【作者】 王小艳；

【导师】 李春；

【作者基本信息】 石河子大学 ， 农产品加工及贮藏工程， 2007， 硕士

【摘要】 通过筛选和甘草酸转化结果分析,得到了三种不同转化类型的真菌,利用分子生物学和形态学方法进行了菌株鉴定,并对其进行了催化特性和催化动力学研究,针对定向合成单葡萄糖醛酸基甘草次酸(GAMG)的Penicillium sp. Li-3进行了β-D-葡萄糖醛酸苷酶的克隆与表达,研究结果如下:三种不同转化类型的真菌经分子生物学和形态学方法鉴定为Penicillium purpurogenum Li-3、Aspergillus terreus Li-20和Aspergillus ustus Li-62,其转化甘草酸的产物分别是GAMG、GAMG与甘草次酸(GA)和GA。三株菌所表达的β-D-葡萄糖醛酸苷酶催化特性研究表明,Penicillium purpurogenum Li-3所产β-D-葡萄糖醛酸苷酶的催化反应最适pH为4.2,最适温度为50℃,培养48h后开始产酶;Aspergillus terreus Li-20所产酶的催化反应最适pH为5.8,最适温度为45℃,培养24 h后开始产酶;而Aspergillus ustus Li-62所产酶的催化反应最适pH为6.2,最适温度为55℃,72h后才开始产酶。催化甘草酸的Km分别为0.328μmol·L-1、3.61 mmol·L-1和0.43 mmol·L-1,催化反应的最大速率Vmax分别为0.0035 mmol·L-1·min-1、0.034 mmol·L-1·min-1和0.106 mmol·L-1·min-1。针对定向合成GAMG的Penicillium purpurogenum Li-3生长过程中存在的碳代谢阻遏现象,研究了该菌β-D-葡萄糖醛酸苷酶的高效诱导表达策略。建立了新的诱导产酶方法为,葡萄糖培养基中以32℃摇床(180rpm)培养,在葡萄糖耗尽时加入20%诱导剂(10g/L GL+1.2% Tween80)进行诱导,且每24h添加一次,诱导72h后转入到40℃摇床(180rpm)培养产酶,以该方法诱导产酶表明在5g/L葡萄糖中富集菌体,进行诱导后,酶活性从产酶基础培养基中的646U/mL提高到2356U/mL。利用PCR方法从Penicillium purpurogenum Li-3基因组DNA中成功克隆到编码β-D-葡萄糖醛酸苷酶的基因Pgus,基因全长1815bp,编码604个氨基酸,分子量为67.6KDa。构建了Pgus的原核表达载体pET-28a(+)-Pgus和真核表达载体pPIC9K-Pgus,分别转化E.coli BL21(DE3)和Pichia pastoris GS115,在E.coli BL21(DE3)中诱导表达后形成无酶活性的包涵体,转化Pichia pastoris GS115后已筛选到具有6~10个高拷贝的H is+ M u ts s型毕赤酵母重组菌。更多还原

【Abstract】 Three fungi with the different type of biotransformation of glycyrrhizin were determined by analyzing the products of metabolism, and were identified by morphological observation and gene homology analysis. The catalytic characteristic and kinetics ofβ-D-glucuronidase from the three fungi were also investigated.β-D-glucuronidase from Penicillium sp. Li-3 which could directly biosynthesize Glycyrrhetinic acid monoglucuronide (GAMG) from glycyrrhizin was cloned and expressed. The results showed as follows:According to morphology, phylogenetic analysis and 18SrDNA sequence homology, the results demonstrated that three Fungi were identified as Penicillium purpurogenum Li-3, Aspergillus terreus Li-20 and Aspergillus ustus Li-62. Penicillium purpurogenum Li-3 converted glycyrrhizin to GAMG, Aspergillus terreus Li-20 converted glycyrrhizin to GAMG and GA, and Aspergillus ustus Li-62 converted glycyrrhizin to GA, respectively.Research about the catalytic characteristic and kinetics ofβ-D-glucuronidase from three fungi showed that the optimal reaction temperature ofβ-D-glucuronidase from Penicillium purpurogenum Li-3, Aspergillus terreus Li-20 and Aspergillus ustus Li-62 was 50℃, 45℃and 55℃, respectively. The optimal pH for the catalytic reaction ofβ-D-glucuronidases was 4.2, 5.8 and 6.2, respectively. The Km ofβ-D-glucuronidase from these strains was 0.328μmol·L-1, 3.61 mmol·L–1 and 0.43 mmol·L–1, and the Vmax was 0.0035 mmol·L-1·min-1, 0.034 mmol·L-1·min-1 and 0.106 mmol·L-1·min-1, respectively. For the production of the enzymes, the time of formation was about 48h, 24h and 72h, respectively.Catabolite repression was found when Penicillium purpurogenum Li-3 grew in medium including other carbon sources except of glycyrrhizin, and the inducing strategy ofβ-D-glucuronidase was studied. The results indicated that the enzyme activity was enhanced from 646U/mL to 2356U/mL by adding 20% inducer (1.2% Tween80 and 10% glycyrrhizin) when glucose was completely consumed in the medium including 5g/L of glucose.The genes ofβ-D-glucuronidase (Pgus) were triumphantly cloned by PCR from the genome DNA of Penicillium purpurogenum Li-3. The sequence analysis indicated that the gene of Pgus has 1815 base pairs and encodes 604 amino acids, the molecular weight of subunit was 67.6KDa. The expression plasmids of pET-28a(+)-Pgus and pPIC9K-Pgus were conceived, and then transformed into E.coli BL21(DE3) and Pichia pastoris GS115, respectively. The inclusion body was formed after induced in E.coli BL21(DE3), and the H is+ M u ts- Pichia pastoris with 6~10 gene copy were successfully screened in Pichia pastoris GS115.更多还原