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Breeding And Construction Of The Strain Of High-Producing Beta-mannanase And Its Enzymatic Characteristics

Posted on:2015-12-13Degree:DoctorType:Dissertation
Country:ChinaCandidate:H L LiFull Text:PDF
GTID:1220330434955073Subject:Forest bio-engineering
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Inject fracturing fluid into the oil well is one of the important measures for increasing the production. In order to improve the fracturing effect, we often add thickener(guar gum and konjac gum) into fracturing fluid. High viscosity of fracturing fluid need to degrade quickly after completing fracture. Adding enzyme gel breaker in the fracturing fluid, thickener would be degraded. In this study, we achieved the guar gum-degradated Chryseobacterium sp. HL-28ptoducing beta-mannanase. Additional, we have done a series of experiments about Bacillus subtilis degrating konjac gum saved in microbiology laboratory of Northeast Forestry University. After ultraviolet mutagenesis, Methyl sulfonic acid ethyl ester mutagenesis and60Co mutagenesis, we screened a strain which had good genetic stability. Clone Man gene sequence and complete heterologous expression in Pichia pastoris. Furthermore, enzymology characteristic of recombinant enzyme were studied. Main results of the study were as follows:(1) The screening and identification of bacteria degrading guar gumA guar gum-degradated strain producing beta-mannanase was found from flax liquid. This strain was identified as Flavobacteriaceae Chryseobacterium through morphological observation, physiological-biochemical characteristic and sequence analysis of16s rDNA. We named it Chryseobacterium sp. HL-28.(2) Mutation breeding of strains degrading guar gum and konjac gumCulture Chryseobacterium sp. HL-28, Bacillus subtilis WD23and Bacillus subtilis K. Their bacteria quantity had a maximizing at13h. Enzyme activity of Chryseobacterium sp. HL-28reached maximizing at23h. Enzyme activity of B. subtilis WD23'B. subtilis K reached maximizing at11h.The specific activity of beta-mannanase produced by Chryseobacterium sp. HL-28, B. subtilis WD23and B. subtilis K could be improved by means of ultraviolet mutation, which the enzyme activity improved were426.53%,8.72%and13.97%respectively, the strains after ultraviolet mutation were named after Chryseobacterium sp. H30, B. subtilis W12and B. subtilis K116respectively. Then, the strains of B. subtilis W12and B. subtilis K116were carried out by methyl sulfonic acid ethyl ester mutagenesis, which the enzyme activity improved by1.29%and3.97%respectively, after this the two strains were performed by ray of60Co, two new strains were obtained and named for B. subtilis WLY-12and B. subtilis KLY-116, which their enzyme activity were4181.82U/mL and4095.48U/mL respectively, the ratio improved were16.89%an d7.72%respectively. The result above showed that it is better way by physical mutagenic than chemical one. (3) Clone and expression of Man gene from Bacillus subtilis WLY-12Design the primers through primer premier5.0software according to Man gene sequence published in GenBank. And then amplify Man gene by gradient PCR. It was sequenced1089bp. Its accession number in GenBank was KC979152. Man gene was ligated with the pPIC9K expression vector by T4DNA ligase. Recombinant expression vector pPIC9K/Man was linearized with Sall and then transformed by electroporation into Pichia pastoris GS115competent cell. It indicated that we got pPIC9K/Man/GS115positive strains by direct PCR identification. Target protein expressed successfully after methanol induction.The results of SDS-PAGE shows that molecular weight of target protein is about41kDa. The enzyme activity of beta-mannanase was assayed using konjac gum as the substrate through DNS. It reached5156.742U/mL and raised23.32%than before.(4) Purification of recombinant mannanase and its enzymatic characterizationThe culture of pPIC9K/Man/GS115after centrifugation was crude enzyme. We achieved purified recombinant protein of15ml after salting out, dialysis, PEG20000concentration, DEAE-Sepharose Fast Flow anion-exchange chromatography, Sephadex G-75gel filtration chromatography. The total enzyme activity was54183.9U and total protein was3.75mg. Specific activity of recombinant enzyme was14449.04U/mg. Yield coefficient reached10.51%and purification fold was9.70. Recombinant protein by SDS-PAGE electrophoresis to detect. There was a single protein band in the result of SDS-PAGE electrophoresis. It meaned we had got electrophoretically pure protein. There is99%homology between joint peptides of flight mass spectrometry and mannanase published in NCBI. Purification steps are reasonable, the result is reliable.Optimum pH of recombinant mannanase playing a catalytic role is6.3. Optimum temperature is65℃. Al3+, Hg+, Fe2+, Fe3+have a strong inhibiting effect on recombinant enzyme activity. K+, NH4+, Li+, Zn2+, Mn2+, SDS, EDTA perform at different extent as inhibitors.The effect of K+, NH4+, Li+is much weaker. Mg2+, Ba2+have no effect on the recombinant rmannanase activity. Co2+play a role as a promoter. Promote effect of Na+、Ca2+is weaker. When NaCl concentration was below150mg/mL, the relative enzyme activity was above100%.
Keywords/Search Tags:beta-mannanase, strain, recombination, enzymatic characteristics
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