印尼热泉菌产高温卡拉胶酶的分离纯化、酶学性质及其降解特性研究

发布时间：2018-02-09 19:02

  本文关键词： 热泉菌 卡拉胶酶 芽孢杆菌 培养优化 分离纯化 酶学性质 降解特性　出处：《青岛科技大学》2014年硕士论文　论文类型：学位论文

【摘要】：本论文目的是从印尼热泉菌中筛选具有产卡拉胶酶活性的菌株，并对高活性菌株的发酵条件进行优化；纯化该热泉菌所产卡拉胶酶，系统研究其酶学性质；分析该酶的降解特性，阐明其降解产物及其类型，以期为卡拉胶酶和卡拉寡糖的工业化生产提供理论和技术支持。 产卡拉胶酶热泉菌株的筛选。从印尼泥样中通过富集培养、稀释涂布及划线分离的方法获得了94株菌，其中14株具有卡拉胶降解活性。并进一步通过平板划线与卢戈氏碘液染色相结合的方式，对这14株菌进行复筛，最终筛选出菌株Lc50-1的产酶活性最高，其活性为3.56U/mL。经过形态学观察及16S rDNA序列等分析，最终确定菌株Lc50-1为芽孢杆菌属（Bacillus sp.）。 菌株Bacillus sp. Lc50-1产酶发酵条件的优化。分别以装液量、接种量、转速、温度、pH、碳源、氮源和金属离子作为单一变量进行单因素实验，，筛选出对菌株的酶活具有显著影响的单因素的取值范围；用Design-Expert软件对所有单因素进行Plackett-Burman实验设计，筛选出影响菌株产酶活力的4个最主要因素：温度、碳源、氮源和金属离子；用最陡爬坡实验逼近最大响应区域，再对这4种因素用Box-Behnken设计及响应面分析法进行回归分析。经过多次实验确定菌株Bacillus sp. Lc50-1的最佳产酶条件为：500mL三角瓶装入200mL发酵培养基、摇床转速150r/min、接种量1%、pH7.0、培养温度47.5℃；最佳培养基组成为：蛋白胨0.25%、卡拉胶0.3%、KCl21.55mmol/L，优化后发酵上清液的酶活达到8.9U/mL，比优化前提高了1.5倍。 菌株Lc50-1产卡拉胶酶的纯化。菌株Lc50-1进行液体发酵培养24h，离心去菌体得到发酵上清液；通过30%-80%硫酸铵分级沉淀获得粗酶，然后采用Q-Sepharose Fast Flow离子交换层析和Sephacryl S-200HR凝胶过滤层析技术对粗酶进行分离纯化，纯化后的样品经SDS-PAGE电泳检测显示为单一条带，经计算确定此卡拉胶酶的分子量为38kDa。 菌株Lc50-1所产卡拉胶酶的酶学性质研究。研究结果表明，该卡拉胶酶的最适pH范围为9.0左右，说明该酶在碱性环境中有助于该卡拉胶酶行使其生物学功能；最适反应温度为75℃，并且在30min之内仍可以保持其活性在20%以上，具有较好的高温耐受性；当酶液中加入氯化铁和氯化镁时，卡拉胶酶活性丧失；酶液中加入EDTA、氯化锶、氯化锰、氯化钾、氯化镉对卡拉胶酶的活性具有抑制作用；而酶液中加入氯化钴、氯化钙、氯化铜、氯化钠对卡拉胶酶的活性具有不同程度的增强作用，其中氯化钙、氯化铜的作用尤为显著使酶活分别提高了1.7倍和2.2倍；酶的专一性实验表明该酶对λ-卡拉胶的降解具有强烈的专一性，对κ-卡拉胶、ι-卡拉胶、琼胶和褐藻胶没有水解作用。 菌株Lc50-1所产卡拉胶酶的降解特性。采用薄层色谱对菌株Bacillus sp.Lc50-1所产卡拉胶酶的降解特性进行研究，结果表明，降解产物主要是λ-新卡拉二糖，与现已报道细菌的λ-卡拉胶酶的酶解产物有所不同，说明该卡拉胶酶一种新型卡拉胶酶。
[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

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