Department of Bioscience and Bioinformatics, Myongji-Ro 116, Yongin, Gyeoggido, 17058, South Korea.
Karakoram International University, Gilgit, Gilgit-Baltistan, Pakistan.
Appl Biochem Biotechnol. 2019 Aug;188(4):1077-1095. doi: 10.1007/s12010-019-02963-w. Epub 2019 Feb 21.
Agar, a major polysaccharide of red algal cells, is degraded by β-agarases into neoagarobiose, which is further hydrolyzed into the monomers, D-galactose and 3,6-anhydro-L-galactose, by 1,3-α-3,6-anhydro-L-galactosidases including α-1,3-L-neoagarooligasaccharide hydrolase (α-NAOSH). A novel cold-adapted alkaline α-NAOSH, Ahg558, consisting of 359 amino acids (40.8 kDa) was identified from Gayadomonas joobiniege G7. It was annotated as a glycosyl hydrolase family 43 based on genomic sequence analysis, showing 84% and 74% identities with the characterized α-NAOSHs from Agarivorans gilvus WH0801 and Saccharophagus degradans 2-40, respectively. The recombinant Ahg558 (rAhg558) purified from Escherichia coli formed dimers and cleaved α-1,3 glycosidic bonds at the non-reducing ends of the neoagarobiose, neoagarotetraose, and neoagarohexaose, which was confirmed by thin-layer chromatography and mass spectrometry. The optimum pH and temperature for rAhg558 activity were 9.0 and 30 °C, respectively. Unusually, it retained over 93% activity in a broad range of temperatures between 0 and 40 °C and over 73% in a broad range of pH between pH 6.0 and pH 9.0, indicating it is a unique cold-adapted alkaline exo-acting α-NAOSH. Its enzymatic activity was dependent on Mn ions. K and V values toward neoagarobiose were 2.6 mg/mL (8.01 mM) and 133.33 U/mg, respectively.
琼脂是红藻细胞的主要多糖，被β-琼脂酶降解为新琼二糖，然后再被 1,3-α-3,6-脱水-L-半乳糖苷酶，包括α-1,3-L-新琼寡糖水解酶（α-NAOSH），水解为单体 D-半乳糖和 3,6-脱水-L-半乳糖。从乔氏Gayadomonas G7 中鉴定出一种新型的耐冷碱性α-NAOSH，即 Ahg558，由 359 个氨基酸（40.8 kDa）组成。根据基因组序列分析，它被注释为糖苷水解酶家族 43，与已鉴定的 Agarivorans gilvus WH0801 和 Saccharophagus degradans 2-40 的α-NAOSH 分别具有 84%和 74%的同一性。从大肠杆菌中纯化的重组 Ahg558（rAhg558）形成二聚体，并在新琼二糖、新琼四糖和新琼六糖的非还原端切割α-1,3 糖苷键，这通过薄层层析和质谱得到了证实。rAhg558 的最适 pH 和温度分别为 9.0 和 30°C。不同寻常的是，它在 0 到 40°C 的宽温度范围内保留了超过 93%的活性，在 pH 6.0 到 pH 9.0 的宽 pH 范围内保留了超过 73%的活性，表明它是一种独特的耐冷碱性外切α-NAOSH。它的酶活性依赖于 Mn 离子。rAhg558 对新琼二糖的 K 和 V 值分别为 2.6mg/mL（8.01mM）和 133.33U/mg。
