蜜蜂球囊菌几丁质酶纯化及酶学性质的研究
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摘要
蜜蜂白垩病(bee chalkbrood disease)是由蜜蜂球囊菌(Ascosphaera apis Olive & Spiitoir)引起的蜜蜂幼虫的真菌传染病。目前为止，还没有治疗该病的有效方法。在无法控制的条件下，该病仍呈自然流行状态。几丁质酶在昆虫病原真菌的入侵中有重要作用。我们从10株蜜蜂球囊菌中筛选出一株高产几丁质酶的菌株，并且对其几丁质酶进行了诱导、分离、纯化和酶学性质的研究。经过硫酸铵盐析、SephadexG-75凝胶层析、DE-52离子交换层析得到纯化倍数为6.7的几丁质酶。该几丁质酶的最适反应温度为40℃，在35℃～45℃之间能保持40％以上的活力，但是这种几丁质酶对高温比较敏感；在pH7.5时，蜜蜂球囊菌几丁质酶的活性最高，在pH4.5以下几乎检测不到活力，在pH9.0以上酶活力在20％以下，在pH6～9范围内几丁质酶活力都在40％以上；在0～100mmol／L范围内，一价金属离子Na~+、K~+对几丁质酶活力影响均表现为先促进后抑制；在浓度为1～10mmol／L范围内，二价金属离子Mg~(2+)、Mn~(2+)对其活力起抑制作用，Zn~(2+)对几丁质酶活力基本没有影响，Ca~(2+)、Fe~(2+)对几丁质酶活力起促进作用；在浓度1～10mmol／L范围内，三价金属离子Al~(3+)对蜜蜂球囊菌几丁质酶活力起促进作用，Fe~(3+)对几丁质酶活力基本没有影响。
Bee chalkbrood disease was a fungal epidemic disease of apian larva which was caused by Ascosphaera apis Olive&Spiltoir. Up to now , there were no effective methods to cure this disease. Under this conditions, this disease was still natural popular. Chitinase has a important role when pathogenetic fungi infected insects. One strain was selected from a total of ten stains Ascosphaera apis Olive & Spiltoir that could produce chitinase. The chitinase was purified , and its Properities was studied. After salt out of (NH4)_2SO_4, ion-exchange chromat-ography on DE-52 and Gel-filtration chromatography on Sephadex G-75, the fold was 6. 7. The chitinase exhibited optimum catalytic activity 40℃. It was retained above 40% activity between 35℃～45℃, but it was sensitive to high temperature; Its optimal pH was 7. 5, its activity could not be detected when pH below 4. 5 and above pH9. 0 its activity below 20%, and its activity was above 40% when pH between pH 6～9. When concentration between 1～100mmol/L, metal metal ions Na~+、K~+ enhanced the enzyme activity at first and then inhibited the enzyme activity. When concentration between 1～10mmol/L, Ca~(2+)、Fe~(2+)、Al~(3+) enhanced the enzyme activity, Mg~(2+)、Mn~(2+) inhibited the enzyme activity, Zn~(2+)、Fe~(3+) has no effect to the enzyme activity.
Bee chalkbrood disease was a fungal epidemic disease of apian larva which was caused by Ascosphaera apis Olive&Spiltoir. Up to now , there were no effective methods to cure this disease. Under this conditions, this disease was still natural popular. Chitinase has a important role when pathogenetic fungi infected insects. One strain was selected from a total of ten stains Ascosphaera apis Olive & Spiltoir that could produce chitinase. The chitinase was purified , and its Properities was studied. After salt out of (NH4)_2SO_4, ion-exchange chromat-ography on DE-52 and Gel-filtration chromatography on Sephadex G-75, the fold was 6. 7. The chitinase exhibited optimum catalytic activity 40℃. It was retained above 40% activity between 35℃～45℃, but it was sensitive to high temperature; Its optimal pH was 7. 5, its activity could not be detected when pH below 4. 5 and above pH9. 0 its activity below 20%, and its activity was above 40% when pH between pH 6～9. When concentration between 1～100mmol/L, metal metal ions Na~+、K~+ enhanced the enzyme activity at first and then inhibited the enzyme activity. When concentration between 1～10mmol/L, Ca~(2+)、Fe~(2+)、Al~(3+) enhanced the enzyme activity, Mg~(2+)、Mn~(2+) inhibited the enzyme activity, Zn~(2+)、Fe~(3+) has no effect to the enzyme activity.
引文
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