【作者】 邸胜苗；

【导师】 阚振荣；

【作者基本信息】 河北大学 ， 微生物学， 2007， 硕士

【摘要】 α-乙酰乳酸脱羧酶（α-acetolactate decarboxylase；简称α-ALDC,EC.4.1.1.5）可以去除啤酒中的双乙酰前体,缩短啤酒熟化期,提高啤酒产量和质量,在啤酒工业中有着广泛的应用前景。因此对α-乙酰乳酸脱羧酶产生菌的育种尤为重要。本文以产α-乙酰乳酸脱羧酶的枯草芽孢杆菌（BacilluS subtilis.）W195为出发菌株,进行诱变育种,以期获得稳定高产菌株,并通过优化培养基组成、产酶条件等,进一步提高酶产量,旨在为α-乙酰乳酸脱羧酶的工业化生产提供一定的理论依据。主要包括以下几个方面：1、微波的诱变效应采用不同强度微波对枯草芽孢杆菌W195进行不同时间的诱变处理,研究微波对枯草芽孢杆菌的致死效应和诱变效应,考察不同剂量下的致死效果和诱变效果,并根据结果确定微波诱变的最佳剂量。结果表明,微波诱变剂量对菌体的致死率有较大的影响,相同强度微波辐射处理不同时间时,菌体致死率随着辐射时间的延长而增加；不同强度微波辐射处理相同时间时,菌体致死率随着辐射强度的增大而增加。微波的热效应十分明显,间歇处理可以明显降低热效应的影响。诱变剂量对菌体的诱变效应也有较大影响,菌株总突变率随着低强度微波或中等强度微波辐射时间的增加而增加；但正突变率在上升到一定程度后都呈下降趋势。微波诱变的最佳剂量为：低强度微波,辐射时间为30～40s。2、复合诱变与筛选通过对出发菌株进行微波、紫外线复合诱变处理,以提高目的产物—α-乙酰乳酸脱羧酶的产量,并考察获得的高产变异菌株的遗传稳定性。菌株W195经过微波单因子诱变、紫外线和微波复合诱变、微波单因子反复诱变等多次诱变处理,及大量变异菌株的发酵筛选试验后,得到一株酶活力有了明显提高的变异菌株CM91,该菌株酶活力达到了237.66u／g,相对出发菌株酶活力提高了174%,遗传稳定性试验表明该菌株的产酶能力保持稳定。3、发酵条件的初步优化对变异菌株CM91的摇瓶发酵条件进行了初步研究,包括发酵培养基组成和产酶条件的优化,以使其表现出更高的生产性能。通过单因子和多因子反复试验,最终确定菌株CM91的最佳发酵培养基为：葡萄糖25g／L、酵母膏12.5g／L、牛肉膏7.5g／L、硫酸铵8g/L、NaCl0.4g/L、K₂HPO₄0.03g/L、Mg²⁺0.030g/L、Mn²⁺0.00076g/L、Zn²⁺0.003g/L;通过对菌株CM91的培养条件进行优化,确定发酵产酶的最佳条件为：接种量9%、初始pH9.0、培养温度37℃,摇床转速220rpm、发酵时间19h。在优化发酵条件下菌株的酶活力比原始培养基提高了16.5%。更多还原

【Abstract】 a-acetolactate decarboxylase（a-ALDC, EC.4.1.1.5） is used widely in beer industry. It can remove diacetyl from the beer, reduce the production cycle and increase the quality of the beer. So radioactive breeding of a-acetolactate decarboxylase producing strain is very important. To obtain a strain with higher production, Bacillus subtilis W195 with a-acetolacetate decarboxylase activity as the initial strain was induced by radioactive breeding. Through optimizing microbial culture conditions and studying extract condition, the enzyme yield was reincreased. The main concent are showed as following aspects: 1. Mutagenic effect of microwave radiationTo research mutagenic effect of the microwave, and definite the best radioactive dosage of microwave, Bacillus subtilis W195 with a-acetolacetate decarboxylase activity was induced by different microwave intensity and different time. The result indicated that, the effect of dying by different microwave irradiation dosage was tremendous. When the strain was induced by the same microwave intensity but different time, the lethality of the strain increased along with the radiation time; When the strain was induced by the same time but different intensity, the lethality of the strain increased along with the intensity. Thermal reaction of microwave was extremely obvious, but it can be obviously reduced by batch treaded. The rate of total mutating increased when the radiactive time of low intensity microwave or medium intensity microwave increasing. The rate of positive mutating rised to a certain degree first, but then dropped. Based on the comparison and analysis, the optimal dosage of microwave irradiation was obtained, that is low intensity microwave of 30-40s. 2. Compound mutation and filtrationTo increase the enzyme yield of a-acetolactate decarboxylase, the initial strain was induced by microwave, ultraviolet radiation, and the genetic stability of the mutants was determined. After Bacillus subtilis W195 was induced by microwave, ultraviolet+microwave, microwave radiation, the mutant CM91 which a-acetolacetate decarboxylase yield was 174% higher than that of the initial strain was obtained, the enzyme yield of CM91 achieves 237.66u/g. The genetic stability trial indicated that the enzyme yield of the strain was quite stablable.3. Preliminary research of fermentation conditionsThrough the preliminary research of cultivation condition of the mutant strain CM91, the culturing conditions such as the optimal carbon and nitrogen source and their optimal concentration and so on, were determined. Through single factor and multi-factorial experiments, the best fermentation medium and the best ferment condition of CM91 was finally determined. The best ferment culture medium:glucose 25g/L, yeast extract 12.5g/L, beef extract 7.5g/L, （NH4）2SO₄8g/L, NaCl 0.4g/L, K2HPO₄ 0.03g/L, Mg²⁺0.030g/L, Mn²⁺ 0.00076g/L, Zn2+0.003g/L; The best ferment condition: inoculum 9%, initial pH9.0, temperature 37℃,220rpm, fermentation time 19h. At last the integration experiment showed the optimization culture was very good, which improved on enzyme activity a lot.更多还原