Institute of Technical Microbiology, Hamburg University of Technology (TUHH), 21073, Hamburg, Germany.
Clariant Produkte (Deutschland) GmbH, Group Biotechnology, 81477, Munich, Germany.
Protein J. 2018 Oct;37(5):454-460. doi: 10.1007/s10930-018-9787-5.
From a biogas reactor metagenome an ORF (bp_cel9A) encoding a bacterial theme C glycoside hydrolase family 9 (GH9) enzyme was recombinantly produced in E. coli BL21 pQE-80L. BP_Cel9A exhibited ≤ 55% identity to annotated sequences. Subsequently, the enzyme was purified to homogeneity by affinity chromatography. The endo-beta-glucanase BP_Cel9A hydrolyzed the beta-1,3-1,4-linked barley beta-glucan with 24 U/mg at 30 °C and pH 6.0. More than 62% of activity was measured between 10 and 40 °C. Lichenan and xyloglucan were hydrolyzed with 67% and 40% of activity, respectively. The activity towards different substrates varied with different temperatures. However, the enzyme activity on CMC was extremely low (> 1%). In contrast to BP_Cel9A, most GH9 glucanases act preferably on crystalline or soluble cellulose with only side activities towards related substrates. The addition of calcium or magnesium enhanced the activity of BP_Cel9A, especially at higher temperatures. EDTA inhibited the enzyme, whereas EGTA had no effect, suggesting that Mg may adopt the function of Ca. BP_Cel9A exhibited a unique substrate spectrum when compared to other GH9 enzymes with great potential for mixed-linked glucan or xyloglucan degrading processes at moderate temperatures.
从沼气反应器宏基因组中，通过在大肠杆菌 BL21 pQE-80L 中重组表达，得到一个编码细菌主题 C 糖苷水解酶家族 9（GH9）酶的 ORF（bp_cel9A）。BP_Cel9A 与注释序列的同一性≤55%。随后，通过亲和层析将该酶纯化为均一性。内切-β-葡聚糖酶 BP_Cel9A 在 30°C 和 pH 6.0 下以 24 U/mg 的酶活水解大麦 β-葡聚糖，β-1,3-1,4 键连接。在 10-40°C 之间测量到超过 62%的活性。几丁质和木葡聚糖的水解活性分别为 67%和 40%。不同温度下，不同底物的活性不同。然而，BP_Cel9A 对 CMC 的活性极低（>1%）。与 BP_Cel9A 相反，大多数 GH9 葡聚糖酶更喜欢作用于结晶或可溶性纤维素，仅对相关底物具有侧活性。添加钙或镁可增强 BP_Cel9A 的活性，特别是在较高温度下。EDTA 抑制酶，而 EGTA 没有影响，表明 Mg 可能具有 Ca 的功能。与其他 GH9 酶相比，BP_Cel9A 具有独特的底物谱，在中温下具有用于混合连接葡聚糖或木葡聚糖降解过程的巨大潜力。
