Paenibacillus sp. 598K 6-α-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from α-(1→4)-glucan
Paenibacillus sp. 598K 6-α-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from α-(1→4)-glucan
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类芽孢杆菌属 sp. 598K 6-α-葡萄糖基转移酶对于从 α-(1→4)-葡聚糖合成环异麦芽寡糖至关重要
DOI：
10.1007/s00253-017-8174-z
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发表时间：
2017
影响因子：
5
通讯作者：
Kazumi Funane
中科院分区：
文献类型：
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作者：
Hitomi Ichinose;Ryuichiro Suzuki;Takatsugu Miyazaki;Keitarou Kimura;Mitsuru Momma;Nobuhiro Suzuki;Zui Fujimoto;Atsuo Kimura;Kazumi Funane
拟青霉属Paenibacillussp.598K即使在没有葡聚糖的情况下也能从淀粉中产生环异麦芽低聚糖(环糊精)。环异麦芽低聚糖葡聚糖转移酶专门从至少由四个分子组成的α-(1-→-6)-连续的葡萄糖链合成环异麦芽低聚糖。淀粉不是这种酶的底物。因此，我们预测该菌具有从淀粉中延伸α-(1-→-6)连接的葡萄糖的另一种酶系统，可以作为环异麦芽低聚糖葡糖基转移酶的底物，并鉴定了转糖酶Ps6GT31a。我们从细菌培养上清液中纯化了Ps6GT31a，克隆了相应的基因，并对重组酶进行了鉴定。Ps6GT31A属于糖苷水解酶家族31，它按以下活性顺序从底物的非还原末端释放葡萄糖：α-(1)→(4)->；α-(1)→(2)-α-(1)→(3)-α-(1)→(6)-葡萄糖和麦芽五糖。Ps6GT31a既能催化水解，又能催化葡萄糖转移。用高效液相色谱和质谱仪对产物进行了分析。13C核磁共振分析表明，Ps6GT31A具有较强的α-(1-→-4)到α-(1-→-6)葡萄糖转移活性。Ps6GT31a通过一系列的转糖反应将α-(1-→-6)连接的低聚糖延长到至少10的聚合度。最终，环异麦芽低聚糖葡聚糖转移酶以Ps6GT31A产生的转糖产物为底物合成环异麦芽低聚糖。我们的数据表明，Ps6GT31a是在无葡聚糖环境中合成α-(1-→-6)-葡聚糖的关键酶，用于生产环异麦芽低聚糖。
Paenibacillussp. 598K produces cycloisomaltooligosaccharides (cyclodextrans) from starch even in the absence of dextran. Cycloisomaltooligosaccharide glucanotransferase synthesizes cycloisomaltooligosaccharides exclusively from an α-(1 → 6)-consecutive glucose chain consisting of at least four molecules. Starch is not a substrate of this enzyme. Therefore, we predicted that the bacterium possesses another enzyme system for extending α-(1 → 6)-linked glucoses from starch, which can be used as the substrate for cycloisomaltooligosaccharide glucanotransferase, and identified the transglucosylation enzyme Ps6GT31A. We purified Ps6GT31A from the bacterial culture supernatant, cloned its corresponding gene, and characterized the recombinant enzyme. Ps6GT31A belongs to glycoside hydrolase family 31, and it liberates glucose from the non-reducing end of the substrate in the following order of activity: α-(1 → 4)-> α-(1 → 2)- > α-(1 → 3)- > α-(1 → 6)-glucobiose and maltopentaose > maltotetraose > maltotriose > maltose. Ps6GT31A catalyzes both hydrolysis and transglucosylation. The resulting transglucosylation compounds were analyzed by high-performance liquid chromatography and mass spectrometry. Analysis of the initial products by13C nuclear magnetic resonance spectroscopy revealed that Ps6GT31A had a strong α-(1 → 4) to α-(1 → 6) transglucosylation activity. Ps6GT31A elongated α-(1 → 6)-linked glucooligosaccharide to at least a degree of polymerization of 10 through a successive transglucosylation reaction. Eventually, cycloisomaltooligosaccharide glucanotransferase creates cycloisomaltooligosaccharides using the transglucosylation products generated by Ps6GT31A as the substrates. Our data suggest that Ps6GT31A is the key enzyme to synthesize α-(1 → 6)-glucan for cycloisomaltooligosaccharide production in dextran-free environments.