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[目的]以甲醛降解菌宛氏拟青霉菌F1-23为实验菌株,分离纯化得到甲醛脱氢酶的酶液,并考察其酶学性质。[方法]从液体培养液中收集菌体,通过细胞粉碎,采用(NH4)2SO4沉淀、透析、DFAE-Sepharose离子交换层析及Superdex-200凝胶过滤层析,纯化得到电泳纯的FADH。[结果]该酶的分子量约为112.81 k Da;亚基分子量分别为65k Da和42 k Da;纯酶液活力为336.10 U/mg,较纯化前提高了4.02倍。对该酶的酶学性质分析结果表明,该酶最适反应温度为37℃,在25~45℃时稳定性很好;最适反应p H为8.0,在p H 6.5~8.0范围内酶活力较为稳定。底物特异性研究表明FADH最适底物为甲醛,相对偏好短链醛类。金属离子对酶活性的影响试验表明,Zn~(2+)、Mn~(2+)、Ag~+、Fe~(3+)和Hg~(2+)几乎完全抑制FADH酶活力;Ca~(2+)对酶活有促进作用。纯酶液在4℃环境下,在24h内可保存78%的活性。[结论]FADH结构较为特殊,且活性较大,为后期表达和深入研究其生物学功能提供理论和数据支持。
[Objective] The study aimed to study formaldehyde degrading bacteria Paecilomyces variotii F1-23 as experimental strain, isolating and purifying enzyme solution of formaldehyde dehydrogenase, and studying its enzymatic properties. [Method] The bacterial cells were collected from the liquid culture medium and purified by cell crushing, precipitation with (NH4) 2SO4, dialysis, DFAE-Sepharose ion exchange chromatography and gel filtration chromatography. [Result] The molecular weight of this enzyme was about 112.81 kDa; the molecular weights of subunits were 65 kDa and 42 kDa; the activity of pure enzyme liquid was 336.10 U / mg, 4.02 times higher than that before purification. The results of enzymatic analysis showed that the optimal reaction temperature was 37 ℃ and the stability was good at 25 ~ 45 ℃. The optimum reaction pH was 8.0, and the enzyme activity in the range of pH 6.5 ~ 8.0 More stable. Substrate specificity studies have shown that the most suitable substrate for FADH is formaldehyde, with a relative preference for short chain aldehydes. The effect of metal ions on enzyme activity showed that FADH activity was almost completely inhibited by Zn ~ (2 +), Mn ~ (2 +), Ag ~ +, Fe ~ (3+) and Hg ~ (2+) on the enzyme has a role in promoting. Pure enzyme solution at 4 ℃ environment, can save 78% of the activity within 24h. [Conclusion] The FADH structure is more specific and more active, providing theoretical and data support for the subsequent expression and further study of its biological functions.