Toshizo Isemura

8 Bacterial and Mold Amylases

Publisher Summary Bacterial and mold amylases have been the objects of organic chemical and physicochemical studies as they are available in large quantities and can be easily purified and crystallized. A large number of bacterial and mold amylases have been isolated in crystalline form from various sources. Bacillus subtilis α -amylase was the first to be crystallized, which was later followed by B. subtilis , Aspergillus oryzae , B. coagulans , A. candidus , Pseudomonas saccharophila , B. polymyxa , B. macerans , A. niger , B. amyloliquefaciens , and B. stearothermophilus . Crystallization of amylases requires the presence of divalent cations, especially calcium ions. When crystallization is repeated, it becomes increasingly difficult as loss of the cations results in apparent high solubility of the enzymes. Most of the crystalline amylases have been shown to be homogeneous in sedimentation and electrophoretic analysis. It was found, however, that even after repeated crystallization, crystalline Taka-amylase A is still contaminated by traces of proteases that can only be removed by chromatography. Polyacrylamide gel electrophoresis and analytical ion exchange chromatography are used to examine the homogeneity of an amylase preparation. It has been shown by polyacrylamide gel electrophoresis that crystalline amylases from B. macerans , B. amyloliquefaciens , B. stearothermophilus , and A. oryzae are homogeneous.

The β-structure of poly-S-carbobenzoxymethyl-l-cysteine in solution

Poly-S-carbobenzoxymethyl- L -cysteine has been shown from a study of polarized infrared spectra to exist in oriented films in the cross-β-conformation with an anti-parallel arrangement of polypeptide chains. Solutions of the polymer in chloroform-dichloroacetic acid mixtures were investigated by measurements of optical rotatory dispersion, viscosity and infrared spectra; their properties suddenly changed at solvent compositions near 2% dichloroacetic acid content. The polymer was found to assume the random coil form at higher diehloroacetic acid contents but to associate into the intermolecular β-conformation at lower dichloroacetic acid contents. At the solvent composition where the transition between random coil and β-form occurred, the β-form was partly converted into the random coil as the temperature was raised or the polymer concentration was diluted. The rotatory dispersion of the β-form of the polymer is characterized by a large positive a0 value but a small b0 value.

Optical rotatory dispersion and circular dichroism of d-amino acid oxidase

Abstract The optical rotatory dispersion and circular dichroism of d -amino acid oxidase ( d -amino acid: O 2 oxidoreductase, EC 1.4.3.3, pig kidney) were measured. In the visible region, the holoenzyme showed anomalous dispersion curves with characteristic patterns depending on the state of the coenzyme (FAD or FADH 2 ). The presence of optically active absorption bands was confirmed by measurement of circular dichroism. The anomaly in the disperison curves can be explained by assuming that weak Cotton effects, due to chromophores of the bound coenzyme, are superimposed on the intrinsic dispersion curves of the protein moiety of the enzyme. A plain dispersion curve was observed with the apoenzyme, and it differed significantly from the dispersion curve presumed for the protein moiety of the holoenzyme. In the far-ultraviolet region, the apoenzyme and holoenzyme exhibited identical optical rotatory dispersion and circular dichroism. It is concluded that the two forms of holoenzyme have almost the same conformation, and removal of the coenzyme produces a reversible conformational change in the non-α-helical portion of the protein moiety of the enzyme.

Inversion and enhancement of the circular dichroic spectrum of flavin adenine dinucleotide by the combination to D-amino acid oxidase

Abstract The optical rotatory dispersion (ORD) of D-amino acid oxidase (EC 1.4.3.3; pig kidney) has been reported by Aki et al. (1966) . The bound coenzyme, flavin adenine dinucleotide (FAD), displays Cotton effects centered about the wavelengths of the corresponding absorption maxima. Measurements of circular dichroic (CD) spectra of the bound FAD clearly indicated the presence of CD absorption bands. In this paper, a marked change of CD spectrum of FAD by the combination to D-amino acid oxidase is reported. This phenomenon can be taken as an evidence of a conformational change of FAD as the result of the binding to the apoenzyme.

Extent of renaturation of reduced Taka-amylase a before reformation of disulfide bonds☆

Abstract The conformation of reduced Taka-amylase A(α-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1 from Aspergillus oryzae ) was investigated in comparison with those of native Taka-amylase A and reduced-carboxymethylated Taka-amylase A. Sedimentation, viscocity, optical rotatory dispersion, and ultraviolet difference spectra were measured. The object was to determine whether intramolecular non-covalent interactions in the reduced Taka-amylase A molecule can bring about reformation of a ‘native-like’ conformation. It was found that the reduced enzyme molecule has an effective molecular volume 3–6 times as large as that of the enzyme molecule. Clearly, reduced Taka-amylase A is partially refolded, but further refolding requires the reformation of disulfide bonds through re-oxidation.

Monolayer Films of Retinal1 and the Effects of Light on Them

SEVERAL stereoisomers of retinal1 (vitamin A1 aldehyde) are known and their interconversions can easily be promoted by the action of light1. The photoconversion of 11-cis to all-trans retinal1 takes place with the highest efficiency2,3 and is involved in the first step of the visual process4. Studies of vitamin A and its derivatives have suggested that their surface-active properties are closely related to their physiological functions5,6. The purpose of the present investigation was to study the properties of monolayer films of all-trans and 11-cis retinal1 at air–water interfaces, and to determine the effect of light on the films.