Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli I: purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium

化学 枯草芽孢杆菌 巨芽孢杆菌 大肠杆菌 分子质量 色谱法 荧光 离解常数 脱氢酶 离解(化学) 生物化学 细菌 生物 有机化学 物理 受体 基因 量子力学 遗传学
作者
Wolfgang Hilt,Gerhard Pfleiderer,Peter Fortnagel
出处
期刊:Biochimica Et Biophysica Acta - Proteins And Proteomics [Elsevier BV]
卷期号:1076 (2): 298-304 被引量:49
标识
DOI:10.1016/0167-4838(91)90281-4
摘要

Escherichia coli containing the Bacillus subtilis glucose dehydrogenase gene on a plasmid (prL7) was used to produce the enzyme in high quantities. Glue-DH-S was purified from the cell extract by (NH4)2SO4-precipitation, ion-exchange chromatography and Triazine-dye chromatography to a specific activity of 375 U/mg. The enzyme was apparently homogenous on SDS-PAGE with a subunit molecular mass of 31.5 kDa. Investigation of Glue-DH-S was performed for comparison with the corresponding properties of Glue-DH-M. The limiting Michaelis constant at pH 8.0 for NAD+ is Ka = 0.11 mM and for d-glucose Kb = 8.7 mM. The dissociation constant for NAD+ is Kia = 17.1 mM. Similar to Gluc-DH-M, Gluc-DH-S is inactivated by dissociation under weak alkaline conditions at pH 9.0. Complete reactivation is attained by readjustment to pH 6.5. Ultraviolet absorption, fluorescence and CD-spectra of native Gluc-DH-S, as well as fluorescence- and CD-backbone-spectra of the dissociated enzyme were nearly identical to the corresponding spectra of Gluc-DH-M. The aromatic CD-spectrum of dissociated Gluc-DH-S was different, representing a residual ellipticity of tryptophyl moieties in the 290–310 nm region. Density gradient centrifugation proved that this behaviour is due to the formation of inactive dimers in equilibrium with monomers after dissociation. In comparison to Gluc-DH-M, the kinetics of inactivation as well as the time-dependent change of fluorescence intensity at pH 9.0 of Gluc-DH-S showed a higher velocity and a changed course of the dissociation process.
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