ENDO-BETA-1,4-D-GALACTANASE FROM ASPERGILLUS-NIGER VAR ACULEATUS - PURIFICATION AND SOME PROPERTIES

An endo-1,4-beta-D-galactanase from the commercial preparation SP 249 (Novo Industri) originating from Aspergillus niger var. aculeatus was purified using affinity chromatography on laboratory cross-linked Sepharose 6B with divinyl sulfone, gel filtration on Sephadex G 75, and ion-exchange chromatography on DEAE-Sephadex A 50. Galactanase-apparent specific activity was increased 114-fold after purification and the enzyme is devoid of endopolygalacturonase, pectin-, pectate-lyase, arabinanase, beta-D-galactosidase, and alpha-L-arabinofuranosidase activities. It migrates as a single band in sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis (PAGE) and has a molecular weight of 38 000. It is an acidic enzyme of pHi 2.8 containing a low amount of a putative isoenzyme of pHi 2.7 which also migrates as a minor band on PAGE without SDS. Enzymatic activity is optimum at pH 3.5-4.0 and at 50-55-degrees-C and the enzyme is most stable between approximately pH 4.0-7.0 and below 30-degrees-C. It does not attack other galactans such as agarose, kappa- and iota-carrageenan nor type II arabinogalactans. Activity is inhibited by excess substrate and dissociation constants K(D) (0.6 mg ml-1) and K'(D) (10.7 mg ml-1), and maximal velocity V (8500 nkat) were obtained from Line-weaver-Burk and Khun plots. Endo-galactanase specific activity is 17.0 mkat mg-1. Galactose and galactobiose are the end products of galactan hydrolysis.