产植酸酶木霉菌的遗传基因改良
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摘要
植酸酶(myo-inositol hexakisphosphate phosphohydrolases),是催化植酸及植酸盐水解成肌醇与磷酸的一类酶的总称。植酸酶主要存在于植物、微生物和一些动物组织中。单胃动物如猪、鱼和家禽类不能消化植酸,饲料中加入植酸酶分解植酸,促进单胃动物对磷和金属元素的吸收和利用,另外还可以降低环境中的磷污染。
本文以木霉为研究对象,以各地采集木霉为基础筛选高产植酸酶菌株。通过植酸钙透明圈筛选法,再经过植酸酶液体发酵酶活最终选择11号菌株为出发菌株进行基因组改组(Genome shuffling)技术。11号菌株通过紫外诱变和亚硝基胍诱变,筛选优良诱变子,将两种诱变子的原生质体分别进行热灭活和紫外灭活,然后随机融合,筛选融合子。
紫外诱变和亚硝基胍诱变均以出发菌株为诱变菌株,选着合适的剂量进行诱变改良。从大量的诱变子中通过初筛和复筛筛选出合适的诱变子。紫外诱变子Ⅱ的酶活为最高,且5代后培养未发生回复突变,酶活为1.21±0.013 U/L,将此菌株编号为UV-Ⅱ(p<0.05)。亚硝基胍诱变中NTG-4酶活为0.94±0.014 U/L(p<0.05)。
对木霉的原生质体制备和再生条件进行了研究。分别考察了菌体菌龄、酶浓度、酶解时间、酶解温度等因素对木霉原生质制备和再生的影响。霉菌细胞壁主要成分是多糖,大多数是几丁质、少数种类是纤维素。以UV-Ⅱ出发菌株,以蜗牛酶为溶菌酶制备木霉原生质体。研究得到木霉原生质体制备条件:蜗牛酶浓度2.0mg/mL,菌龄为20h,酶解时间2.0 h,酶解温度30℃。
基因组改组技术将UV-Ⅱ和NTG-4的原生质体分别进行热灭活和紫外灭活,然后随机结合,使用PEG为融合剂进行原生质体融合。两两结合进行4轮基因组改组,筛选到融合子Fu-②,酶活为2.4±0.016 U/L,比原始菌株分别高出320%。融合子遗传特性稳定。
对融合子Fu-②进行液体培养基优化实验,得到优化培养基Ⅱ培养基:葡萄糖3.0 g ,可溶性淀粉3.0 g,麸皮0.05 g,NH4NO30.5 g,MgSO4·7H2O 0.05 g,MnSO4·7H2O 0.003 g,FeSO4·7H2O 0.003 g,CaCO3 0.5 g,pH值5.5-6.0,115℃30min灭菌。同时对融合子Fu-②进行液体发酵条件优化:转速115r/min,装液量100 ml(500mL三角瓶),接种量为3%,温度30℃,发酵时间120h,酶活3.68 U /L,酶活提高33.3%。
Phytases (myo-inositol hexakisphosphate phosphohydrolases) catalyze the hydrolysis of phytates to myoinositol and phosphates. Phytases are varied and widespread in nature, which occurr in plants, microorganisms, and animal tissues. Phytases, in general, are known to enhance phosphate and mineral uptake in monogastric animals such as human beings, poultry, swine, and fish, which cannot metabolize phytate besides reducing environmental pollution significantly.
In this paper, phytase of Trichoderma was studied, and Trichoderma strains of high phytase yield were selected from those strains collected from multiple areas.NO.11 strain of high phytase yield was isolated from soil samples and, which was selected by transparent circle method and the phytase activity method of liquid fermentation. The research is aimed to improve phytase activity by genome shuffling, and the results are as follows. NO.11 strain was treated with ultraviolet radiation and NTG, respectively.Then good mutants were obtained.The protoplasts of goog mutants were heat-killed and UV-inactivated, and combined randomly.At last fusants which were high phytase yield were selected.
Wiedtype strain was treated with ultraviolet radiation and NTG, respectively. Then the optimal dose for mutant were selected. Lots of mutants were selected with phytase activity method. The high phytase yield strain of UV radiation and NTG mutant was UV-Ⅱand NTG-4, the phytase activity of which were 1.21±0.013 U / L (p <0.05) and 0.94±0.014 U / L (p <0.05), respectively.
The conditions for protoplast preparation and regeneration were studied. The effects of strain age, the lysozyme concentration, the lysozyme treating time and temperature on preparation and regeneration of protoplast were studied. The chief constituent of fungal cell wall is polysaccharide, which is made chiefly of chitin and few cellulose. It was prepared for Trichoderma lysozyme protoplasts with UV-Ⅱstrain and snail enzyme. The optimum conditions of Trichoderma protoplast are as follow: snail enzyme concentration 2.0mg/mL, strain age 20h, lysozyme time 2.0 h, lysozyme temperature 30℃.
UV-Ⅱand NTG-4 protoplasts were heat-killed and UV-inactivated, and combined randomly with PEG as the integration agents of protoplast fusion. Fu-②was obtained from genome reorganization, phytase activity of which was 2.4±0.016 U / L and higher 320% compared with the original strains . The fusant is genetically stable.
The result was found thatⅡliquid medium was the best medium. The mediumⅡ: Glucose 3.0 g, soluble starch 3.0 g, wheat bran 0.05 g, NH4NO30.5 g, MgSO4 ? 7H2O0.05 g, MnSO4 ? 7H2O 0.003 g, FeSO4 ? 7H2O 0.003 g, CaCO30.5 g, pH value of 5.5 -6.0,115℃30min sterilization. The conditions of Fu-②optimization: shaking speed 115r/min, medium volume 100 ml (500 ml volumetric flask), inoculation number 3%, temperature 30℃, fermentation time 120h, phytase activity 3.68 U / L and the enzyme activity was increased 33.3%.
本文以木霉为研究对象,以各地采集木霉为基础筛选高产植酸酶菌株。通过植酸钙透明圈筛选法,再经过植酸酶液体发酵酶活最终选择11号菌株为出发菌株进行基因组改组(Genome shuffling)技术。11号菌株通过紫外诱变和亚硝基胍诱变,筛选优良诱变子,将两种诱变子的原生质体分别进行热灭活和紫外灭活,然后随机融合,筛选融合子。
紫外诱变和亚硝基胍诱变均以出发菌株为诱变菌株,选着合适的剂量进行诱变改良。从大量的诱变子中通过初筛和复筛筛选出合适的诱变子。紫外诱变子Ⅱ的酶活为最高,且5代后培养未发生回复突变,酶活为1.21±0.013 U/L,将此菌株编号为UV-Ⅱ(p<0.05)。亚硝基胍诱变中NTG-4酶活为0.94±0.014 U/L(p<0.05)。
对木霉的原生质体制备和再生条件进行了研究。分别考察了菌体菌龄、酶浓度、酶解时间、酶解温度等因素对木霉原生质制备和再生的影响。霉菌细胞壁主要成分是多糖,大多数是几丁质、少数种类是纤维素。以UV-Ⅱ出发菌株,以蜗牛酶为溶菌酶制备木霉原生质体。研究得到木霉原生质体制备条件:蜗牛酶浓度2.0mg/mL,菌龄为20h,酶解时间2.0 h,酶解温度30℃。
基因组改组技术将UV-Ⅱ和NTG-4的原生质体分别进行热灭活和紫外灭活,然后随机结合,使用PEG为融合剂进行原生质体融合。两两结合进行4轮基因组改组,筛选到融合子Fu-②,酶活为2.4±0.016 U/L,比原始菌株分别高出320%。融合子遗传特性稳定。
对融合子Fu-②进行液体培养基优化实验,得到优化培养基Ⅱ培养基:葡萄糖3.0 g ,可溶性淀粉3.0 g,麸皮0.05 g,NH4NO30.5 g,MgSO4·7H2O 0.05 g,MnSO4·7H2O 0.003 g,FeSO4·7H2O 0.003 g,CaCO3 0.5 g,pH值5.5-6.0,115℃30min灭菌。同时对融合子Fu-②进行液体发酵条件优化:转速115r/min,装液量100 ml(500mL三角瓶),接种量为3%,温度30℃,发酵时间120h,酶活3.68 U /L,酶活提高33.3%。
Phytases (myo-inositol hexakisphosphate phosphohydrolases) catalyze the hydrolysis of phytates to myoinositol and phosphates. Phytases are varied and widespread in nature, which occurr in plants, microorganisms, and animal tissues. Phytases, in general, are known to enhance phosphate and mineral uptake in monogastric animals such as human beings, poultry, swine, and fish, which cannot metabolize phytate besides reducing environmental pollution significantly.
In this paper, phytase of Trichoderma was studied, and Trichoderma strains of high phytase yield were selected from those strains collected from multiple areas.NO.11 strain of high phytase yield was isolated from soil samples and, which was selected by transparent circle method and the phytase activity method of liquid fermentation. The research is aimed to improve phytase activity by genome shuffling, and the results are as follows. NO.11 strain was treated with ultraviolet radiation and NTG, respectively.Then good mutants were obtained.The protoplasts of goog mutants were heat-killed and UV-inactivated, and combined randomly.At last fusants which were high phytase yield were selected.
Wiedtype strain was treated with ultraviolet radiation and NTG, respectively. Then the optimal dose for mutant were selected. Lots of mutants were selected with phytase activity method. The high phytase yield strain of UV radiation and NTG mutant was UV-Ⅱand NTG-4, the phytase activity of which were 1.21±0.013 U / L (p <0.05) and 0.94±0.014 U / L (p <0.05), respectively.
The conditions for protoplast preparation and regeneration were studied. The effects of strain age, the lysozyme concentration, the lysozyme treating time and temperature on preparation and regeneration of protoplast were studied. The chief constituent of fungal cell wall is polysaccharide, which is made chiefly of chitin and few cellulose. It was prepared for Trichoderma lysozyme protoplasts with UV-Ⅱstrain and snail enzyme. The optimum conditions of Trichoderma protoplast are as follow: snail enzyme concentration 2.0mg/mL, strain age 20h, lysozyme time 2.0 h, lysozyme temperature 30℃.
UV-Ⅱand NTG-4 protoplasts were heat-killed and UV-inactivated, and combined randomly with PEG as the integration agents of protoplast fusion. Fu-②was obtained from genome reorganization, phytase activity of which was 2.4±0.016 U / L and higher 320% compared with the original strains . The fusant is genetically stable.
The result was found thatⅡliquid medium was the best medium. The mediumⅡ: Glucose 3.0 g, soluble starch 3.0 g, wheat bran 0.05 g, NH4NO30.5 g, MgSO4 ? 7H2O0.05 g, MnSO4 ? 7H2O 0.003 g, FeSO4 ? 7H2O 0.003 g, CaCO30.5 g, pH value of 5.5 -6.0,115℃30min sterilization. The conditions of Fu-②optimization: shaking speed 115r/min, medium volume 100 ml (500 ml volumetric flask), inoculation number 3%, temperature 30℃, fermentation time 120h, phytase activity 3.68 U / L and the enzyme activity was increased 33.3%.
引文
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[2] Haefner S, Knietsch A, Scholten E, et al. Biotechnological production and applications of phytases. Appl Microbiol Biotechnol[J], 2005, 68: 588-597
[3] Kies AK, De Jonge LH, Kemme PA, Jongbloed AW. 2006. Interaction between protein, phytate, and microbial phytase. In vitro studies. Agric Food Chem[J] 54:1753–1758.
[4] Bebot-Brigaud A, Dange C, Fauconnier N, Gerard C. 1999. 31P NMR, potentiometric and spectrophotometric studies of phytic acid ionizationand complexation properties toward Co2+, Ni2+, Cu2+, Zn2+ and Cd2+. Inorg Biochem 75:71–78.
[5] Mullaney, E. J., & Ullah, A. H. J. (2003). Biochemical and Biophysical Research Communication[J]s, 312, 179–184.
[6] Wodzinski RJ., Ullah AH.. phytase Adv App l Microbiol[J] ,1996 ,42 :263～302
[7] Mullaney EJ., Daly CB. Advancesin phytase research. Adv Appl Microbiol [J],2000 ,47 :157～199
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