1887

Abstract

Two carboxymethylcellulases (CMCase, 1,4-1,4---glucan glucanohydrolase, EC 3.2.1.4), designated E-H and E-L, were purified to homogeneity from a culture filtrate of the alkalophilic sp. KSM-635, by chromatography on DEAE-Toyopearl 650S and gel filtration on Bio-Gel A-0·5m. The purified CMCases both contained approximately 2–3% (w/w) glucosamine. Molecular masses deduced from SDS-PAGE were 130 kDa for E-H and 103 kDa for E-L. The pH optima of the enzymes were both about 9·5, and their optimum temperatures were around 40 °C. Activities of both enzymes were inhibited by Hg, Cu, Fe and Fe, but sulphydryl inhibitors, such as -ethylmaleimide, monoiodoacetate and 4-chloromercuribenzoate, had either no effect or a slightly inhibitory effect. -Bromosuccinimide was strongly inhibitory, suggesting that a tryptophan residue is essential for the activity of the CMCases from . In addition, the activities of both E-H and E-L were stimulated by Co, and they required Mg, Ca, Mn or Co for stabilization. Both enzymes efficiently hydrolysed carboxymethylcellulose (-1,4-linkage) and lichenan (-1,3; 1,4-linkage), but crystalline cellulosic substrates, curdlan (-1,3-linkage), laminarin (-1,3; 1,6-linkage) and 4-nitrophenyl---glucopyranoside were hydrolysed very little, if at all. 4-Nitrophenyl---cellobioside was hydrolysed by both enzymes to liberate 4-nitrophenol, and their hydrolysis rates were higher at neutral pH than at alkaline pH.

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1990-10-01
2021-08-04
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