Lee Areum, Choi Kyoung-Hwa, Yoon Dahye, Kim Suhkmann, Cha Jaeho
Department of Microbiology, Genetic Engineering Institute, Pusan National University, Busan 46241, Republic of Korea.
Department of Chemistry, Center for Proteome Biophysics, Chemistry Institute for Functional Materials, Pusan National University, Busan 46241, Republic of Korea.
J Biosci Bioeng. 2017 Sep;124(3):289-295. doi: 10.1016/j.jbiosc.2017.04.011. Epub 2017 May 5.
The putative gene cluster involved in the degradation of the raffinose family oligosaccharides (RFO) was identified in Caldicellulosiruptor bescii. Within the cluster, the gene encoding a putative α-galactosidase (CbAga36) was cloned and expressed in Escherichia coli. Size exclusion chromatography of the purified rCbAga36 indicated that the native form was a tetramer. Its primary sequence was similar to the family of glycoside hydrolase 36. The purified recombinant CbAga36 (rCbAga36) was optimally active at pH 5.0 and 70°C and had a half-life of 15 h and 10 h at 70°C and 80°C, respectively. rCbAga36 showed high activity with the artificial substrate (p-nitrophenyl α-d-galactopyranoside, pNPαGal) exhibiting lower K and higher k than natural substrates such as melibiose and raffinose. Although rCbAga36 demonstrated preferential activity toward the hydrolysis of RFO such as raffinose and stachyose, it did not degrade the polymeric galactomannans. Our results imply that CbAga36 may play a role in the degradation of RFO, transported into the cytoplasm via a transporter into galactose, which is further utilized as an energy source in C. bescii. Furthermore, its ability to synthesize novel oligosaccharides by transglycosylation renders this enzyme potentially useful for the production of dietary oligosaccharides with novel function.
在嗜热栖热放线菌中鉴定出了与棉子糖家族低聚糖（RFO）降解相关的假定基因簇。在该基因簇中，编码假定α-半乳糖苷酶（CbAga36）的基因被克隆并在大肠杆菌中表达。纯化的rCbAga36的尺寸排阻色谱表明其天然形式为四聚体。其一级序列与糖苷水解酶36家族相似。纯化的重组CbAga36（rCbAga36）在pH 5.0和70°C时活性最佳，在70°C和80°C下的半衰期分别为15小时和10小时。rCbAga36对人工底物（对硝基苯基α-D-吡喃半乳糖苷，pNPαGal）表现出高活性，与诸如蜜二糖和棉子糖等天然底物相比，其Km较低，kcat较高。尽管rCbAga36对棉子糖和水苏糖等RFO的水解表现出优先活性，但它不会降解聚合的半乳甘露聚糖。我们的结果表明，CbAga36可能在RFO的降解中发挥作用，通过转运蛋白进入细胞质后转化为半乳糖，进而在嗜热栖热放线菌中作为能量来源被进一步利用。此外，其通过转糖基化合成新型低聚糖的能力使该酶在生产具有新功能的膳食低聚糖方面具有潜在用途。
