The Enzymatic Characteristics Of Intracellular ?-Glucosidase From Aspergillus Niger

?-glucosidase is the significant component of cellulase,which can hydrolyze cellobiose to glucose.When the cellobiose was accumulated it can inhibit the function of CBH and EG,while the ?-glucosidase could relieve this situation,so ?-glucosidase was the key enzyme and limit speed of cellulose degradation.Glucose that the production of ?-glucosidase,which could can inhibition the function of ?-glucosidase.So the research direction is find a ?-glucosidase that can tolerance the glucose.Aspergillus niger is a filamentous,black spored fungus that has been used in many industrial processes,including production of enzymes,it could secretion lots of ?-glucosidase,When the complete genome sequence for both the industrial strain CBS513.88 and the citric acid-producing strain ATCC-1015 were published in the internet.Pick the gene of ?-glucosidase out and according to the prediction of signal peptide,we found that A.niger has many intracellular ?-glucosidase genes,the cell transporter glucose from extracellular to intracellular was active transport,so we conjectures the intracellular ?-glucosidase can tolerance glucose of high concentration.For verification this idea,we studied the enzymatic characteristics of intracellular ?-glucosidase,the result as follow:?according to the genome sequence,A.niger contains 15 putative BGLs,three of them were belong to GH 1 family,twelve of them were belong to GH 3 family.According to the prediction of signal peptide,6 bgl genes were predicted to location intracellular,one of them belong to GH 1 and five of them belong to GH 3.?when A.niger exposure to different carbon sources,the intracellular beta-glucosidase has different response.The activity of intracellular of beta-glucosidases were found to vary depending on the carbon sources,lignocellulosic biomasses induced higher beta-glucosidase activity higher than other carbon sources?The q PCR data shown that the expression level of GH 1-1 always great majority in vary carbon source,GH 1-1 and GH 3-12 were found to be highly induced by wheat straw,the response of other intracellular genes to vary carbon source were different with GH 1-1,not obvious induced by wheat straw.GH 1 and GH 3-12 have same expression pattern.?There were two different transformants when cloning the GH 1,one lose about 72 bp than normal one.This case was not random appear,the probability of approximately about 1/3,homology alignment show that 72 bp was a small ?-helical located protein surface,but the function was not know.The intracellular bgls were expression in E.coli,most of the expressed BG protein was present as insoluble,when change the concentration of IPTG and temperature of induced,after crushing supernatant presence soluble protein,however,it did not have any catalytic activity.?The intracellular bgl were expressed in P.pastoris,if secretion to out the cell,there is not protein strip in the supernatant,but we could detect the catalytic activity to the p NPG,the optimum p H and optimum temperature of the rude enzyme were p H 6.6 and 50? respectively,when incubation in 55?,almost no activity.The enzymatic products were analyzed with HPLC,when cellobiose was used as substrate,the obvious hydrolysis ability was GH 1-4,while GH 3-12'S transglycosylation was more higher than GH 1-4 and GH 1-7.Compared GH 1-7 with GH 1-4 shown that the ability of hydrolysis cellobiose was decrease,however the ability of transglycosylation almost change.In this study,why those protein did not secrete to exteacellular,we do not know why.?the intracellular bgl from A.niger was expressed intracellular in P.pastoris,after cell disrupted by glass microsphere and purified by Ni-NTA system,we found the enzyme activity was improved much better than secretion expression in P.pastoris.When the p NPG as substrate,the optimum p H of the pure enzyme were from 6.6 to 7.0 and the intracellular BG would lossed activity at p H4,the optimum temperature was 50?.Intracellular BG were not sensitive to p H,thermal stability test shows that half-life of approximately 300 mins,but that were very sensitive to temperature,especially GH 1-7,the half-life only have about 60 mins.The Km(p NPG)of GH 1-4,GH 1-7,GH 3-12 were 0.277?0.434?0.361 m M respectively,these enzyme were tolerant to glucose with a Ki of 165?138?170 m M respectively.