Le Gal-Coëffet M F, Jacks A J, Sorimachi K, Williamson M P, Williamson G, Archer D B
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Eur J Biochem. 1995 Oct 15;233(2):561-7. doi: 10.1111/j.1432-1033.1995.561_2.x.
Glucoamylase 1 from Aspergillus niger is an economically important enzyme in many industrial processes. It hydrolyses granular starch and comprises two distinct domains, a catalytic and a starch-binding domain (SBD). We have transformed A. niger with an expression vector for the secretion of SBD for physico-chemical studies. This was achieved by introducing into the glucoamylase gene a short sequence encoding an endoproteolytic cleavage recognition site such that free SBD was secreted at yields up to 200 mg/l. Free SBD was also obtained by proteolytic digestion of full-length glucoamylase 1. Electrospray mass spectroscopy was used to determine the carbohydrate content of both SBDs. It revealed that the engineered one is more glycosylated: an average of three mannose residues compared to one for the proteolytically derived SBD. Sequencing results also suggest partial glycosylation for the three Thr residues involved (510, 511, 513). It is probable that the engineered SBD represents the true glycosylation level of the SBD in native glucoamylase. Binding of beta-cyclodextrin to the SBD was investigated. It was found that the stoichiometry and the spectral perturbation of Trp residues were identical for both SBDs, but the engineered SBD bound less strongly to the ligand. At high concentrations of beta-cyclodextrin relative to the estimated Kd values, the maximum absorbance changes were identical. The observed difference at low beta-cyclodextrin levels was probably due to the higher level of glycosylation of the expressed SBD. We conclude that the proteolytically derived and expressed starch binding domains both bind 2 mol beta-cyclodextrin/mol protein, but that the pattern of glycosylation and strength of binding are different.
来自黑曲霉的葡糖淀粉酶1在许多工业过程中都是一种具有重要经济价值的酶。它能水解颗粒淀粉，由两个不同的结构域组成，一个催化结构域和一个淀粉结合结构域（SBD）。我们用一个用于分泌SBD的表达载体转化了黑曲霉，以便进行物理化学研究。这是通过在葡糖淀粉酶基因中引入一个编码内切蛋白酶切割识别位点的短序列来实现的，从而使游离的SBD以高达200 mg/l的产量分泌出来。游离的SBD也可通过对全长葡糖淀粉酶1进行蛋白酶消化获得。采用电喷雾质谱法测定了两种SBD的碳水化合物含量。结果表明，工程改造后的SBD糖基化程度更高：平均有三个甘露糖残基，而通过蛋白酶消化得到的SBD平均只有一个。测序结果还表明，所涉及的三个苏氨酸残基（510、511、513）存在部分糖基化。工程改造后的SBD可能代表了天然葡糖淀粉酶中SBD的真实糖基化水平。研究了β-环糊精与SBD的结合情况。发现两种SBD的化学计量比和色氨酸残基的光谱扰动相同，但工程改造后的SBD与配体的结合较弱。在相对于估计的Kd值较高的β-环糊精浓度下，最大吸光度变化相同。在低β-环糊精水平下观察到的差异可能是由于表达的SBD糖基化水平较高所致。我们得出结论，通过蛋白酶消化得到的和表达的淀粉结合结构域都能与每摩尔蛋白质结合2摩尔β-环糊精，但糖基化模式和结合强度不同。
