霉菌甲醛脱氢酶活性的分光光度法测定

Determination of Activity of Formaldehyde Dehydrogenase by Spectrophotometry

甲醛在甲醛脱氢酶的作用下与辅酶Ⅰ(NAD)形成甲酸或甲酸盐,根据其生成的还原型辅酶Ⅰ二钠盐(β-NADH)的量与吸光度成正比,建立了测定3株霉菌在3个处理方法中的甲醛脱氢酶活性的分光光度法。以绿色木霉(Trichoderma viride)、爪哇青霉(Penicillium javanicum)、黄曲霉(Aspergillus flavus)3株甲醛降解霉菌为材料,通过全温振荡培养,pH7.5的0.05 mol/L的磷酸钾缓冲溶液洗涤、离心、提取,并在37℃、pH7.5、340 nm处测定甲醛脱氢酶活性。结果表明:细胞破碎处理1(600 W,工作5 s、间歇7 s,60次)破碎细胞不完全,只能测得部分酶活性;细胞破碎处理3(800 W,工作5 s、间歇7 s,60次)破碎细胞完全,但破坏了部分酶活性;细胞破碎处理2(700 W,工作5 s、间歇7 s,60次)能很好地破碎细胞且对酶活性的破坏最少,测得最高酶活性。

In presence of formaldehyde dehydrogenase, formaldehyde react with coenzyme Ⅰ （NAD） to produce formate and β-NADH. Based on the linear relationship between the produced amount of β-NADH and absorbance （A）, the activity of formaldehyde dehydrogenase of three fungi treated under three experimental conditions were studied by spectrophotometry. Three formaldehydedegrading fungi included Trichoderma viride , Penicillium javanicum , Aspergillus flavus , were shaking cultured under constant temperature, washed and centrifuged with pH 7.5 0. 05 mol/L potassium phosphate buffer solution several times. The histiocyte was extracted by ultrasonic method. The enzymatic activity of formaldehyde dehydrogenase was measured at 340 nm, pH of 7.5 at 37 ℃. The experimental results showed, under the conditon Ⅰ （600 W, working time of 5 s with the intermission of 7 s, repeated times of 60）, a part of enzymatic activity was detected due to the imcompletion of the histiocyte cataclase. Under the condition Ⅲ （800 W, working time of 5 s with the intermission of 7 s, repeated times of 60 ）, the part of enzymatic activity was destroyed. The highest enzymatic activity was achieved under the condition Ⅱ （700 W, working time of 5 s with the intermission of 7 s, repeated times of 60）.