本文關(guān)鍵詞： β-木糖苷酶 Enterobacter cloacae 發(fā)酵優(yōu)化 克隆表達　出處：《華南理工大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：半纖維素作為應(yīng)用潛力巨大的可再生資源,其主要成分是木聚糖。木聚糖的完全降解需要組成復(fù)雜的木聚糖降解酶系。β-木糖苷酶是木聚糖降解酶系的關(guān)鍵酶,其酶催化功能是從低聚木糖和木二糖的非還原性末端開始水解而產(chǎn)生木糖單糖。研究β-木糖苷酶對于半纖維素的降解應(yīng)用意義重大。本文以從森林土壤樣品中篩選出的一株高產(chǎn)β-木糖苷酶菌株為基礎(chǔ),測定發(fā)酵產(chǎn)酶的最高酶活力值和相應(yīng)比酶活力值分別為1.29 U/mL和1.05 U/mg,并通過測定胞外液和胞內(nèi)液確定該菌所產(chǎn)β-木糖苷酶為胞內(nèi)酶。經(jīng)16S rDNA系統(tǒng)進化樹分析、形態(tài)學(xué)觀察和生理生化實驗鑒定,確定該菌株為Enterobacter cloacae,并命名為Enterobacter cloacae LY-62。以初始發(fā)酵培養(yǎng)條件為基礎(chǔ),對Enterobacter cloacae LY-62進行發(fā)酵產(chǎn)酶條件的初步優(yōu)化,考察不同碳源及其濃度、不同氮源及其濃度、培養(yǎng)基起始pH值和培養(yǎng)溫度對發(fā)酵產(chǎn)酶的影響。經(jīng)優(yōu)化發(fā)酵條件后,Enterobacter cloacae LY-62發(fā)酵產(chǎn)酶水平最高可達到的酶活力值和相應(yīng)比酶活力值分別為2.2 U/mL和2.87 U/mg,產(chǎn)酶水平提高了170%左右。研究優(yōu)化發(fā)酵條件下該菌的產(chǎn)酶曲線和生長曲線,發(fā)現(xiàn)該菌株具有生長快速、木聚糖利用效率高、產(chǎn)酶快和酶活力高的特點。為進一步研究Enterobacter cloacae LY-62的β-木糖苷酶,設(shè)計引物成功對其β-木糖苷酶基因進行了克隆表達以及功能基因的分子鑒定。以該酶基因序列和氨基酸序列為基礎(chǔ),應(yīng)用生物信息學(xué)方法獲得了其蛋白質(zhì)氨基酸組成、疏水性和相對分子量等一級結(jié)構(gòu)的相關(guān)信息,并對其二級結(jié)構(gòu)元件和蛋白質(zhì)的三級空間立體結(jié)構(gòu)進行預(yù)測。隨后,對Enterobacter cloacae LY-62的β-木糖苷酶進行表達,重組蛋白的N端攜帶組氨酸標簽,重組蛋白His-Xyl62經(jīng)鎳柱親和層析純化后進行酶活性測定并研究其酶學(xué)特性:該β-木糖苷酶的最適pH值為7.0,酶活性在弱酸性和弱堿性的條件下較穩(wěn)定;最適酶反應(yīng)溫度為40℃,熱穩(wěn)定性相對較差;以PNPX為底物測定的His-Xyl62的米氏常數(shù)Km、最大反應(yīng)速率Vmax和催化速度常數(shù)Kcat分別為1.55 mmoL/L、38.61 umoL/(min·mg)和8.51 s~(-1)。
[Abstract]:Hemicellulose is a renewable resource with great application potential. The main component is xylan. The complete degradation of xylan requires the formation of complex xylanase system. 尾 -xylosidase is the key enzyme of xylan degradation enzyme system. The catalytic function of 尾 -xylosidase is to hydrolyze xylose and xylose from the non-reducing end of xylose and to produce xylose monosaccharide. It is important to study the application of 尾 -xylosidase in hemicellulose degradation. A strain with high 尾 -xylosidase production was screened. The highest enzyme activity and the corresponding specific enzyme activity were 1.29 U / mL and 1.05 U / mg, respectively. The 尾 -xylosidase produced by the strain was identified as intracellular enzyme by measuring the extracellular fluid and intracellular fluid. The phylogenetic tree analysis of 16s rDNA, morphological observation and physiological and biochemical experiments were performed to identify the 尾 -xylosidase. The strain was identified as Enterobacter cloacae. And named Enterobacter cloacae LY-62.Based on the initial fermentation conditions. The fermentation conditions of Enterobacter cloacae LY-62 were optimized, and different carbon sources and their concentrations, different nitrogen sources and their concentrations were investigated. The effects of the initial pH value and the culture temperature on the fermentation enzyme production were studied and the fermentation conditions were optimized. The highest enzyme activity and corresponding specific enzyme activity of Enterobacter cloacae LY-62 were 2.2 U / mL and 2.87 U / mL, respectively. U/mg. The enzyme production level was increased by about 170%. The enzyme production curve and growth curve of the strain were studied under optimized fermentation conditions. It was found that the strain had rapid growth and high Xylan utilization efficiency. In order to further study the 尾 -xylosidase of Enterobacter cloacae LY-62. The 尾 -xylosidase gene was cloned and expressed successfully with primers, and the functional gene was identified based on the sequence and amino acid sequence of the 尾 -xylosidase gene. Bioinformatics was used to obtain the information about the amino acid composition, hydrophobicity and relative molecular weight of the protein. The third order spatial stereoscopic structure of its secondary structure element and protein was predicted. Then, the 尾 -xylosidase of Enterobacter cloacae LY-62 was expressed. The N-terminal of the recombinant protein carries a histidine label. The recombinant protein His-Xyl62 was purified by nickel column affinity chromatography and its enzymatic properties were studied. The optimum pH value of the 尾 -xylosidase was 7.0. The enzyme activity was stable under the condition of weak acidity and weak alkalinity. The optimum reaction temperature is 40 鈩，

本文編號：1482616