【作者】 陈小龙；

【导师】 郑裕国；

【作者基本信息】 浙江工业大学 ， 生物化工， 2002， 硕士

【摘要】 腺苷蛋氨酸的生产制备方法主要有化学合成法、发酵法和酶促转化法3种。化学合成法采用S-腺苷同型半胱氨酸（S-adenosylhomocysteine）和甲基供体（CH₃I）合成，腺苷蛋氨酸有（+）和（-）两种构型，只有（-）构型才有生物活性，但合成产物中含有少量（+）腺苷蛋氨酸，且难以分离。发酵法采用在培养基中添加L-甲硫氨酸，用酵母发酵生产（-）型腺苷蛋氨酸，是60年代至80年代生产腺苷蛋氨酸的主要方法。酶促转化法与化学合成法、发酵法相比，具有终产物积累量高、分离提纯容易、反应周期短以及无污染等优点，因而是较为有效的工业化生产方法。酶促转化法主要利用腺苷蛋氨酸合成酶（S-adenosylmethionine synthetase，又称ATP：L-methionine S-adenosyltransferase，EC2.5.1.6），催化底物L-甲硫氨酸和ATP生成（-）型腺苷蛋氨酸。 本实验通过从土壤中对产腺苷蛋氨酸菌种的分离筛选，挑取了587株酵母菌，得到了8株产腺苷蛋氨酸较高的菌株，尤其是千-95，腺苷蛋氨酸的产量达到了956μg／ml发酵液。 对菌株千-65进行了6次诱变处理，其中5次为紫外线诱变，最后1次为γ射线诱变。经六次诱变后的A5，其腺苷蛋氨酸产量提高了86.9％。 在培养条件优化过程中，着重考察了初始pH值、发酵温度和加入底物浓度。发酵培养基的最适初始pH值为5.0；最佳发酵温度为30℃；最适底物浓度为1％；在培养基优化过程中，研究了培养基中的碳源、氮源、磷源和无机离子。碳源以麦芽糖、L-果糖和蔗糖为最好，由于前二者在价格上比蔗糖贵，且蔗糖易得，故选用蔗糖为碳源；碳源浓度试验表明8％蔗糖为最佳碳源浓度；氮源选择了0.5％的NH₄NO₃；磷源选择了0.08％KH₂PO₄和0.016％K₂HPO₄；考察了MnCl₂，MgSO₄·7H₂O，CaCl₂，ZnSO₄·7H₂O，FeSO₄·7H₂O，Na₂MoSO₄·2H₂O和LiCl对腺苷蛋氨酸发酵的影响，选择了有促进作用的MgSO₄·7H₂O、CaCl₂、FeSO₄·7H₂O和LiCl，并其浓度进行了单因素试验和正交试验。结果表明，在0.01％MgSO₄·7H₂O、0.03％CACl₂、0.04％FeSO₄·7H₂O和0.01％LiCl浓度下，腺苷蛋氨酸的产量最高；并进行了气升式生物反应器和机械搅拌式发酵罐的上罐试验对比试验，结果表明，气升式生物反应器优于机械搅拌式发酵罐。一 考察了破碎酵母细胞三种方法中，以超声波破碎法最好，条件为22KHZ和15分钟，得到粗酶液的酶活力为 0．934U／IIl；最佳超声波粗酶液的酶促反应时间为20分钟，最适反应温度为28习0C，最适反应pH值为7．8七．3，最佳底物浓度为spML蛋氨酸，spMATP。ATP浓度过高会对腺昔蛋氨酸合成起抑制作用；酵母细胞通透性的改变可通过加入表面活性剂SDS和吐温80。其中吐温明显好于SDS，且最佳浓度为0．10gisg酵母泥，释放率可达到62．3％；采用包埋法固定经改变通透性的酵母细胞。固定化酵母细胞的最适酶促反应温度为30℃，最适 反应pH值为7．8士．2。但是同游离酶相比，对反应环境变化敏感性不强；在室 温下，游离超声波粗酶液的半衰期为32天。 建立了腺苦蛋氨酸的初步提取工艺，得到了腺苦蛋氨酸对甲苯磺酸盐实验室 小样。更多还原

【Abstract】 There are three ways to produce adenosyl-L-methionine, including chemical synthesis, microbial fermentation and enzymatic synthesis. Chemical synthesis is carried out with S-adenosylhomocysteine and CHsI as the substrates. Adenosyl-L-methionine can be divided into (-)-diasterioisomer and (+)-diasterioisomer according to the configuration. Only (-)-diasterioisomer has the active activity. In the product of chemical synthesis, there is a little (+)-diasterioisomer and it is not easy to be separated. Microbial fermentation was carried out with L-methionine as the substrate in the medium. It had been the main way to produce (-)-diasterioisomer with the fermentation of yeast. Compared to chemical synthesis and microbial fermentation, Enzymatic synthesis has the virtues like high aim product concentration, easily separated, short reacting time and no pollution. Therefore, it has the effective production way hi the industry. Enzymatic synthesis is to use the enzyme, S-adenosylmethionine synthetase (also called ATP: L-methionineS-adenosyltranserase, EC 2.5.1.6), with L-methionine and ATP as the its substrates.In this experiment, 587 strains, which could produce SAM, were screened from the soil picked from all over Zhejiang Province. In these strains, 8 strains were selected with high yield of SAM, especially Qian-95, with 956 y g/ml broth.In the optimization of culture conditions, the initial broth pH, fermentation temperature and the concentration of L-methionine in the broth were examined. The optimized conditions were pH 5.0, temperature 30℃ and substrate concentration 1%. In the optimization of media, the C source, N source, P source and mineral ion were studied. The optimal C source was 8% sucrose, N source 0.5% NRtNOs, P source 0.08% KH2PO4 and 0.016%K2HP04 and mineral ion 0.01%MgSO4 ?7H20, 0.03%CaCl2,0.04%FeSO4 ’7H20 and 0.01%LiCl. Also the experiments were carried to investigate the fermentation of SAM in the airlift bioreactior and mechanically stirred fermentor. The results showed the yield of SAM in the airlift bioreactor washigher than that in the mechanically stirred fermentor.The yeast cell disruptions were researched with abrasives, ultrasonic and toluene. The results of disruption was the best with ultrasonic. The operation conditions were 22 KHz and 15 minutes and 0.934 U/ml was obtained as the enzyme activity. It was investigated that the optimal reaction conditions of S-adenosylmethionine synthetase were reacting time 20 min, temperature 28-30℃, pH7. 8-8. 3, L-methionine 5u M and ATP 5 u M. But with the increase of the concentration of ATP, the enzyme activity was inhibited. The penetrating ability of the yeast cell walls could be increased by adding detergents, such as SDS and Tween-80. The results of Tween-80 was better than that of SDS. The optimal concentration of Tween-80 was 0.10g/5g cells and the rate of SAM penetration was 62.3%. The treated cells were immobilized with entrapment of calcium alginate. The optimal enzyme reacting conditions of immobilized cells were temperature 30 ℃ andpH 7.8-8.2. Compared to the free enzyme, the immobilized cells were less sensitive the environment. The half life period of free enzyme was 32 days at the room temperature.The isolation of SAM was established and a experimental sample of SAM salts of sulphuric acid and p-toluenesulphonic acid, which had the purity of 98.2%, was obtained.更多还原