Production, Recycle, Gene Clone And Expression Of β-glucosidase From Aspergillus Niger

In the process of hydrolysis of lignocellulosic material with cellulase produced by Trichoderma viride,supplying additionalβ-glucosidase was an effective way of increasing the yield of fermentable monosaccharide,decreasing the consumption and lowering the cost of enzyme.So the supplementaryβ-glucosidase activity and its cost are the significant factors for application in industries.Studies in this dissertation were focused on the fermentation techniques for production ofβ-glucosidase at a high level by Aspergillus niger,purification and characterization the intracellular and extracellularβ-glucosidase from Aspergillus niger,recycleβ-glucosidase techniques of ultrafiltration,acetone precipitation and enzyme immobilization,and Clone the and expression gene ofβ-glucosidase.The main results are as follows:(1)The highβ-glucosidase producing strain A.niger-n1-1 was selected from the experimental strains and its highest activity,up to 4.7 U/ml,was synthesized by A.niger-n1-1 at 96 h.Besidesβ-glucosidase,the higher exoglucanases and endoglucanases were synthesized by the strain during the same fermentation.The filter paper activity(FPA)reached 0.62 U/ml.(2)The initial pH value of enzyme production was 5.0.Regulated pH value around 4.0 in the fermentation period the enzyme activity reached 6.12 U/ml at 120 h.and it would be favorable for the secretion ofβ-glucosidase.(3)β-glucosidase activity increased by nearly 3 times after fourth batch feed bran during the fermentation.It worked the best when the initial concentration of bran was 3%and fed with descending mode.Under this conditions,the enzyme activity reached 7.0U/ml.the results showed that feed-batch culture was a favorable way to enhance the enzyme production.(4)Two kinds of extracellularβ-glucosidase were purified to homogeneity from an Aspergillus niger by salting out,hydrophobic interaction chromatography,negative ion exchange chromatography,gel layer chromatography and other processes.Their single subunit molecular weight was 114.6 KD and 70.3 KD respectively.The one with higher molecular weight was highly glucose-tolerant.Its K_i was 41.01mmol/l using p-nitrophenyl-β-D-glucopyranoside as substrates.Metal ions had no effects on the two enzymes.It could be activated by a certain concentration of ethanol,methanol,1-butanol and acetoacetate.(5)Obtained a kind of intracellularβ-glucosidase by purification and its molecular weight was 122.7 KD.It could be obviously activated by methanol,ethanol,1-butanol,acetone and acetoacetate.The relative enzyme activities were 153%,162%,184%,146%with additions of 30%methanol,30%acetoacetate,40%1-butanol,40%acetone.This kind of enzyme will have favorable applicative prospects in the synthetical industries.(6)The 30 KD membrane was selected to recoverβ-glucosidase from cellobiose hydrolytic liquor when using ultrafiltration technique.In the signal batch,theβ-glucosidase recovery rate and the average permeation flux were 99.5%and 109.4%L/m².h respectively. The amount of recoveredβ-glucosidase from the 20^thbatches of cellobiose hydrolytic liquor would still be maintained 90%of the initial total enzyme activity and the average permeation flux reached 79.2 L/m².h.When using organic solvent precipitation technique,it was suitable that the acetone and cellobiose hydrolytic liquor at the volume ratio of 0.75:1 to 1:1.The enzyme recovery rate was up to 95.7%by using -20℃acetone as precipitator for 2h.50% enzyme activity could still be maintained and 78.97%amount of required enzyme was saved when recycleβ-glucosidase 15 batches by acetone precipitation.(7)β-glucosidase was immobilized by sodium alginate as carrier.The yield of enzymatic hydrolysis kept higher than 90%during 20 batches.When the rate were 1.5ml/min and 1.0ml/min,the yield reached 96.65%and 99%respectively.The synergetic hydrolysis processes on filter paper and Avicel were carrier out by the cellulase of T.viride and the immobilizedβ-glucosidase.On the condition of the ratio of totalβ-glucosidase activity to filter paper activity was 0.5 and FPA=2.0 IU/ml,at 60h,the degradation rate of filter paper and Avicel was increased by 20.4%and 29.3%respectively compared to that using T.viride only.(8)Cloned theβ-glucosidase gene of A.niger that had 2582bp.Construct the gene expression vector pPICZα-A-bg11 ofβ-glucosidase and obtained the recombinant Pichia pastoris by transformation.(9)Optimized the conditions to induce the recombinant Pichia pastoris to express.In the one-step method the activity reached 11.29U/ml on 5^thday which had high enzyme productivity than the two-step method and the enzyme yielding period was greatly shorten.Obtained the recons GS115 bg11-38 with relaxed copy by using 2^thelectric transformation.It was showed that the expression amount of GS115 bg11-38 was increased by 2.7 times compared with the excellent recons GS115 bg11-3 got by 1^thelectric transformation by shake-flask cultivation,5 times of that of the primary strain.So actualize the expression with high efficiency.There are fruits in four aspects to summarize:(1)Obtained the highestβ-glucosidase producing strain from the 17 strains of Trichoderma,Basidiomycetes and Aspergillus. Established the techniques of pH regulation and feed-batch fermentation in the yielding process. (2)Provided a reference and direction for selecting more suitableβ-glucosidase for the enzyme hydrolysis of lignocellulosic resources and broadeningβ-glucosidase application by purification and characterization of three kind ofβ-glucosidase from A.niger.(3)Established the techniques of recycleβ-glucosidase using ultrafiltration,acetone precipitation and immobilized enzyme method.Provided theoretical basis for building new techniques or modes of hydrolyzing lignocellulosic material with an aid ofβ-glucosidase.(4)Cloned theβ-glucosidase gene of A. niger successfully and expressed in Pichia pastoris with high efficiency.So it showed great applicative potential in industries.