海洋溶菌酶产生菌YJ007的选育、鉴定和发酵条件优化
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摘要
海洋微生物溶菌酶与动植物来源的溶菌酶及其它微生物来源的溶菌酶相比具有独特的优势，应用前景广泛。本论文对一株产溶菌酶的海洋芽孢杆菌进行了选育、鉴定和发酵条件优化的研究，为海洋微生物溶菌酶的研究和应用奠定了基础。
以一株产溶菌酶的海洋芽孢杆菌S-12-86为出发菌株，对其进行原生质体的制备和再生。结果表明原生质体的最佳制备条件为：以菌株S-12-86培养18h，采用溶菌酶液与菌液1：1配比，溶菌酶浓度为1．0mg／mL，在35℃下，酶解30min，原生质体形成率为97．6％，再生率为23．6％。将此条件下获得的原生质体于30W紫外灯下80cm照射120s进行诱变处理。诱变处理后初筛复筛，并结合产酶性能稳定性分析，获得了一株产溶菌酶活性比原始菌株提高46．8％且性能稳定的突变菌株YJ007。
对筛选出的高产菌株YJ007进行其形态观察和部分生理生化指标的测定以及系统发育学分析。结果表明，该菌株具有典型的芽孢杆菌属的形态学特征；YJ007的16S rDNA序列在GenBank的注册号为EU016215，与Bacillus licheniformisDSM13~T(X68416)同源性高达99．68％，确定为地衣芽孢杆菌(B.licheniformis)。
采用单因素实验对突变株YJ007的产酶条件进行了研究，得到其最佳培养基成份：玉米粉、蛋白胨、牛肉膏、NaCl、MgSO_4；最佳培养条件：初始pH值为7．5，温度为30℃，转速为200r／min，接种量为6％，接种时间为18～20h，发酵培养时间为20～22h。在此基础上采用二次回归正交设计对突变株YJ007的发酵培养基进行了优化。得到理想最优培养基条件：玉米粉0．200％、蛋白胨0．773％、牛肉膏0．132％、NaCl 0．320％、MgSO_4 0．759×10~(-5)mol／L。
The lysozyme from marine microorganism has unique advantages compared with those from various animals, plants and other microorganisms. Because of its promising application prospect, breeding, identification of one marine Bacillus producing lysozyme and optimization of fermentation conditions were investigated as a basis of further study and application on lysozyme from marine microorganism.
Preparation and regeneration of strain S-12-86 producing lysozyme were studied. The results showed that optimal conditions of the protoplast preparation and regeneration were as follows: incubation time 18h, proportion of the strain to lysozyme 1:1, concentrations of lysozyme 1.0mg/ml, and enzymolysis 30min at 35℃. Under the optimal conditions the rates of protoplast preparation and regeneration were 97.6% and 23.6%, respectively. When the protoplasts of strain S-12-86 were irradiated 120s by a 30w UV lamp from 80cm distance , the optimal condition of protoplasts mutagenesis was achieved. After irradiation, a genetically stable mutant strain YJ007 was obtained by primary and secondary screens and genetic stability analysis, lysozyme activity for the new strain was 46.8% higher than that for the original strain S-12-86.
The lysozyme-producing strain YJ007 was identified as a member of the genus Bacillus based on phenotypic and phylogenetic analysis. 16S rDNA gene sequence has been registered in GenBank (number EU016215), which analysis of YJ007 showed that similarity of sequence is 99.68% with Bacillus licheniformis DSM13~T (X68416), so YJ007 was identified as B. licheniformis..
The fermentation conditions of strain YJ007 were investigated by unifactor experiment. The results indicated that medium ingredients were corn flour, peptone, beef extract, NaCl, and MgSO_4, and the fermentation conditions were incubation temperature 30℃, initial pH7.5, inoculum size 6%, rotationl speed 200 r/min, inoculum time 18-20h, and fermentation time 20-22h. Quadratic regression orthogonal design was applied to optimize the composition of medium used on shaking flask fermentation. The optimized medium ingredients were corn flour 0.200%, peptone 0.773%, beef extract 0.132%, NaCl 0.320%, and MgSO_40.759×10~(-5) mol/L.
以一株产溶菌酶的海洋芽孢杆菌S-12-86为出发菌株，对其进行原生质体的制备和再生。结果表明原生质体的最佳制备条件为：以菌株S-12-86培养18h，采用溶菌酶液与菌液1：1配比，溶菌酶浓度为1．0mg／mL，在35℃下，酶解30min，原生质体形成率为97．6％，再生率为23．6％。将此条件下获得的原生质体于30W紫外灯下80cm照射120s进行诱变处理。诱变处理后初筛复筛，并结合产酶性能稳定性分析，获得了一株产溶菌酶活性比原始菌株提高46．8％且性能稳定的突变菌株YJ007。
对筛选出的高产菌株YJ007进行其形态观察和部分生理生化指标的测定以及系统发育学分析。结果表明，该菌株具有典型的芽孢杆菌属的形态学特征；YJ007的16S rDNA序列在GenBank的注册号为EU016215，与Bacillus licheniformisDSM13~T(X68416)同源性高达99．68％，确定为地衣芽孢杆菌(B.licheniformis)。
采用单因素实验对突变株YJ007的产酶条件进行了研究，得到其最佳培养基成份：玉米粉、蛋白胨、牛肉膏、NaCl、MgSO_4；最佳培养条件：初始pH值为7．5，温度为30℃，转速为200r／min，接种量为6％，接种时间为18～20h，发酵培养时间为20～22h。在此基础上采用二次回归正交设计对突变株YJ007的发酵培养基进行了优化。得到理想最优培养基条件：玉米粉0．200％、蛋白胨0．773％、牛肉膏0．132％、NaCl 0．320％、MgSO_4 0．759×10~(-5)mol／L。
The lysozyme from marine microorganism has unique advantages compared with those from various animals, plants and other microorganisms. Because of its promising application prospect, breeding, identification of one marine Bacillus producing lysozyme and optimization of fermentation conditions were investigated as a basis of further study and application on lysozyme from marine microorganism.
Preparation and regeneration of strain S-12-86 producing lysozyme were studied. The results showed that optimal conditions of the protoplast preparation and regeneration were as follows: incubation time 18h, proportion of the strain to lysozyme 1:1, concentrations of lysozyme 1.0mg/ml, and enzymolysis 30min at 35℃. Under the optimal conditions the rates of protoplast preparation and regeneration were 97.6% and 23.6%, respectively. When the protoplasts of strain S-12-86 were irradiated 120s by a 30w UV lamp from 80cm distance , the optimal condition of protoplasts mutagenesis was achieved. After irradiation, a genetically stable mutant strain YJ007 was obtained by primary and secondary screens and genetic stability analysis, lysozyme activity for the new strain was 46.8% higher than that for the original strain S-12-86.
The lysozyme-producing strain YJ007 was identified as a member of the genus Bacillus based on phenotypic and phylogenetic analysis. 16S rDNA gene sequence has been registered in GenBank (number EU016215), which analysis of YJ007 showed that similarity of sequence is 99.68% with Bacillus licheniformis DSM13~T (X68416), so YJ007 was identified as B. licheniformis..
The fermentation conditions of strain YJ007 were investigated by unifactor experiment. The results indicated that medium ingredients were corn flour, peptone, beef extract, NaCl, and MgSO_4, and the fermentation conditions were incubation temperature 30℃, initial pH7.5, inoculum size 6%, rotationl speed 200 r/min, inoculum time 18-20h, and fermentation time 20-22h. Quadratic regression orthogonal design was applied to optimize the composition of medium used on shaking flask fermentation. The optimized medium ingredients were corn flour 0.200%, peptone 0.773%, beef extract 0.132%, NaCl 0.320%, and MgSO_40.759×10~(-5) mol/L.
引文
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