Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.
Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.
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DOI：
10.1007/s007920050031
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发表时间：
1997-11-01
期刊：
影响因子：
2.9
通讯作者：
Antranikian, G
中科院分区：
文献类型：
--
作者：
Kozianowski, G;Canganella, F;Antranikian, G
从意大利的一家温泉中分离到一株能够在柑橘果胶和聚半乳糖醛酸(果胶)上生长的新型嗜热厌氧芽胞形成微生物(AB9菌株)。该菌株最适生长温度为70℃，以果胶为底物的生长速率为0.23h(-1)，以果胶为底物的生长速率为0.12h(-1)。木聚糖、淀粉和糖原也被用作碳源，并形成热活性木聚糖酶(最高活性在70-75摄氏度)、淀粉酶以及幼芽分解酶(最高活性在80-85摄氏度)。从菌株AB9在柑橘果胶上生长的300-L培养上清液中分离到两个具有热活性的果胶裂解酶。经硫酸铵处理、阴离子交换层析、疏水层析、制备性凝胶电泳法纯化得到两种酶(裂解酶a、b)。经十二烷基硫酸钠(SDS)凝胶电泳法测定，裂解酶a为单一多肽，相对分子质量为135 000 Da，裂解酶b由两个亚基组成，相对分子质量分别为93 000 Da和158 000 Da。这两种酶具有相似的催化性能，在pH 9.0和80℃时活性最高，在70℃和80℃时非常稳定，半衰期超过60min。果胶裂解酶以果胶酸橙(100%)和果胶钠盐(90%)活性最高，而果胶受到攻击的程度较小(50%)。两种裂解酶对果胶和柑橘果胶的K-m值分别为0.5g.L(-1)和5.0g.L(-1)。与聚半乳糖醛酸孵育后，最终产物为一、二、三半乳糖醛酸脂。当果胶裂解酶在1 mM的Ca~(2+)中孵育时，酶活提高了2.5倍。加入1 mM乙二胺四乙酸乙酯(EDTA)可完全抑制酶活性。这些耐热的酶是从嗜热厌氧细菌中分离和鉴定的第一种果胶裂解酶。根据16S rRNA序列比较的结果和观察到的表型差异，我们提出AB9菌株为高温厌氧细菌的一个新种，即意大利高温厌氧细菌sp.11月
A novel thermophilic spore-forming anaerobic microorganism (strain Ab9) able to grow on citrus pectin and polygalacturonic acid (pectate) was isolated from a thermal spa in Italy. The newly isolated strain grows optimally at 70 degrees C with a growth rate of 0.23 h(-1) with pectin and 0.12 h(-1) with pectate as substrates. Xylan, starch, and glycogen are also utilized as carbon sources and thermoactive xylanolytic (highest activity at 70 degrees-75 degrees C), amylolytic as well as pullulolytic enzymes (highest activity at 80 degrees-85 degrees C) are formed. Two thermoactive pectate lyases were isolated from the supernatant of a 300-l culture of isolate Ab9 after growth on citrus pectin. The two enzymes (lyases a and b) were purified to homogeneity by ammonium sulfate treatment, anion exchange chromatography, hydrophobic chromatography and finally by preparative gel electrophoresis. After sodium dodecylsulfate (SDS) gel electrophoresis, lyase a appeared as a single polypeptide with a molecular mass of 135 000 Da whereas lyase b consisted of two subunits with molecular masses of 93 000 Da and 158 000 Da. Both enzymes displayed similar catalytic properties with optimal activity at pH 9.0 and 80 degrees C. The enzymes were very stable at 70 degrees C and at 80 degrees C with a half-life of more than 60 min. The maximal activity of the purified lyases was observed with orange pectate (100%) and pectate-sodium salt (90%), whereas pectin was attacked to a much lesser extent (50%). The K-m values of both lyases for pectate and citrus pectin were 0.5 g.l(-1) and 5.0 g.l(-1), respectively. After incubation with polygalacturonic acid, mono-, di-, and tri-galacturonate were detected as final products. A 2.5-fold increase of activity was obtained when pectate lyases were incubated in the presence of 1 mM Ca2+. The addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These heat-stable enzymes represent the first pectate-lyases isolated and characterized from a thermophilic anaerobic bacterium. On the basis of the results of the 16S rRNA sequence comparisons and the observed phenotypic differences, we propose strain Ab9 as a new species of Thermoanaerobacter, namely Thermoanaerobacter italicus sp. nov.