产几丁质酶菌株的筛选与鉴定及几丁质酶酶学性质研究
摘要
几丁质为自然界丰富的天然资源,具有可再生性,生物相容性,生物可降解性,生物功能性等特点,是许多高新技术领域中不可替代的生物医用和环境友好材料,具有广泛的用途。几丁质酶是几丁质专一性降解所需的酶。开展几丁质酶的研究,对几丁质资源的转化,已成为全球性热门科研课题。
本文从湖南养虾场,河流等地筛选到一株产几丁质酶的真菌CS-01,并对其进行了分类鉴定;对该菌株产的几丁质酶进行了分离纯化,研究了温度和pH对几丁质酶降解胶体几丁质的酶活性的影响以及葡萄糖和胶体几丁质等底物对该菌株几丁质酶表达的影响。
(1) 12份样品经富集培养后,采用平板透明圈法进行初筛,得到7株可在以几丁质为唯一碳源的选择性固体平板上形成明显透明圈的菌株,对该7株菌进行复筛,获得一株产酶活力相对较高的真菌CS-01。
(2)利用形态学和分子生物学手段对该菌株进行了鉴定,通过对其菌落特征、个体形态的观察和ITS rDNA序列分析,最终确定该菌株为烟曲霉。
(3)采用盐析,凝胶过滤层析,透析,聚乙二醇浓缩,离子交换层析,冷冻干燥等方法分离纯化几丁质酶。Native-PAGE和SDS-PAGE分析确定了该几丁质酶为45 kDa的单体酶。
(4)以浓缩纯酶为材料,研究了温度和pH对几丁质酶表观活力的影响。结果表明:在pH 5的缓冲液中,当酶反应温度达到55℃时表观活性最大;在45-65℃间相对表观活性都在80%以上,说明该初酶液具有宽的高温适应范围,这对直接利用该菌株进行发酵降解胶体几丁质具有重要意义。
(5)将烟曲霉菌株CS-01培养于不同碳源的培养基中,发现,当以1%葡萄糖为碳源培养时,在整个培养过程中始终没有检测到几丁质酶活力;而以1%胶体几丁质作为碳源进行培养时,培养到12h就有几丁质酶活力产生,并在36h达到最大(0.128 U/mL);在以胶体几丁质和葡萄糖为混合碳源的培养基中进行培养时,在培养早期没有检测到几丁质酶活力的产生。通过对混合碳源中还原糖的测定,发现只有当培养基中葡萄糖耗尽时,才能检测到几丁质酶活力的产生。上述结果表明,CS-01产几丁质酶属于诱导型表达。
Chitin is one kind of widespread natural resource, with a series of excellent properties viz. biorenewability, biocompatibility, biodegradability, which offers application potentials in diversified areas, especially applied in preparation of biorelated and environment-friendly materials. Chitinase selectively splits chitin. The significance of realizing chitin conversion by chitinase is subject of significant concern to many people all over the world.
In this dissertation, a chitinase-producing strain CS-01 from Hunan Province of China was isolated and identified. Chitinase secreted by the strain was purified and its partial properties were investigated. Then, effects of pH and temperature on the activity of the concentrated purified enzyme were studied. Additionally, effect of substrates viz. glucose and colloidal chitin on the CS-01 chitinase expression was also studied. The main results are listed as follows.
(1) After the twelve samples cultured in the enrichment medium, the transparent flat circle was adopted to do the primary screening and 7 strains were got when chitin was used as the sole carbon source of selective solid medium to form clear transparent circle. After secondary screening of the 7 strains, a high-activity fungi strain named CS-01 was selected and maintained on colloidal chitin agar slant for further study.
(2) Morphological and molecular biology methods were used to identify the colony characteristics, individual forms of observation and ITS rDNA sequence analysis of CS-01, and ultimately classified the strain as Aspergillus fumigatus.
(3) Purification of the CS-01 chitinase was carried out by salting out, gel filtration chromatography, dialysis, PEG concentration, ion-exchange chromatography, freeze-drying, followed with electrophoresis analysis. Native-PAGE and SDS-PAGE indicated that the chitinase from A. fumigatus CS-01 was monomer with 45 kDa.
(4) The effects of pH and temperature on the apparent activity of the concentrated purified enzyme were investigated. It was found that the maximum activity of the concentrated crude enzyme was appeared at pH 5 and 55℃. In the buffer pH 5, over 80% of the maximum activity was shown at 45-65℃. The advantage of its high adaptability to a wide range of high temperatures makes the strain useful in direct chitin hydrolysis by its fermentation broth.
(5) A. fumigatus CS-01 is cultivated by different carbon sources. Chitinase activity present in the supernatant of 1% glucose cultivation could not detected all the time, while the culture supernatant from 1% colloidal chitin cultivation possessed enzymatic activity after incubated for 12 h and chitinase activity reached to its maximum (0.128 U/mL) at 36 h,. A. fumigatus CS-01 cultivated by mixed carbon source shows chitinase activity at late stage of growth. The above results indicate that the chitinase expression is chitin-inducible.
本文从湖南养虾场,河流等地筛选到一株产几丁质酶的真菌CS-01,并对其进行了分类鉴定;对该菌株产的几丁质酶进行了分离纯化,研究了温度和pH对几丁质酶降解胶体几丁质的酶活性的影响以及葡萄糖和胶体几丁质等底物对该菌株几丁质酶表达的影响。
(1) 12份样品经富集培养后,采用平板透明圈法进行初筛,得到7株可在以几丁质为唯一碳源的选择性固体平板上形成明显透明圈的菌株,对该7株菌进行复筛,获得一株产酶活力相对较高的真菌CS-01。
(2)利用形态学和分子生物学手段对该菌株进行了鉴定,通过对其菌落特征、个体形态的观察和ITS rDNA序列分析,最终确定该菌株为烟曲霉。
(3)采用盐析,凝胶过滤层析,透析,聚乙二醇浓缩,离子交换层析,冷冻干燥等方法分离纯化几丁质酶。Native-PAGE和SDS-PAGE分析确定了该几丁质酶为45 kDa的单体酶。
(4)以浓缩纯酶为材料,研究了温度和pH对几丁质酶表观活力的影响。结果表明:在pH 5的缓冲液中,当酶反应温度达到55℃时表观活性最大;在45-65℃间相对表观活性都在80%以上,说明该初酶液具有宽的高温适应范围,这对直接利用该菌株进行发酵降解胶体几丁质具有重要意义。
(5)将烟曲霉菌株CS-01培养于不同碳源的培养基中,发现,当以1%葡萄糖为碳源培养时,在整个培养过程中始终没有检测到几丁质酶活力;而以1%胶体几丁质作为碳源进行培养时,培养到12h就有几丁质酶活力产生,并在36h达到最大(0.128 U/mL);在以胶体几丁质和葡萄糖为混合碳源的培养基中进行培养时,在培养早期没有检测到几丁质酶活力的产生。通过对混合碳源中还原糖的测定,发现只有当培养基中葡萄糖耗尽时,才能检测到几丁质酶活力的产生。上述结果表明,CS-01产几丁质酶属于诱导型表达。
Chitin is one kind of widespread natural resource, with a series of excellent properties viz. biorenewability, biocompatibility, biodegradability, which offers application potentials in diversified areas, especially applied in preparation of biorelated and environment-friendly materials. Chitinase selectively splits chitin. The significance of realizing chitin conversion by chitinase is subject of significant concern to many people all over the world.
In this dissertation, a chitinase-producing strain CS-01 from Hunan Province of China was isolated and identified. Chitinase secreted by the strain was purified and its partial properties were investigated. Then, effects of pH and temperature on the activity of the concentrated purified enzyme were studied. Additionally, effect of substrates viz. glucose and colloidal chitin on the CS-01 chitinase expression was also studied. The main results are listed as follows.
(1) After the twelve samples cultured in the enrichment medium, the transparent flat circle was adopted to do the primary screening and 7 strains were got when chitin was used as the sole carbon source of selective solid medium to form clear transparent circle. After secondary screening of the 7 strains, a high-activity fungi strain named CS-01 was selected and maintained on colloidal chitin agar slant for further study.
(2) Morphological and molecular biology methods were used to identify the colony characteristics, individual forms of observation and ITS rDNA sequence analysis of CS-01, and ultimately classified the strain as Aspergillus fumigatus.
(3) Purification of the CS-01 chitinase was carried out by salting out, gel filtration chromatography, dialysis, PEG concentration, ion-exchange chromatography, freeze-drying, followed with electrophoresis analysis. Native-PAGE and SDS-PAGE indicated that the chitinase from A. fumigatus CS-01 was monomer with 45 kDa.
(4) The effects of pH and temperature on the apparent activity of the concentrated purified enzyme were investigated. It was found that the maximum activity of the concentrated crude enzyme was appeared at pH 5 and 55℃. In the buffer pH 5, over 80% of the maximum activity was shown at 45-65℃. The advantage of its high adaptability to a wide range of high temperatures makes the strain useful in direct chitin hydrolysis by its fermentation broth.
(5) A. fumigatus CS-01 is cultivated by different carbon sources. Chitinase activity present in the supernatant of 1% glucose cultivation could not detected all the time, while the culture supernatant from 1% colloidal chitin cultivation possessed enzymatic activity after incubated for 12 h and chitinase activity reached to its maximum (0.128 U/mL) at 36 h,. A. fumigatus CS-01 cultivated by mixed carbon source shows chitinase activity at late stage of growth. The above results indicate that the chitinase expression is chitin-inducible.
引文
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