Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
Int J Biol Macromol. 2019 Oct 1;138:1044-1055. doi: 10.1016/j.ijbiomac.2019.07.135. Epub 2019 Jul 23.
In the present study, out of three isoforms of α-mannosidase identified in the crude extract of defatted Custard apple seed powder, isoform III has been purified to homogeneity by two-step chromatography: hydrophobic interaction and gel filtration. The purified Custard apple α-mannosidase isoform III (CAM) hydrolyzed both chromogenic (p-nitrophenyl-α-D-mannopyranoside) and fluorescent (4-methylumbelliferyl α-D-mannopyranoside) substrates. Custard apple α-mannosidase migrated as a single band in native PAGE, showed about 220 kDa molecular mass in gel filtration and in SDS PAGE, dissociated into four bands (Mr ~ 75, 68, 56 and 50 kDa respectively). Temperature and pH optima were found to be 50 °C and 4.0-5.0 respectively and CAM was stable up to 60-70 °C. The enzymatic activity of CAM was inhibited by EDTA, Ag, Hg, Ni and swainsonine (IC value of 1.5 μM). CAM was observed to be a metallo enzyme requiring zinc for its activity. Kinetic parameters K and Vmax were found to be 1.75 mM and 0.068 U/mL respectively. The CD spectral analysis at far UV region (190-300 nm) shows that purified CAM exists as helix (30.4%), β turns (18%) and random coils (29.7%) in its secondary structure. Chemical modification studies with N-Bromosuccinimide revealed the presence of tryptophan in its active site.
在本研究中，从脱脂番木瓜籽粉粗提物中鉴定出的三种α-甘露糖苷酶同工酶中，同工酶 III 通过两步层析法（疏水相互作用和凝胶过滤）纯化至均一性。纯化的番木瓜 α-甘露糖苷酶同工酶 III（CAM）水解了显色（对硝基苯-α-D-甘露吡喃糖苷）和荧光（4-甲基伞形酮-α-D-甘露吡喃糖苷）底物。番木瓜 α-甘露糖苷酶在天然 PAGE 中迁移为单一带，在凝胶过滤和 SDS-PAGE 中显示约 220 kDa 的分子量，在 SDS-PAGE 中解离成四个带（Mr 分别为 75、68、56 和 50 kDa）。发现最适温度和 pH 值分别为 50°C 和 4.0-5.0，CAM 在 60-70°C 下稳定。CAM 的酶活性被 EDTA、Ag、Hg、Ni 和 swainsonine（IC 值为 1.5 μM）抑制。CAM 被观察为一种金属酶，其活性需要锌。发现动力学参数 K 和 Vmax 分别为 1.75 mM 和 0.068 U/mL。远紫外区（190-300nm）的 CD 光谱分析表明，纯化的 CAM 在其二级结构中存在螺旋（30.4%）、β 转角（18%）和无规卷曲（29.7%）。用 N-溴代丁二酰亚胺进行的化学修饰研究表明，其活性位点存在色氨酸。
