云南红豆杉内生真菌的固定化及其GGPP酶的研究
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摘要
筛选和应用红豆杉内生真菌发酵生产紫杉醇已成为当前医药领域研究的热点之一。GGPP合成酶是紫杉醇生物合成途径中的关键酶,能够催化合成紫杉烷类化合物的共同前体——牻牛儿牻牛儿焦磷酸酯(简称GGPP)。利用固定化技术发酵生产代谢产物,由于固定化酶具有可长期重复使用,连续发酵和简化代谢产物分离过程等优点,是一种很有发展前景的新型技术。因此,本文以产紫杉烷类化合物的云南红豆杉内生真菌菌株12.3.2作为研究对象,对其生理特性、固定化条件和GGPP合成酶活性进行了一系列初步研究。
首先,考察了培养基与培养条件对其生理特性的影响,研究发现:温度对菌丝体生长具有明显的影响,最适温度为30℃;pH值在前24小时呈下降趋势,然后开始逐步升高,在5天后趋于稳定,综合pH的变化,菌株在中性条件时生长良好,在接种后3天菌丝体干质量可达到最大值;蔗糖对菌丝体的生长最为有利,对有机氮的利用要优于对无机氮的利用;菌丝体干质量与培养基质量浓度呈正向关系,而且较高的培养基质量浓度对其生长没有抑制作用;对维生素依赖性不强,但添加适量的维生素可以促进其生长。
其次,建立TTC法测定红豆杉内生真菌分生孢子的脱氢酶酶活,并以此来考察固定化效果。用海藻酸钙包埋云南红豆杉内生真菌12.3.2分生孢子制成固定化孢子的微胶珠。通过TTC法测定红豆杉内生真菌分生孢子的脱氢酶活力,并以此来考察固定化粒径、海藻酸钠的浓度、CaCl_2浓度、单位凝胶分生孢子量、固定化时间以及温度等六个因素对固定化效果的影响。通过实验确定,当海藻酸钠浓度为4.0%,CaCl_2浓度为2.6%,固定化时间为6 h,温度为40℃,单位凝胶中分生孢子量应为1 mL/10g凝胶(孢子悬液浓度为2.3×10~9个/mL),此条件下包埋率为46%,此时内生真菌12.3.2活力最强,固定化效果最佳。因此,TTC法可以准确反应红豆杉内生真菌分生孢子的固定化效果,固定的分生孢子具有更好的发酵特性。
最后,初步探讨了GGPP合成酶活性的检测。该酶属于异戊烯基转移酶,催化FPP与IPP发生亲电子耦合形成GGPP。对产紫杉烷类化合物的菌株12.3.2的GGPP合成酶活性进行检测,与标准品对比进行定性分析,确定其内含有GGPP合成酶。
Endophytic fungus strain 12.3.2 were isolated from the Taxus Yunnanensis, which had the ability to produce the taxol or its derivates, were the study subjects in this paper.
The effects of medium compositions and culture conditions on the growth of strain 12.3.2 were investigated. The temperature, carbon sources, nitrogen sources, pH, the concentration of the media, saccharides and the requirement of some general vitamins as well as the relationship of the aim strain were studied. The result showed that temperature had significant effect on the growth of the aim endophytes. In the neutral conditions, it was suitable for the growth of the aim endophytes. The dry weight of mycelium had positive correlation with the concentration of culture medium.
To determine the immobilization efficiency of conidia in Taxus yunnanensis, the TTC method was established. Conidia of Fungal Endophytes in Taxus yunnanensis 12.3.2 were immobilized with sodium alginate. Then, the TTC method was applied to examine the viability of conidia. And the effects of diameter of microbeads sodium, alginate concentration, calcium chloride concentration, amount of condia unit Calcium Alginate gel, time and temperature in immobilization were also analyzed. The results showed that the under the conditions of microbeads diameter 2 mm , 40°C, 4.0 % sodium alginate, 2.6 % calcium chloride, 6 hour incubation, 2.3×10~9 condia /10 g Calcium Alginate gel and 46 % the embedded rate the immobilization efficiency was the highest. The TTC method can be used for assaying the immobilization efficiency. And the immobilization conidia exhibited the better fermentation characteristics.
Geranylgeranyl diphosphate synthase (GGPP synthase) supplied the essential acyclic precursor for Taxol biosynthesis. This enzyme belonged to the isopentenyl transferase, which occurred electronic nucleophilic coupling, catalyzed FPP and IPP to form GGPP. Through using the function, controlled diterpene and in tetraterpene biosynthesis way branching point, and started the taxol biosynthesis. The GGPP synthase in the aim endophytic fungus was qualitative analyzed .
首先,考察了培养基与培养条件对其生理特性的影响,研究发现:温度对菌丝体生长具有明显的影响,最适温度为30℃;pH值在前24小时呈下降趋势,然后开始逐步升高,在5天后趋于稳定,综合pH的变化,菌株在中性条件时生长良好,在接种后3天菌丝体干质量可达到最大值;蔗糖对菌丝体的生长最为有利,对有机氮的利用要优于对无机氮的利用;菌丝体干质量与培养基质量浓度呈正向关系,而且较高的培养基质量浓度对其生长没有抑制作用;对维生素依赖性不强,但添加适量的维生素可以促进其生长。
其次,建立TTC法测定红豆杉内生真菌分生孢子的脱氢酶酶活,并以此来考察固定化效果。用海藻酸钙包埋云南红豆杉内生真菌12.3.2分生孢子制成固定化孢子的微胶珠。通过TTC法测定红豆杉内生真菌分生孢子的脱氢酶活力,并以此来考察固定化粒径、海藻酸钠的浓度、CaCl_2浓度、单位凝胶分生孢子量、固定化时间以及温度等六个因素对固定化效果的影响。通过实验确定,当海藻酸钠浓度为4.0%,CaCl_2浓度为2.6%,固定化时间为6 h,温度为40℃,单位凝胶中分生孢子量应为1 mL/10g凝胶(孢子悬液浓度为2.3×10~9个/mL),此条件下包埋率为46%,此时内生真菌12.3.2活力最强,固定化效果最佳。因此,TTC法可以准确反应红豆杉内生真菌分生孢子的固定化效果,固定的分生孢子具有更好的发酵特性。
最后,初步探讨了GGPP合成酶活性的检测。该酶属于异戊烯基转移酶,催化FPP与IPP发生亲电子耦合形成GGPP。对产紫杉烷类化合物的菌株12.3.2的GGPP合成酶活性进行检测,与标准品对比进行定性分析,确定其内含有GGPP合成酶。
Endophytic fungus strain 12.3.2 were isolated from the Taxus Yunnanensis, which had the ability to produce the taxol or its derivates, were the study subjects in this paper.
The effects of medium compositions and culture conditions on the growth of strain 12.3.2 were investigated. The temperature, carbon sources, nitrogen sources, pH, the concentration of the media, saccharides and the requirement of some general vitamins as well as the relationship of the aim strain were studied. The result showed that temperature had significant effect on the growth of the aim endophytes. In the neutral conditions, it was suitable for the growth of the aim endophytes. The dry weight of mycelium had positive correlation with the concentration of culture medium.
To determine the immobilization efficiency of conidia in Taxus yunnanensis, the TTC method was established. Conidia of Fungal Endophytes in Taxus yunnanensis 12.3.2 were immobilized with sodium alginate. Then, the TTC method was applied to examine the viability of conidia. And the effects of diameter of microbeads sodium, alginate concentration, calcium chloride concentration, amount of condia unit Calcium Alginate gel, time and temperature in immobilization were also analyzed. The results showed that the under the conditions of microbeads diameter 2 mm , 40°C, 4.0 % sodium alginate, 2.6 % calcium chloride, 6 hour incubation, 2.3×10~9 condia /10 g Calcium Alginate gel and 46 % the embedded rate the immobilization efficiency was the highest. The TTC method can be used for assaying the immobilization efficiency. And the immobilization conidia exhibited the better fermentation characteristics.
Geranylgeranyl diphosphate synthase (GGPP synthase) supplied the essential acyclic precursor for Taxol biosynthesis. This enzyme belonged to the isopentenyl transferase, which occurred electronic nucleophilic coupling, catalyzed FPP and IPP to form GGPP. Through using the function, controlled diterpene and in tetraterpene biosynthesis way branching point, and started the taxol biosynthesis. The GGPP synthase in the aim endophytic fungus was qualitative analyzed .
引文
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