菊粉酶高产菌株的生物学特性研究
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摘要
本论文以菊粉酶的高产菌株Asp.niger M89、Asp.niger H8和突变菌株Asp.niger U γ-2为研究对象，对三株菌的主要生物学特性进行了研究。研究内容包括菌株形态学研究、菌株所产酶系的研究和菊粉酶基因序列的研究。
在菌株形态学研究方面，对三株高产菊粉酶菌株在察氏培养基、麦芽汁培养基、PDA培养基、同化亚硝酸盐培养基中的菌落形态；显微镜下的分生孢子头、分生孢子梗、足细胞、顶囊等个体形态；扫描电镜下的分生孢子表面形态等方面做了较系统的研究。根据资料可将菌株Asp.niger M89和Asp.niger H8鉴定为黑曲霉群泡盛酒曲霉种(Aspergillus awamori)，诱变菌株Asp.nigerU γ-2可鉴定到黑曲霉群臭曲霉种(Aspergillus foetides)。诱变使菌落形态发生了很大的变化。并且通过此方面的研究建立起一套简便有效的用于霉菌形态鉴定当中样品的处理方法。
对菌株所产酶系的研究方面，分别测了菊粉酶、蛋白酶、淀粉酶、果胶酶、纤维素酶的产生曲线。此三株菌株除高产菊粉酶外，均产生不同水平的蛋白酶、淀粉酶、果胶酶、纤维素酶，其中菊粉酶的产酶水平较高，产其他酶系的活力相对较低。诱变菌株除菊粉酶活力大幅度提高以外，淀粉酶和果胶酶也有明显提高。对于同一菌株来说酶系中不同酶的产酶高峰不同，酶量高峰出现的早晚不同。其酶活力高峰随发酵时间依次是蛋白酶、淀粉酶、菊粉酶和果胶酶，纤维素酶因不同菌株有一定差异。
针对GenBank所报道的四种来源于黑曲霉菌株的菊粉酶序列，分别设计引物，以本研究菌株基因组DNA为模版，进行PCR扩增，并进行了核苷酸序列的测序。测序结果与原序列进行了同源性比较分析，同源性并不高，可以推测本研究菌株所产菊粉酶基因序列与所报道的有所不同。
Aspergillus niger M89, Asp. niger H8 and Aspergillus niger U y - 2 are all Inulinase high- yielding strains ,whose major biological characteristics have been studied in this paper . The research content includes the studies on morphology, major enzyme systems of the strains and the preliminary study of inulinase genes.
For researching the morphologies of the strains.I had turned the strains respectively in Czapek, MEA, PDA and culture media that could assimilate nitrous acid salt to observe the characters of the morphologies of the colonies. At the same time the observation of the microscopical feature of the individual strain and the observation of the conidium of the strain by the scanning electronic microscope are done. According to the information we divide Asp.niger M89 and Asp. niger H8 into Aspergillus Genus , Aspergillus niger section Aspergillus awamori species. The mutant strain Asp. nigerU gamma - 2 can be divided into Aspergillus Genus , Aspergillus niger section Aspergill'i.s foetides species. The mutant makes the morphology of the colony changed greatly. A set of easy and high efficient methods used to identify the morphologies of the mould were set up through those studies.
On the research of enzyme system produced by the strains, we had measured the growing curves of the inulinase, protease, amylase, amylase, and cellulose enzyme. Excepting the high yielding inulinase ,this three strains also have different levels in protease, amylase, pectinase, and cellulose enzyme. Among these , the production of the inulinase is higher than the others. Besides the inulinase activity of mutant strain Asp. niger U gamma -2 raised substantially, amylase and pectinase also have been obviously raised. For different enzyme of the same strain, the time is difference that reaches the highest enzyme activity. Followed to ferment time, protease, amylase, inulinase and pectinase reaches the highest enzyme activity. Cellulose enzyme has certain discrepancy because of different strains.
According to the four kinds of sequence from Aspergillus niger strains reported by GenBank , primer are designed respectively, genomic DNA of these three strains are taken as mould, then PCR is carried out and nucleus sequence is to be measured next. The
result of measurement and original sequence are analyzed, which proved that nucleotide sequence identities are not high , it is possible to draw a conclusion that gene sequence of mulinase from Aspergillus niger is different from that reported.
在菌株形态学研究方面，对三株高产菊粉酶菌株在察氏培养基、麦芽汁培养基、PDA培养基、同化亚硝酸盐培养基中的菌落形态；显微镜下的分生孢子头、分生孢子梗、足细胞、顶囊等个体形态；扫描电镜下的分生孢子表面形态等方面做了较系统的研究。根据资料可将菌株Asp.niger M89和Asp.niger H8鉴定为黑曲霉群泡盛酒曲霉种(Aspergillus awamori)，诱变菌株Asp.nigerU γ-2可鉴定到黑曲霉群臭曲霉种(Aspergillus foetides)。诱变使菌落形态发生了很大的变化。并且通过此方面的研究建立起一套简便有效的用于霉菌形态鉴定当中样品的处理方法。
对菌株所产酶系的研究方面，分别测了菊粉酶、蛋白酶、淀粉酶、果胶酶、纤维素酶的产生曲线。此三株菌株除高产菊粉酶外，均产生不同水平的蛋白酶、淀粉酶、果胶酶、纤维素酶，其中菊粉酶的产酶水平较高，产其他酶系的活力相对较低。诱变菌株除菊粉酶活力大幅度提高以外，淀粉酶和果胶酶也有明显提高。对于同一菌株来说酶系中不同酶的产酶高峰不同，酶量高峰出现的早晚不同。其酶活力高峰随发酵时间依次是蛋白酶、淀粉酶、菊粉酶和果胶酶，纤维素酶因不同菌株有一定差异。
针对GenBank所报道的四种来源于黑曲霉菌株的菊粉酶序列，分别设计引物，以本研究菌株基因组DNA为模版，进行PCR扩增，并进行了核苷酸序列的测序。测序结果与原序列进行了同源性比较分析，同源性并不高，可以推测本研究菌株所产菊粉酶基因序列与所报道的有所不同。
Aspergillus niger M89, Asp. niger H8 and Aspergillus niger U y - 2 are all Inulinase high- yielding strains ,whose major biological characteristics have been studied in this paper . The research content includes the studies on morphology, major enzyme systems of the strains and the preliminary study of inulinase genes.
For researching the morphologies of the strains.I had turned the strains respectively in Czapek, MEA, PDA and culture media that could assimilate nitrous acid salt to observe the characters of the morphologies of the colonies. At the same time the observation of the microscopical feature of the individual strain and the observation of the conidium of the strain by the scanning electronic microscope are done. According to the information we divide Asp.niger M89 and Asp. niger H8 into Aspergillus Genus , Aspergillus niger section Aspergillus awamori species. The mutant strain Asp. nigerU gamma - 2 can be divided into Aspergillus Genus , Aspergillus niger section Aspergill'i.s foetides species. The mutant makes the morphology of the colony changed greatly. A set of easy and high efficient methods used to identify the morphologies of the mould were set up through those studies.
On the research of enzyme system produced by the strains, we had measured the growing curves of the inulinase, protease, amylase, amylase, and cellulose enzyme. Excepting the high yielding inulinase ,this three strains also have different levels in protease, amylase, pectinase, and cellulose enzyme. Among these , the production of the inulinase is higher than the others. Besides the inulinase activity of mutant strain Asp. niger U gamma -2 raised substantially, amylase and pectinase also have been obviously raised. For different enzyme of the same strain, the time is difference that reaches the highest enzyme activity. Followed to ferment time, protease, amylase, inulinase and pectinase reaches the highest enzyme activity. Cellulose enzyme has certain discrepancy because of different strains.
According to the four kinds of sequence from Aspergillus niger strains reported by GenBank , primer are designed respectively, genomic DNA of these three strains are taken as mould, then PCR is carried out and nucleus sequence is to be measured next. The
result of measurement and original sequence are analyzed, which proved that nucleotide sequence identities are not high , it is possible to draw a conclusion that gene sequence of mulinase from Aspergillus niger is different from that reported.
引文
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