诺卡氏菌形放线菌β甘露聚糖酶的纯化和性质

PURIFICTION AND PROPERTIES OF βD MANNANASE FROM NOCARDIOFORM ACTINOMYCETES

产β１ ，４Ｄ甘露聚糖酶的诺卡氏菌形放线菌（ Ｎｏｃａｒｄｉｏｆｏｒｍ ａｃｔｉｎｏｍｙｃｅｔｅｓ） 菌株ＮＡ３５４０ ，发酵培养７２ｈ ，发酵液离心去菌体，上清经硫酸铵沉淀，９５ ％ 乙醇沉淀，ＣＭＳｅｐｈａｄｅｘ Ａ５０ 柱层析、羟基磷灰石柱层析、ＤＥＡＥ纤维素离子交换及Ｓｅｐｈａｄｅｘ Ｇ１００ 分子凝胶过滤柱等步骤，β甘露聚糖酶的比活提高了１３７ 倍，获得凝胶电泳均一的蛋白样品。经ＳＤＳＰＡＧＥ 和凝胶过滤法分别测定β甘露聚糖酶分子量为４１ｋＤ和４０ｋＤ，证明该酶为单聚体；用等电聚焦电泳测得其等电点为４－８ ；经氨基酸组成分析，发现蛋白中有较高含量的Ｇｌｙ、Ａｓｐ、Ａｌａ 及Ｇｌｕ 残基。该酶的最适反应温度为７５ ℃，在不超过６０ ℃时酶活较稳定；酶反应的最适ｐＨ 为８－０ ，ｐＨ 稳定范围为６－５～１１－０ 。重金属离子Ｈｇ２＋ 、Ｃｕ２＋ 、Ｐｂ２ ＋ 、Ｆｅ３＋ 、Ｃｏ２＋ 、Ｚｎ２ ＋ 能强烈抑制该酶活性，而Ｍｎ２＋ 、Ｆｅ２＋ 、Ａｇ＋ 对该酶有部分的抑制作用，低浓度的Ｎａ＋ 、Ｋ＋ 、Ｌｉ＋ 对该酶基本没有影响。

After cultivating in liquid medium for 3 days, an extracellular endo\|1,4 β D mannanase (EC\|3.2.1.78) from a \%Nocardioform actinomycetes\% strain NA3\|540 was purified 137\|fold to electrophoretic homogeneity by ammonium sulfate precipitation, 95% alcohol precipitation, CM\|sephadex A\|50, hydroxyaptite column chromatography, DEAE\|cellulose anion\|exchange column chromatography and Sephadex G\|100 column chromatography. The enzyme had a molecular mass of 41 kDa(by SDS\|PAGE) and 40 kD (by gel filtration), a pI of 4.8 (PAGEIEF) and carbohydrates content of 3.5%. The enzyme was optimally active at pH 8 and 75℃ and showed stability at pH range of 6.5 to 12 at the temperature below 60℃. The amino acid composition analysis of the enzyme proved that there were large amount of Gly, Asp, Ala and Glu. The enzyme was strongly inhibited by Hg 2+ 、Cu 2+ 、Pb 2+ 、Fe 3+ 、Co 2+ 、Zn 2+ and partly inhibited by Mn 2+ 、Fe 2+ 、Ag + and slightly inhibited by Na\++\,K\++\,Li\++, Mg 2+ , Ca 2+ ,Ba 2+ .