Kozianowski G, Canganella F, Rainey F A, Hippe H, Antranikian G
Department of Biotechnology/Technical Microbiology, Technical University of Hamburg-Harburg, Hamburg, Germany.
Extremophiles. 1997 Nov;1(4):171-82. doi: 10.1007/s007920050031.
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 135000 Da whereas lyase b consisted of two subunits with molecular masses of 93000 Da and 158000 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 Km values of both lyases for pectate and citrus pectin were 0.5 g(-1) and 5.0 g(-1), respectively. After incubation with polygalacturonic acid, mono-, di-, and trigalacturonate 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.
从意大利的一个温泉中分离出一种新型嗜热形成芽孢的厌氧微生物（菌株Ab9），它能够在柑橘果胶和聚半乳糖醛酸（果胶酸盐）上生长。新分离的菌株在70℃时生长最佳，以果胶为底物时生长速率为0.23 h⁻¹，以果胶酸盐为底物时生长速率为0.12 h⁻¹。木聚糖、淀粉和糖原也被用作碳源，并且会形成热活性木聚糖酶（在70℃ - 75℃时活性最高）、淀粉酶以及支链淀粉酶（在80℃ - 85℃时活性最高）。在菌株Ab9于柑橘果胶上生长后，从300升培养物的上清液中分离出两种热活性果胶酸裂解酶。这两种酶（裂解酶a和b）通过硫酸铵处理、阴离子交换色谱、疏水色谱，最后通过制备性凝胶电泳纯化至同质。十二烷基硫酸钠（SDS）凝胶电泳后，裂解酶a呈现为一条分子量为135000 Da的单一多肽，而裂解酶b由分子量分别为93000 Da和158000 Da的两个亚基组成。两种酶都表现出相似的催化特性，在pH 9.0和80℃时活性最佳。这些酶在70℃和80℃时非常稳定，半衰期超过60分钟。纯化后的裂解酶对橙果胶酸盐（100%）和果胶酸钠盐（90%）表现出最大活性，而对果胶的作用程度要小得多（50%）。两种裂解酶对果胶酸盐和柑橘果胶的Km值分别为0.5 g⁻¹和5.0 g⁻¹。与聚半乳糖醛酸孵育后，检测到单半乳糖醛酸、二半乳糖醛酸和三半乳糖醛酸作为最终产物。当果胶酸裂解酶在1 mM Ca²⁺存在下孵育时，活性提高了2.5倍。添加1 mM乙二胺四乙酸（EDTA）会导致酶完全抑制。这些热稳定酶是首次从嗜热厌氧细菌中分离和表征的果胶酸裂解酶。基于16S rRNA序列比较结果和观察到的表型差异，我们提议将菌株Ab9作为嗜热栖热菌属的一个新物种，即意大利嗜热栖热菌（Thermoanaerobacter italicus sp. nov.）。
