克拉维酸产生菌的研究
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摘要
本论文对克拉维酸(clavulanic acid，CA)产生菌-棒状链霉菌NR3585(Streptomyces clavuligerus NR3585)进行了深入的研究，目的是提高其生物合成克拉维酸的能力，以适用于工业化生产的需要。
为了能够对棒状链霉菌的代谢产物克拉维酸进行定量和定性测定，本论文首先建立了检测发酵样品中克拉维酸含量的生物效价测定法，薄层层析-生物显影法和高效液相色谱法(HPLC)。在对棒状链霉菌的培养条件、菌落特征、及对各种诱变剂的敏感性进行了研究的基础上，利用UV、X—ray、DES、HNO_2及NTG等物理和化学诱变剂对棒状链霉菌进行了诱变处理。为了提高菌种选育的效率、加快菌种选育的进程，本研究建立了耐受高浓度甘油的菌种筛选模型和琼脂块固体发酵的筛选方法，经过考察确定了适宜的固体发酵培养基。经过多代的诱变处理结合筛选模型的应用，本研究选育出了克拉维酸高产菌株-棒状链霉菌U_7D_5XH-45(S.clauligerus U_7D_5XH-45)，经摇瓶发酵，其克拉维酸生产能力达1139.4μ／ml，大约是原始出发菌株发酵单位的20倍；以HPLC法测定，克拉维酸含量从大约70％提高到接近100％。
本论文还对克拉维酸产生菌的发酵培养基进行了考察，通过对碳、氮源的考察确定了发酵培养基最适碳源为麦芽糖，最适氮源为黄豆粉；通过试验发现添加5，10，15 mM的各种氨基酸对克拉维酸的生物合成都没有明显的促进作用；但添加同样量的赖氨酸、缬氨酸、酪氨酸、苯丙氨酸、半胱氨酸或色氨酸则对克拉维酸的生物合成有显著的抑制作用。当培养基中加入5～15mM的半胱氨酸后，克拉维酸的生物合成被完全抑制，与此同时在发酵液中积累了一个浓度较高的未知组分。通过考察无机盐、微量元素对克拉维酸生物合成的影响，发现KNO_3、Mn~(2+)以及低浓度的Mg~(2+)对克拉维酸的生物合成有一定的促进作用。试验还发现氧化还原剂吩嗪甲酯硫酸盐(PMS)在极低的浓度下(1.0μM)就能促进克拉维酸的生物合成。
通过对发酵培养基的考察，利用均匀设计试验方法对克拉维酸的
沈阳药科大学硕士学位论文 中文摘要
发酵培养基组成进行了忧化，用优化后的培养基进行克拉维酸发酵，
发酵单位提高了25．2％。
通过对克拉维酸发酵条件的考察确定了最适的克拉维酸发酵工
艺条件：种子及发酵摇瓶的培养温度为 26 C；发酵周期控制在 80～
84h：接种量控制在 10％；装瓶量为 40ml／250 ml摇瓶；发酵培养基消
前PH为6．5。
采用优化后的发酵培养基和发酵工艺条件，本文还对克拉维酸发
酵过程中的各种参数进行了检测，并通过绘制代谢曲线对发酵过程中
各种参数之间的相互关系进行了分析。
通过研究发现磷酸盐对克拉维酸的生物合成有显著的调节作用，
适合克拉维酸生物合成的磷酸盐浓度在5～10nunol几范围内，高于
10nunol／L的磷酸盐强烈抑制克拉维酸的生物合成；糖的代谢途径对克
拉维酸的生物合成也有显著的影响，当添加低浓度（0．1。g／100ml）的
碘乙酸改变糖的代谢途径时，明显促进了克拉维酸的生物合成。
In order to improve the production of clavulanic acid for the industrial need, the organism Streptomyces clavuligerus NR3585 was studied in this paper.
Firstly, the bioassay method, TLC-Bio-autograph method and HPLC method of clavulanic acid were developed, so that clavulanic acid in the fermentation samples can be measured qualitatively and quantitatively. On the basis of studies on S. clavuligerus culture characteristic, colonies characteristic and sensitivity to various mutagens, physical and chemical mutagens including UV, X-ray, DES, HNO_(2) and NTG were used in the mutagenesis of Streptomyces clavuligerus. Then, in order to accelerate the process of strain improvement, a glycerol-resistant screening model and an agar column solid fermentation screening method was established.
A clavulanic acid high-yield strain Streptomyces clavuligerus U7D5XH-45 was obtained after several circles of mutagenesis, which produced 1139.4#/ml clavulanic acid in shaking flask fermentation, about 20 times higher than the productivity of initial strain. At the same time the content of clavulanic acid increased from about 70% to almost 100% measured by HPLC.
The fermentation media for clavulanic acid production were also inspected and optimized in this paper. The optimal carbon source and nitrogen source was maltose and soybean powder respectively through the study. Feeding amino acids to fermentation medium from 5 mM to 15 mM at the beginning of fermentation showed no promoting effect on the biosynthesis of clavulanic acid. However Lys, Val, Tyr, Phe, Cyc and Trp showed strong inhibition effect on the biosynthesis of clavulanic acid. An unknown product but no clavulanic acid accumulated when Cys was added into the fermentation medium. The investigation of the effects of
inorganic salts and trace elements showed that KNO_(3), Mn2+ and Mg2+ promoted the production of clavulanic acid. Experiment results also showed that feeding of 1.0#M PMS (a redox agent) to fermentation medium stimulated the biosynthesis of clavulanic acid. Fermentation medium of clavulanic acid was optimized through uniform design. The clavulanic acid titer increased obviously with the optimized fermentation medium.
The optimized fermentation techniques of clavulanic acid production by Streptomyces clavuligerus were as follows: fermentation temperature, 26 癈 ; fermentation period, 80-84h; inoculation volume, 10% (v/v); fermentation volume, 40ml in 250ml shaking flask; pH, 6.5 before sterilization.
Results showed that phosphate exerted obvious regulative effects on the biosynthesis of clavulanic acid. The optimal concentration of phosphate for the biosynthesis of clavulanic acid was 5-10mmol/L. Higher phosphate concentration depressed clavulanic acid production greatly. The catabolism pathway of saccharide also exerted regulative effects on clavulanic acid biosynthesis.When low concentration (0. lmg/100ml)iodoacetic acid was added to fermentation media in the beginning of fermentation to alter the catabolism pathway of saccharide, the biosynthesis of clavulanic acid was promoted obviously.
为了能够对棒状链霉菌的代谢产物克拉维酸进行定量和定性测定，本论文首先建立了检测发酵样品中克拉维酸含量的生物效价测定法，薄层层析-生物显影法和高效液相色谱法(HPLC)。在对棒状链霉菌的培养条件、菌落特征、及对各种诱变剂的敏感性进行了研究的基础上，利用UV、X—ray、DES、HNO_2及NTG等物理和化学诱变剂对棒状链霉菌进行了诱变处理。为了提高菌种选育的效率、加快菌种选育的进程，本研究建立了耐受高浓度甘油的菌种筛选模型和琼脂块固体发酵的筛选方法，经过考察确定了适宜的固体发酵培养基。经过多代的诱变处理结合筛选模型的应用，本研究选育出了克拉维酸高产菌株-棒状链霉菌U_7D_5XH-45(S.clauligerus U_7D_5XH-45)，经摇瓶发酵，其克拉维酸生产能力达1139.4μ／ml，大约是原始出发菌株发酵单位的20倍；以HPLC法测定，克拉维酸含量从大约70％提高到接近100％。
本论文还对克拉维酸产生菌的发酵培养基进行了考察，通过对碳、氮源的考察确定了发酵培养基最适碳源为麦芽糖，最适氮源为黄豆粉；通过试验发现添加5，10，15 mM的各种氨基酸对克拉维酸的生物合成都没有明显的促进作用；但添加同样量的赖氨酸、缬氨酸、酪氨酸、苯丙氨酸、半胱氨酸或色氨酸则对克拉维酸的生物合成有显著的抑制作用。当培养基中加入5～15mM的半胱氨酸后，克拉维酸的生物合成被完全抑制，与此同时在发酵液中积累了一个浓度较高的未知组分。通过考察无机盐、微量元素对克拉维酸生物合成的影响，发现KNO_3、Mn~(2+)以及低浓度的Mg~(2+)对克拉维酸的生物合成有一定的促进作用。试验还发现氧化还原剂吩嗪甲酯硫酸盐(PMS)在极低的浓度下(1.0μM)就能促进克拉维酸的生物合成。
通过对发酵培养基的考察，利用均匀设计试验方法对克拉维酸的
沈阳药科大学硕士学位论文 中文摘要
发酵培养基组成进行了忧化，用优化后的培养基进行克拉维酸发酵，
发酵单位提高了25．2％。
通过对克拉维酸发酵条件的考察确定了最适的克拉维酸发酵工
艺条件：种子及发酵摇瓶的培养温度为 26 C；发酵周期控制在 80～
84h：接种量控制在 10％；装瓶量为 40ml／250 ml摇瓶；发酵培养基消
前PH为6．5。
采用优化后的发酵培养基和发酵工艺条件，本文还对克拉维酸发
酵过程中的各种参数进行了检测，并通过绘制代谢曲线对发酵过程中
各种参数之间的相互关系进行了分析。
通过研究发现磷酸盐对克拉维酸的生物合成有显著的调节作用，
适合克拉维酸生物合成的磷酸盐浓度在5～10nunol几范围内，高于
10nunol／L的磷酸盐强烈抑制克拉维酸的生物合成；糖的代谢途径对克
拉维酸的生物合成也有显著的影响，当添加低浓度（0．1。g／100ml）的
碘乙酸改变糖的代谢途径时，明显促进了克拉维酸的生物合成。
In order to improve the production of clavulanic acid for the industrial need, the organism Streptomyces clavuligerus NR3585 was studied in this paper.
Firstly, the bioassay method, TLC-Bio-autograph method and HPLC method of clavulanic acid were developed, so that clavulanic acid in the fermentation samples can be measured qualitatively and quantitatively. On the basis of studies on S. clavuligerus culture characteristic, colonies characteristic and sensitivity to various mutagens, physical and chemical mutagens including UV, X-ray, DES, HNO_(2) and NTG were used in the mutagenesis of Streptomyces clavuligerus. Then, in order to accelerate the process of strain improvement, a glycerol-resistant screening model and an agar column solid fermentation screening method was established.
A clavulanic acid high-yield strain Streptomyces clavuligerus U7D5XH-45 was obtained after several circles of mutagenesis, which produced 1139.4#/ml clavulanic acid in shaking flask fermentation, about 20 times higher than the productivity of initial strain. At the same time the content of clavulanic acid increased from about 70% to almost 100% measured by HPLC.
The fermentation media for clavulanic acid production were also inspected and optimized in this paper. The optimal carbon source and nitrogen source was maltose and soybean powder respectively through the study. Feeding amino acids to fermentation medium from 5 mM to 15 mM at the beginning of fermentation showed no promoting effect on the biosynthesis of clavulanic acid. However Lys, Val, Tyr, Phe, Cyc and Trp showed strong inhibition effect on the biosynthesis of clavulanic acid. An unknown product but no clavulanic acid accumulated when Cys was added into the fermentation medium. The investigation of the effects of
inorganic salts and trace elements showed that KNO_(3), Mn2+ and Mg2+ promoted the production of clavulanic acid. Experiment results also showed that feeding of 1.0#M PMS (a redox agent) to fermentation medium stimulated the biosynthesis of clavulanic acid. Fermentation medium of clavulanic acid was optimized through uniform design. The clavulanic acid titer increased obviously with the optimized fermentation medium.
The optimized fermentation techniques of clavulanic acid production by Streptomyces clavuligerus were as follows: fermentation temperature, 26 癈 ; fermentation period, 80-84h; inoculation volume, 10% (v/v); fermentation volume, 40ml in 250ml shaking flask; pH, 6.5 before sterilization.
Results showed that phosphate exerted obvious regulative effects on the biosynthesis of clavulanic acid. The optimal concentration of phosphate for the biosynthesis of clavulanic acid was 5-10mmol/L. Higher phosphate concentration depressed clavulanic acid production greatly. The catabolism pathway of saccharide also exerted regulative effects on clavulanic acid biosynthesis.When low concentration (0. lmg/100ml)iodoacetic acid was added to fermentation media in the beginning of fermentation to alter the catabolism pathway of saccharide, the biosynthesis of clavulanic acid was promoted obviously.
引文
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