrates for BglA,BglP with model protein 4IIB.This model data provide reference for ?-glucosidases site-directed mutagenesis in later stage.The recombinant enzymes have substrate specificity.The BglA(a)had the best hydrolyze activity for the substrates as salicin(26.07%),cellobiose(15.35%),arbutin(42.85%).The conversion of geniposide and polydatin for BgIN and BglA was 47.12%and 17.7%,which showed the best hydrolyze activity.Through contrasting research,the performance of BglA was better.High density fermentation was performed in 7 L fermentor,and the BglA enzyme activity reached 289 U/mL.Compared with shaking flask fermentation,this achieved a 3.2-fold increase.