产淀粉酶海洋放线菌的筛选与诱变选育【毕业论文】.docVIP

本科毕业论文 （20 届） 产淀粉酶海洋放线菌的筛选与诱变选育 专业：生物科学 目录 摘要 I Abstract II 引言 1 材料与方法 3 1.1 材料 3 1.1.1仪器与设备 3 1.1.2药品与试剂 3 1.1.3培养基 3 1.1.4诱变源 4 1.2 实验方法 4 1.2.1采样及处理 4 1.2.2菌株的分离培养 4 1.2.3初步定向筛选 4 1.2.4保种 4 1.2.5摇瓶复筛 5 1.2.6酶活力测定 5 1.2.7菌悬液的制备 6 1.2.8物理诱变 6 1.2.9原生质体的制备 6 1.2.10原生质体融合 6 1.2.11原生质体再生 7 1.2.12粗酶特征初步分析 7 实验结果与分析 8 2.1 产淀粉酶放线菌的筛选 8 2.2 产淀粉酶菌株的诱变 8 2.3 粗酶特征初步分析 9 讨论 12 3.1 诱变方式的选择 12 3.2 粗酶特征变量选择 13 小结 14 参考文献 15 致谢 17 【目的】通过合适的诱变方法，选育出一株海洋来源产淀粉酶能力显著提高的菌株。【方法】从浙江舟山附近海域进行多层次取样，利用鉴定培养基观察水解圈大小，比较透明圈直径（C）和菌落直径（H）的比值，初步筛选产淀粉酶的放线菌。用YOO改良法测定酶活力，复筛产酶能力强的菌株作为出发菌株，通过紫外线、水浴热处理和超声波3种物理诱变方式以及原生质体融合技术选育高产淀粉酶菌株。探究了突变株粗酶液在不同温度、pH、Ca2+浓度下的酶活力变化。【结果】从海泥、海水和海藻中分离到产淀粉酶的放线菌43株，取其中产酶能力最高的3株中的一株，编号为A19，其初始酶活力为46.5 u/mL。经诱变选育，发现在15W紫外线，照距30 cm，照射时间30s和功率150W超声波处理45min时，酶活力最高，111.7 u/mL，提高140.21%突变株粗酶液在pH6.0时活性最高，在pH7.5时存在一个峰值经30℃处理后酶活较高，在60℃、70℃时有失活趋势；0.03 mol/L Ca2+处理后，酶活力显著提高。 [Abstract] [Objective] To breed a strain of marine whose amylase production capacity remarkably improved by appropriate mutagenesis method. [Method] Multi-level sampling were taken from waters near Zhoushan in Zhejiang Province. Make the use of identifying medium to observe hydrolysis cycle, compare the ratio of the diameter of transparent circle (C) and the diameter of the bacterial colony (H), preliminary screen actinomycetes which could produce amylase. Make the use of Yoo improved method to isolate the vitality of enzyme, rescreen strain and sort out some good strain with strong ability of producing enzyme which will be starting strain. UV, water bath and ultrasonic wave these 3 physical mutagenesis methods and the technology of protoplast fusion were taken to breed strain with high-producing enzyme. Explored the variation of mutant crude enzyme at different temperatures, pH and Ca2+ concentration. [Outcome] 43 actinomycetes that can produce amylase have isolated from marine mud, sea water and algae. I adopt 1 from 3 strains which have the best capacity of producing enzyme, tag it as A19, and its initial enzym