本文關鍵詞： 熱泉菌 卡拉膠酶 芽孢桿菌 培養(yǎng)優(yōu)化 分離純化 酶學性質(zhì) 降解特性　出處：《青島科技大學》2014年碩士論文　論文類型：學位論文

【摘要】：本論文目的是從印尼熱泉菌中篩選具有產(chǎn)卡拉膠酶活性的菌株，并對高活性菌株的發(fā)酵條件進行優(yōu)化；純化該熱泉菌所產(chǎn)卡拉膠酶，系統(tǒng)研究其酶學性質(zhì)；分析該酶的降解特性，闡明其降解產(chǎn)物及其類型，以期為卡拉膠酶和卡拉寡糖的工業(yè)化生產(chǎn)提供理論和技術支持。 產(chǎn)卡拉膠酶熱泉菌株的篩選。從印尼泥樣中通過富集培養(yǎng)、稀釋涂布及劃線分離的方法獲得了94株菌，其中14株具有卡拉膠降解活性。并進一步通過平板劃線與盧戈氏碘液染色相結合的方式，對這14株菌進行復篩，最終篩選出菌株Lc50-1的產(chǎn)酶活性最高，其活性為3.56U/mL。經(jīng)過形態(tài)學觀察及16S rDNA序列等分析，最終確定菌株Lc50-1為芽孢桿菌屬（Bacillus sp.）。 菌株Bacillus sp. Lc50-1產(chǎn)酶發(fā)酵條件的優(yōu)化。分別以裝液量、接種量、轉速、溫度、pH、碳源、氮源和金屬離子作為單一變量進行單因素實驗，，篩選出對菌株的酶活具有顯著影響的單因素的取值范圍；用Design-Expert軟件對所有單因素進行Plackett-Burman實驗設計，篩選出影響菌株產(chǎn)酶活力的4個最主要因素：溫度、碳源、氮源和金屬離子；用最陡爬坡實驗逼近最大響應區(qū)域，再對這4種因素用Box-Behnken設計及響應面分析法進行回歸分析。經(jīng)過多次實驗確定菌株Bacillus sp. Lc50-1的最佳產(chǎn)酶條件為：500mL三角瓶裝入200mL發(fā)酵培養(yǎng)基、搖床轉速150r/min、接種量1%、pH7.0、培養(yǎng)溫度47.5℃；最佳培養(yǎng)基組成為：蛋白胨0.25%、卡拉膠0.3%、KCl21.55mmol/L，優(yōu)化后發(fā)酵上清液的酶活達到8.9U/mL，比優(yōu)化前提高了1.5倍。 菌株Lc50-1產(chǎn)卡拉膠酶的純化。菌株Lc50-1進行液體發(fā)酵培養(yǎng)24h，離心去菌體得到發(fā)酵上清液；通過30%-80%硫酸銨分級沉淀獲得粗酶，然后采用Q-Sepharose Fast Flow離子交換層析和Sephacryl S-200HR凝膠過濾層析技術對粗酶進行分離純化，純化后的樣品經(jīng)SDS-PAGE電泳檢測顯示為單一條帶，經(jīng)計算確定此卡拉膠酶的分子量為38kDa。 菌株Lc50-1所產(chǎn)卡拉膠酶的酶學性質(zhì)研究。研究結果表明，該卡拉膠酶的最適pH范圍為9.0左右，說明該酶在堿性環(huán)境中有助于該卡拉膠酶行使其生物學功能；最適反應溫度為75℃，并且在30min之內(nèi)仍可以保持其活性在20%以上，具有較好的高溫耐受性；當酶液中加入氯化鐵和氯化鎂時，卡拉膠酶活性喪失；酶液中加入EDTA、氯化鍶、氯化錳、氯化鉀、氯化鎘對卡拉膠酶的活性具有抑制作用；而酶液中加入氯化鈷、氯化鈣、氯化銅、氯化鈉對卡拉膠酶的活性具有不同程度的增強作用，其中氯化鈣、氯化銅的作用尤為顯著使酶活分別提高了1.7倍和2.2倍；酶的專一性實驗表明該酶對λ-卡拉膠的降解具有強烈的專一性，對κ-卡拉膠、ι-卡拉膠、瓊膠和褐藻膠沒有水解作用。 菌株Lc50-1所產(chǎn)卡拉膠酶的降解特性。采用薄層色譜對菌株Bacillus sp.Lc50-1所產(chǎn)卡拉膠酶的降解特性進行研究，結果表明，降解產(chǎn)物主要是λ-新卡拉二糖，與現(xiàn)已報道細菌的λ-卡拉膠酶的酶解產(chǎn)物有所不同，說明該卡拉膠酶一種新型卡拉膠酶。
[Abstract]:The purpose of this paper is to select strains with carrageenan - producing enzyme activity from Indonesian hot spring bacteria , optimize the fermentation conditions of highly active strains , purify the carrageenan enzyme produced by the hot spring bacteria , study its enzymatic properties , analyze the degradation characteristics of the enzyme , clarify its degradation products and their types , so as to provide theoretical and technical support for the industrial production of carrageenan and karaoke . Strain Lc50 - 1 was screened by enrichment culture , dilution coating and line - line separation from Indonesia mud samples . The 14 strains were screened by the method of enrichment culture , dilution coating and line separation . The 14 strains were screened by the method of plate - line and Lugol ' s iodine solution staining . The activity of the strain Lc50 - 1 was highest , and the activity of the strain Lc50 - 1 was 3.56U / mL . The strain Lc50 - 1 was finally determined to be Bacillus sp . Strain Bacillus sp . Lc50 - 1 was optimized for producing enzyme fermentation conditions . One - factor experiment was carried out with liquid loading , inoculation quantity , rotation speed , temperature , pH , carbon source , nitrogen source and metal ion as a single variable . Four of the most important factors affecting the enzyme activity of the strain were selected : temperature , carbon source , nitrogen source and metal ion ; the maximum response region was approached by the most steep slope climbing experiment , and the four factors were analyzed by Box - Behnken design and response surface analysis method . The optimal culture conditions of Lc50 - 1 were as follows : 500 mL flask was loaded into 200 mL of fermentation medium , the shaking table rotation speed was 150r / min , the inoculation quantity was 1 % , pH 7.0 , the culture temperature was 47.5 鈩

本文編號：1498637