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首页> 外文期刊>Applied Microbiology >A C-Terminal Proline-Rich Sequence Simultaneously Broadens the Optimal Temperature and pH Ranges and Improves the Catalytic Efficiency of Glycosyl Hydrolase Family 10 Ruminal Xylanases
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A C-Terminal Proline-Rich Sequence Simultaneously Broadens the Optimal Temperature and pH Ranges and Improves the Catalytic Efficiency of Glycosyl Hydrolase Family 10 Ruminal Xylanases

机译：C末端脯氨酸丰富的序列同时拓宽了最佳温度和pH范围，并提高了糖基水解酶家族10瘤胃木聚糖酶的催化效率。

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Efficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene ( xynA ) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed in Escherichia coli . The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.
机译：瘤胃中植物多糖的有效降解需要具有高催化能力的木聚糖分解酶。在这项研究中，从绵羊瘤胃中获得了全长木聚糖酶基因（xynA）。推导的XynA序列包含推定的信号肽，糖苷水解酶家族10（GH10）的催化基序，以及额外的C端富含脯氨酸的序列，而没有同源物。为了确定其功能，成熟的XynA及其C末端截短的突变体XynA-Tr均在大肠杆菌中表达。 C末端寡肽对XynA的功能和结构具有显着影响。与XynA-Tr相比，XynA表现出提高的比活性（12倍）和催化效率（14倍），更高的最佳温度（50°C对45°C）以及更宽的温度和pH最佳范围（pH 5.0）到7.5和40至60°C（pH 5.5至6.5和40至50°C）。此外，当以山毛榉木木聚糖和小麦阿拉伯木聚糖为底物时，XynA释放的木糖比XynA-Tr多。通过底物结合测定，圆二色性（CD）光谱，等温滴定量热法（ITC）和低聚木糖水解来分析造成这些变化的潜在机制。 XynA没有能力与任何测试的可溶性和不溶性多糖结合。然而，它含有更多的α螺旋，并且对木寡糖具有更高的亲和力和催化效率，这有利于底物的完全降解。当C-末端序列与绵羊瘤胃的另一种GH10木聚糖酶融合时，获得了相似的结果。这项研究揭示了一种改善酶催化性能的工程策略。

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