本文選題：右旋糖酐酶 + 圓弧青霉�。� 參考：《安徽工程大學》2017年碩士論文

【摘要】：右旋糖酐酶(Dextranase;EC 3.2.1.11)可專一性催化裂解右旋糖酐(Dextran)分子中的α-1,6葡萄糖苷鍵,用于制糖工業(yè)降低甘蔗汁粘度、醫(yī)藥工業(yè)中低分子量藥用右旋糖酐制備、牙醫(yī)業(yè)去除牙齦糖苷沉積等領(lǐng)域,工業(yè)應用廣泛,市場需求巨大。當前,右旋糖酐酶生產(chǎn)菌株產(chǎn)酶水平偏低,使得規(guī)模工業(yè)發(fā)酵受限,生產(chǎn)效率不高。為提高右旋糖酐酶的產(chǎn)量以適應實際應用的需要,本研究開展圓弧青霉產(chǎn)右旋糖酐酶菌株的誘變選育工作。首先采用常壓室溫等離子體(ARTP)和紫外輻照(UV-ray)技術(shù),對右旋糖酐酶產(chǎn)生菌圓弧青霉Penicilliumcyclopium(P.cyclopium 4022)開展多輪誘變處理,研究藍色葡聚糖平板透明圈圈徑比與搖瓶酶活的相關(guān)性,建立右旋糖酐酶平板透明圈法檢出突變體的方法,以獲得右旋糖酐酶高產(chǎn)突變株;在此基礎上,對突變株P(guān).cyclopium12p27產(chǎn)右旋糖酐酶的培養(yǎng)基營養(yǎng)條件及產(chǎn)酶培養(yǎng)條件開展優(yōu)化研究,以提升圓弧青霉產(chǎn)右旋糖酐酶的產(chǎn)酶能力;并對P.cyclopium 12p27所產(chǎn)右旋糖酐酶進行分離與純化,考察其溫度、pH穩(wěn)定性及其酶學性質(zhì)。主要研究結(jié)果如下:1.采用ARTP輻照和紫外誘變技術(shù)對P.cyclopium 4022誘變處理,經(jīng)過平板透明圈法初篩及搖瓶復篩,獲得一株遺傳性能穩(wěn)定的右旋糖酐酶高產(chǎn)突變株P(guān).cyclopium 12p27,該菌株較出發(fā)菌株酶活力分別提高了49.64%(ARTP)和 128.18%(UV-ray)。構(gòu)建了 Blue-Dextran 2000 平皿透明圈初篩與搖瓶發(fā)酵復篩相結(jié)合的突變株篩選方法:Blue-Dextran 2000平皿透明圈圈徑比與搖瓶發(fā)酵產(chǎn)酶酶力相關(guān)性良好。2.通過單因素實驗研究碳源、氮源以及表面活性物質(zhì)對酶活的影響,進而結(jié)合數(shù)理統(tǒng)計法對圓弧青霉產(chǎn)右旋糖酐酶發(fā)酵條件進行優(yōu)化研究。采用響應面實驗設計對產(chǎn)酶條件進行優(yōu)化,得到圓弧青霉產(chǎn)右旋糖酐酶最佳發(fā)酵條件為:D70:蔗糖49.5g/L,牛肉膏4.1g/L,山梨醇19.70mmol/L。MgSO4·7H2O0.5g/L、KCl0.5g/L、FeSO4·4H2O0.01g/L、K2HPO41.0g/L、pH6.5。在該條件下右旋糖酐酶產(chǎn)酶酶活達到10.95U/mL,較優(yōu)化提升了 173.07%。3.對產(chǎn)酶培養(yǎng)條件中的不同因素進行優(yōu)化處理,進一步提高經(jīng)誘變選育得到的高產(chǎn)菌株12P27的產(chǎn)酶酶活。包括對發(fā)酵過程中的培養(yǎng)溫度、搖床轉(zhuǎn)速、培養(yǎng)基初始pH、搖瓶裝液量與接種量等培養(yǎng)條件進行擇優(yōu)篩選。獲得最有利于發(fā)酵菌株產(chǎn)酶的培養(yǎng)條件為:在30℃下,控制搖床轉(zhuǎn)速為230r/min,搖瓶裝液量40mL,初始pH為7,接種量0.5mL,產(chǎn)酶酶活達到14.48U/mL,較優(yōu)化前的10.95U/mL,提高了 1.32倍。4.利用75%飽和度的硫酸銨沉淀與DEAE Cellulose DE-52離子交換層析,對P.cyclopium12p27右旋糖酐酶進行分離純化。使右旋糖酐酶的比活力從124.53U/mg提高到1052.71U/mg,純化了 8.45倍,回收率達到10.98%。經(jīng)SDS-PAGE電泳鑒定,純化后的右旋糖酐酶純度已達到電泳純,表觀分子量約65 kDa。5.P.cyclopium 12p27右旋糖酐酶的酶學性質(zhì)研究表明:該酶在溫度30℃時表現(xiàn)出良好的熱穩(wěn)定性,最適催化溫度為40℃;最適pH為6.0,偏堿性條件下右旋糖酐酶活力保留效果優(yōu)于偏酸條件,在pH4～7范圍內(nèi),右旋糖酐酶穩(wěn)定性良好。Fe2+和K+對其酶活性具有促進作用,而Pb2+、Al3+、Ag+、Mn2+、Co2+和Zn2+對酶活性有一定的抑制作用。該酶對于不同分子量右旋糖酐的水解性能有差異,對大分子右旋糖酐的水解能力強于中低分子量右旋糖酐,對D70以上的右旋糖酐水解能力高于D3、D40等小分子右旋糖酐底物。采用米氏方程的轉(zhuǎn)換形式,作Lineweaver-Burk雙倒數(shù)圖,得到米氏常數(shù)Km值為4.4827mmol/L,Vmax值為 1.1461μmol·mL~(-1)·min~(-1)。
[Abstract]:Dextranase (EC 3.2.1.11) can be used to catalyze the catalytic cracking of alpha -1,6 glucoside bonds in the Dextran molecule, which is used in sugar industry to reduce the viscosity of sugarcane juice, the preparation of low molecular weight medicinal dextran in the pharmaceutical industry, and the removal of gingival glycosides from the medical industry. The industry is widely used and the market demand is huge. The production strain of dextran producing strain is low, which makes the industrial fermentation limited and the production efficiency is not high. In order to improve the production of dextran enzyme to meet the needs of practical application, this study carried out the mutagenesis and breeding of the strain of dextran produced by Penicillium arcus. First, the ambient temperature plasma (ARTP) and ultraviolet radiation (UV-ray) were used. Technology, a multi wheel mutagenesis was carried out on the dextran producing strain of Penicillium Penicilliumcyclopium (P.cyclopium 4022), and the correlation between the transparent circle diameter ratio of the blue dextran plate and the enzyme activity of the shake flask was studied. The method of detecting the mutant by the transparent ring method of the dextran enzyme plate was established in order to obtain the mutant strain with high dextran enzyme production. The nutrient conditions of mutant P.cyclopium12p27 producing dextran and the cultivation conditions of enzyme production were optimized to improve the enzyme production capacity of dextran produced by Penicillium ARCIS, and to separate and purify the dextran enzyme produced by P.cyclopium 12p27, and investigate its temperature, pH stability and its enzymology properties. As follows: 1. the mutagenesis of P.cyclopium 4022 was treated by ARTP irradiation and ultraviolet mutagenesis. A mutant strain P.cyclopium 12p27 with high genetic performance and stable dextran enzyme was obtained by a transparent plate screening and shake flask rescreening. The strain was 49.64% (ARTP) and 128.18% (UV-ray) higher than the enzyme activity of the starting strain, and Blue was constructed. Selection of mutant strain screening methods combined with -Dextran 2000 Petri dish transparent ring initial screening and shake flask fermentation: a good correlation between Blue-Dextran 2000 plate transparent circle diameter ratio and shake flask fermentation production of enzyme force.2. through single factor experiment to study the effect of carbon source, nitrogen source and surfactant on enzyme activity, and then combined with mathematical statistics method for circular arc green. Optimization of fermentation conditions of mildew producing dextranase. The optimum fermentation conditions were obtained by the response surface experiment design. The optimum fermentation conditions of the enzyme were as follows: D70: sucrose 49.5g/L, beef paste 4.1g/L, sorbitol 19.70mmol/L.MgSO4. 7H2O0.5g/L, KCl0.5g /L, FeSO4. 4H2O0.01g/L, K2HPO41.0g/L, pH6.5. in this condition The enzyme activity of lower dextran enzyme production reached 10.95U/mL, optimized and improved 173.07%.3. to optimize the different factors in the culture conditions of enzyme production, and further improved the enzyme production of high yield strain 12P27, which was induced by mutagenesis, including the culture temperature of the fermentation process, the rotational speed of the rocking bed, the initial pH of the medium, the volume of the shake bottling liquid and the connection. The optimum conditions for the cultivation of the fermentation conditions were as follows: at 30, the rotation speed of the rocking bed was 230r/min, the volume of the shake bottled liquid was 40mL, the initial pH was 7, the inoculation amount was 0.5mL, the enzyme production reached 14.48U/mL, and the optimized 10.95U/mL was improved by 1.32 times the 75% saturation of the ammonium sulfate precipitation and DE. AE Cellulose DE-52 ion exchange chromatography was used to separate and purify P.cyclopium12p27 dextran. The specific activity of dextran was increased from 124.53U/mg to 1052.71U/mg, purified 8.45 times, and the recovery rate was identified by SDS-PAGE electrophoresis. The purity of the purified dextran has reached pure electrophoresis and the apparent molecular weight is about 65 kDa.5.P. The enzymatic properties of cyclopium 12p27 dextran showed that the enzyme exhibited good thermal stability at 30 C, the optimum catalytic temperature was 40, the optimum pH was 6, and the activity retention of dextran was better than the partial acid condition under the alkaline condition. In the range of pH4 ~ 7, the stability of dextran enzyme was good.Fe2+ and K+ on its enzyme activity. Pb2+, Al3+, Ag+, Mn2+, Co2+ and Zn2+ have a certain inhibitory effect on the enzyme activity. The hydrolysis of dextran with different molecular weight is different, the hydrolysis ability of the macromolecular dextran is stronger than that of low molecular weight dextran, and the hydrolysis ability of dextran above D70 is higher than that of D3, D40 and other small molecules. Using the conversion form of the Michaelis equation, the Lineweaver-Burk double reciprocal graph is used to obtain the Km value of 4.4827mmol/L and the Vmax value of 1.1461 Mu mol. ML~ (-1). Min~ (-1).

【學位授予單位】：安徽工程大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q93

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相關(guān)期刊論文 前10條

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本文編號：1870086