论文标题:灭活原生质体融合选育α-ALDC-高产菌株
Screening of α-ALDC-higher-Producing Strains by Inactivated Protoplast Fusion
论文作者 周海霞
论文导师 阚振荣,论文学位 硕士,论文专业 微生物学
论文单位 河北大学,点击次数 290,论文页数 55页File Size1734k
2004-06-01论文网 http://www.lw23.com/lunwen_1229922/ 枯草芽抱杆菌;Q一乙酞乳酸脱梭酶;灭活原生质体;融合
Bacillus subtilis;a-Acetolactate decarboxylase;inactivated protoplast;fusion
α-乙酰乳酸脱羧酶可以去除啤酒中的双乙酰，在啤酒工业中有着广泛的应用前景。双亲灭活原生质体融合技术优点较多，是原生质体融合育种中针对无遗传标记同种不同株双亲的一种常用的有效方式。因此，对本实验室保存的两株产α-乙酰乳酸脱羧酶的枯草芽孢杆菌(Bacillus subtilis)进行双亲灭活原生质体融合，筛选高酶活菌株。 试验首先确定了比较适合的双亲菌株培养条件。选择使用了完全培养液和发酵培养液两种培养基以及完全液体培养基20mL和40mL装液量。绘制了不同条件下的生长曲线，确定了最佳培养条件是：将菌株由斜面接种到完全固体培养基上，活化培养一天，再将其接种到液体完全培养基，37℃，200rpm，摇床培养14h，最后按5％的接种量接种到20mL的液体完全培养基，继续活化3.5—4h。将培养好的菌体收集起来，进行溶菌酶处理，制备原生质体。本试验选择了多个溶菌酶浓度，在32℃条件下处理双亲菌株。通过绘制形成率和再生率曲线，认为在32℃，用50μg／mL的溶菌酶处理双亲菌株20min，形成率较高，而且又有一定的再生能力，适合融合。将制备的V-20原生质体进行紫外灭活，3226—5原生质体进行热灭活，通过分析灭活后原生质体的融合率确定最佳融合条件是用pH9.0的40％的PEG6000，38℃，作用于混合菌液15min。本试验共得到了900株融合子，将这些融合子传代10次并测酶活。最后筛得3株菌，其酶解后的上清液酶活高于亲株。试验最后通过对融合子与双亲菌株的DNA含量的简单比较，进一步验证了筛得的菌株为融合子。
a -Acetolactate decarboxylase( a -ALDC) which can remove diacetyl from the beer is used widely in beer industry. Inactivated protoplasts fusion method has more advantages in breeding techniques and also it has been used effectively in parents without any heritable labels. Bacillus subtilis V-20 and 3226-5 producing a -ALDC are kept in our laboratory. The inactivated protoplast fusion method of Bacillus subtilis V-20 and 3226-5 is carried out to select some strains with higher enzyme activity of a -ALDC.At the beginning of this test, the proper culture conditions of the parents are determined and the growth curve of two different culture solution (complete culture fluid and fermented broth ) and two different volume of the complete culture fluid(20mL and 40mL) are selected. At first, the strains should be activated in complete culture plates and then transferred into the complete culture fluid to shake cultivate under the condition of 37?,200rpm for 14 hours. At last, ImL bacterial fluid should be inoculated to 20mL complete culture fluid to shake culture for 3.5-4 hours. The collection of bacteria is treated by lysozyme. In the test, several lysozyme concentrations are selected to treat parents in 32? The curve of protoplast formation rates and regeneration rates show that parents should be treated at lysozyme concentration of 50 P g/mL, for 20min. Under above conditions, the formation rate and regeneration rate are better for fusion of Bacillus subtilis V-20 and 3226-5. Before fusion, the strain V-20 is inactivated by ultraviolet and the strain 3226-5 is inactivated by boiling water bath. The result of fusion rates presents the better fusion condition of parents. The two strains are fused by PEG6000 at concentration of 32%, in water bath of 38癈 for 15min. After the fusants are transferred for ten generation, they are tested the enzyme activity of a -ALDC. Three fusants are got and their enzyme activitiesare higher than their parents. At the end of the test, a simple comparison of DNA quantity between fusants and parents shows these strains are fusants.
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