脂肪酶产生菌的细胞融合选育及其产酶条件优化
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摘要
脂肪酶(Lipase)是一种特殊的酯键水解酶,它可作用于甘油三酯的酯键,使甘油三脂降解为甘油二酯、单甘油酯、甘油和脂肪酸。脂肪酶是最早被人们研究的酶类之一,也是一种重要的工业用酶,被广泛应用于轻工领域。微生物脂肪酶被发现以来,由于其种类多,高度的底物专一性,宽范围的作用pH和温度,容易工业化生产应用等优点,选育脂肪酶高产菌株成了国内外相关研究的热点。
本文利用化学PEG融合法使白地霉(Geotrichum candidum NS3)原生质体和假丝酵母(Candida valida T2)原生质体融合,结合抗药性标记筛选方法,成功筛选出一株产脂肪酶能力较高并且生长能力较强的融合菌株,并对融合株的固态发酵产脂肪酶条件进行初步研究。主要研究工作和结果如下：
(1)脂肪酶双亲本菌株抗药性标记的选择。实验利用药物浓度梯度平板法和医学给药原则,选用12种药物分别对白地霉(Geotrichum candidum NS3)和假丝酵母(Candida valida T2)进行抗药性测定,成功筛选出白地霉和假丝酵母的抗药性标记。白地霉抗性而假丝酵母非抗性的药物标记为曲咪新乳膏浓度为2×103μg/ml,假丝酵母抗性而白地霉非抗性的药物标记则为硫酸铜浓度为2×103μg/ml。
(2)双亲本菌株原生质体制备和再生条件的研究。利用单因素实验探讨影响白地霉和假丝酵母原生质体形成率和再生率的各种因素,结果表明制备原生质体的优化条件为：双亲本菌龄为对数生长中期、1.5%的纤维素酶酶解1小时、酶解温度30℃、脱壁促进剂为0.05%的半胱氨酸、渗透压稳定剂为0.7MNaCl。
(3)双亲本原生质体融合及融合子的鉴定。利用化学PEG方法使双亲本原生质体融合,通过影印法筛选得到50株融合子。连续传代10次,获得8株性状稳定的融合子,再通过油脂同化平板法筛选得到一株融合菌株,命名为CG16。通过比较双亲本及融合子CG16的菌落表型、显微结构,以及对融合子CG16全细胞可溶性蛋白电泳分析,结果表明融合子CG16是由双亲本原生质体融合得到的新菌株。
(4)融合子CG16固态发酵生产脂肪酶条件初步优化研究。通过单因素比较实验,对融合子CG16进行了固态发酵培养基成分和发酵培养条件的初步优化。结果表明固态发酵的优化培养基为：12g固体物质(麦麸：大豆粉=3：2),8 ml营养盐溶液(g/L：KH2PO47.07, Na2HPO422.06, MgSO4·7H2O 2.0, Na2CO3 2.0, NaNO37.07);发酵条件为：接种量为2%,30℃,初始pH7.0,培养72 h,产酶酶活达到82.22 U/g干物质,比亲本假丝酵母和白地霉的产酶活力分别提高了32%和171%。
Lipase was a special ester bond hydrolase, which can act on the ester bond of triglyceride and cause triglyceride degrade into diacylglycerol, monoglyceride, glycerol and fatty acids. Lipase was an important industrial enzyme and widely used in various industrial fields, which was one of the earliest enzymes being studied. Since the microbial lipase was discovered, screening high-yield microbial strains of lipase has become a research focus at home and abroad because of its following advantages such as diversity, highly specificity of substrate, wide range action pH and temperature, convenient to realize industrialization, easy to obtain and so on.
In this thesis, the protoplast fusion of Geotrichum candidum NS3 protoplast and Candida valida T2 protoplast was studied by chemical PEG fusion method. In the meanwhile, the resistance of drugs was also used to screen a fusion strain with high-yield of lipase and strong growth ability. Moreover, the solid-state fermentation conditions of fusant for producing lipase were studied preliminary. The specific methods and research results are as follows:
(1) The resistant marker was selected from the parents stains produced lipase. Twelve kinds of drugs were chosen as the sign of resistance to antibiotics of Geotrichum candidum NS3 and Candida valida T2, respectively by the method of concentration gradient and principles of medical administration. The resistant markers of the two stains were obtained. One of the medical tags is Qumixi slave at a concentration of 2×103μg/ml when Geotrichum candidum NS3 is resistance and Candida valida T2 non-resistance. The other one is CuSO4 at the concentration of 2×103μg/ml when Candida valida T2 is resistance and Geotrichum candidum NS3 is non-resistance.
(2) The parents of the strains protoplast preparation and regeneration conditions were studied. The various factors that affect the formation rate and regeneration rate of Geotrichum candidum NS3 protoplast and Candida valida T2 protoplast were investigated by means of single factor experiment. The results showed the optimum preparation conditions for protoplasts of Candida valida T2 and Geotrichum candidum NS3 were as following:using middle exponential phase cells, the action of 1.5% cellulase enzyme at 30℃for 1 h, taking 0.05% cysteine as the reagent of promoting cell wall degradation and using 0.7M NaCl as osmotic stabilizer.
(3) The protoplast fusion of the parents and identification of fusant. The protoplast fusion of parents strains protoplasts was studied by chemical PEG fusion method. Fifty individual fusants have been got by replica platting method, and eight individual plants were isolated from the first generation, a steady fusant was selected from the tenth generation culture, then the fusant named CG16. By comparing the colonial morphology and microstructure of the parents and fusant CG16, and the full-cell solubility protein electrophoresis, the result showed that CG16 fusant was a new strain derived from the protoplast fusion of parents.
(4) The invesitigetion and optimization of solid-fermentation conditions of fusant CG16 have been carry out by single factor comparison experiment. The results showed that the optimal medium composition for producing lipase by fusant CG16 was:12 g solid matter (wheat bran: soybean meal=3:2),8 ml nutrient solution (g/L: KH2PO4 7.07, Na2HPO4 22.06, MgSO4·7H2O 2.0, Na2CO3 2.0, NaNO3 7.07). The lipase activity produced by CG16 reached 82.22 U/g dry matter under the fermentation conditions with 30℃, initial pH 7.0 and culture 72 h. The lipase activity of CG16 was higher 32% and 171% than that of the parent strains produced, respectively.
本文利用化学PEG融合法使白地霉(Geotrichum candidum NS3)原生质体和假丝酵母(Candida valida T2)原生质体融合,结合抗药性标记筛选方法,成功筛选出一株产脂肪酶能力较高并且生长能力较强的融合菌株,并对融合株的固态发酵产脂肪酶条件进行初步研究。主要研究工作和结果如下：
(1)脂肪酶双亲本菌株抗药性标记的选择。实验利用药物浓度梯度平板法和医学给药原则,选用12种药物分别对白地霉(Geotrichum candidum NS3)和假丝酵母(Candida valida T2)进行抗药性测定,成功筛选出白地霉和假丝酵母的抗药性标记。白地霉抗性而假丝酵母非抗性的药物标记为曲咪新乳膏浓度为2×103μg/ml,假丝酵母抗性而白地霉非抗性的药物标记则为硫酸铜浓度为2×103μg/ml。
(2)双亲本菌株原生质体制备和再生条件的研究。利用单因素实验探讨影响白地霉和假丝酵母原生质体形成率和再生率的各种因素,结果表明制备原生质体的优化条件为：双亲本菌龄为对数生长中期、1.5%的纤维素酶酶解1小时、酶解温度30℃、脱壁促进剂为0.05%的半胱氨酸、渗透压稳定剂为0.7MNaCl。
(3)双亲本原生质体融合及融合子的鉴定。利用化学PEG方法使双亲本原生质体融合,通过影印法筛选得到50株融合子。连续传代10次,获得8株性状稳定的融合子,再通过油脂同化平板法筛选得到一株融合菌株,命名为CG16。通过比较双亲本及融合子CG16的菌落表型、显微结构,以及对融合子CG16全细胞可溶性蛋白电泳分析,结果表明融合子CG16是由双亲本原生质体融合得到的新菌株。
(4)融合子CG16固态发酵生产脂肪酶条件初步优化研究。通过单因素比较实验,对融合子CG16进行了固态发酵培养基成分和发酵培养条件的初步优化。结果表明固态发酵的优化培养基为：12g固体物质(麦麸：大豆粉=3：2),8 ml营养盐溶液(g/L：KH2PO47.07, Na2HPO422.06, MgSO4·7H2O 2.0, Na2CO3 2.0, NaNO37.07);发酵条件为：接种量为2%,30℃,初始pH7.0,培养72 h,产酶酶活达到82.22 U/g干物质,比亲本假丝酵母和白地霉的产酶活力分别提高了32%和171%。
Lipase was a special ester bond hydrolase, which can act on the ester bond of triglyceride and cause triglyceride degrade into diacylglycerol, monoglyceride, glycerol and fatty acids. Lipase was an important industrial enzyme and widely used in various industrial fields, which was one of the earliest enzymes being studied. Since the microbial lipase was discovered, screening high-yield microbial strains of lipase has become a research focus at home and abroad because of its following advantages such as diversity, highly specificity of substrate, wide range action pH and temperature, convenient to realize industrialization, easy to obtain and so on.
In this thesis, the protoplast fusion of Geotrichum candidum NS3 protoplast and Candida valida T2 protoplast was studied by chemical PEG fusion method. In the meanwhile, the resistance of drugs was also used to screen a fusion strain with high-yield of lipase and strong growth ability. Moreover, the solid-state fermentation conditions of fusant for producing lipase were studied preliminary. The specific methods and research results are as follows:
(1) The resistant marker was selected from the parents stains produced lipase. Twelve kinds of drugs were chosen as the sign of resistance to antibiotics of Geotrichum candidum NS3 and Candida valida T2, respectively by the method of concentration gradient and principles of medical administration. The resistant markers of the two stains were obtained. One of the medical tags is Qumixi slave at a concentration of 2×103μg/ml when Geotrichum candidum NS3 is resistance and Candida valida T2 non-resistance. The other one is CuSO4 at the concentration of 2×103μg/ml when Candida valida T2 is resistance and Geotrichum candidum NS3 is non-resistance.
(2) The parents of the strains protoplast preparation and regeneration conditions were studied. The various factors that affect the formation rate and regeneration rate of Geotrichum candidum NS3 protoplast and Candida valida T2 protoplast were investigated by means of single factor experiment. The results showed the optimum preparation conditions for protoplasts of Candida valida T2 and Geotrichum candidum NS3 were as following:using middle exponential phase cells, the action of 1.5% cellulase enzyme at 30℃for 1 h, taking 0.05% cysteine as the reagent of promoting cell wall degradation and using 0.7M NaCl as osmotic stabilizer.
(3) The protoplast fusion of the parents and identification of fusant. The protoplast fusion of parents strains protoplasts was studied by chemical PEG fusion method. Fifty individual fusants have been got by replica platting method, and eight individual plants were isolated from the first generation, a steady fusant was selected from the tenth generation culture, then the fusant named CG16. By comparing the colonial morphology and microstructure of the parents and fusant CG16, and the full-cell solubility protein electrophoresis, the result showed that CG16 fusant was a new strain derived from the protoplast fusion of parents.
(4) The invesitigetion and optimization of solid-fermentation conditions of fusant CG16 have been carry out by single factor comparison experiment. The results showed that the optimal medium composition for producing lipase by fusant CG16 was:12 g solid matter (wheat bran: soybean meal=3:2),8 ml nutrient solution (g/L: KH2PO4 7.07, Na2HPO4 22.06, MgSO4·7H2O 2.0, Na2CO3 2.0, NaNO3 7.07). The lipase activity produced by CG16 reached 82.22 U/g dry matter under the fermentation conditions with 30℃, initial pH 7.0 and culture 72 h. The lipase activity of CG16 was higher 32% and 171% than that of the parent strains produced, respectively.
引文
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