Partial Purification and Properties of an Induced β‐d‐Xylosidase of Bacillus pumilus 12

Abstract

A highly specific 8-D-xylosidase with absolute glycon-substrate specificity was induced in Bacillus pumilus 12 by xylose. It was purified 34-fold by a procedure involving lysis of the cells with lysozyme, ammonium sulfate fractionation, G-75 Sephadex gel filtration and hydroxylapatite chromatography. The optimal activity was in the pH region 7.0-7.3. The isoelectric point was found to be 4.4. I n contrast to its broad complexing affinity, the hydrolytic activity was restricted to aryl @-D-xylopyranosides and lower xylo-oligosaccharide derivatives. From the dependence of K , and V,,, on pH, it could be deduced that two dissociable groups were necessary for catalytic activity. The enzyme was competitively inhibited by Tris at pH 7.2 and proved sensitive to ionic environment. p-Chloromercuribenzoate and other SH-reagents were inhbitory but substrate analogs and competitive inhibitors protected the enzyme against inactivation by p-chloromercuribenzoate. The results indicate that a histidine residue or imidazolium group and a sulfhydryl group might participate in the enzymatic hydrolysis of p-nitrophenyl 8-D-xylopyranoside. It has been reported that plants [l-61 and fungi [7-111 produce 8-D-xylosidases which are distinct from @-D-ghcosidases.A @-D-xylosidase from a commercial mold hemicellulase sample has been extensively studied in this laboratory reported the production of a pentosanase in Bacillus pumilus 12. This paper describes the induction, partial purification and some properties of a highly specific @-Dxylosidase in the same strain. The results were compared with those obtained for the fungal @-D-xY~o-sidase. Methyl B-D-lyxopyranoside was a gift from Dr. EXPERIMENTAL PROCEDURE Materials Kjolberg (Oslo).Pentoses, hexoses and xylitol were commercial products (Fluka, Merck). Lysozyme, dithiothreitol and Biogel P-150 were purchased from Calbiochem. ; deoxyribonuclease I from Worthington and streptomycin sulfate from Pfizer. Hydroxylapatite was prepared according to the method of Siegelman et al. [28]. Sephadex G-25 and G-75 were obtained from Pharmacia (Uppsala). Beef extract, yeast extract, agar and peptone were Oxoid products. All buffer materials and other reagents were analytical grade. MethodsRoutine enzymic assays were performed with p-nitrophenyl @-D-xylopyranoside as substrate a t a concentration of 4 mM in 0.05 M phosphate buffer, pH 7.2. The hydrolysis was followed by measuring the increase in absorbance a t 400 nm in a Beckman DB-G spectrophotometer. The sample compartment was thermostated a t 25" and the spectrophotometer was equipped with a linear-log Ten-Inch recorder.pa-Nitrophenyl 8-D-xylopyranoside hydrolysis as a function of pH was followed discontinuously by adding equal volumes of a 15 o/o Na2C0, solution to the reaction mixture [lo].Hydrolysis of phenyl and alkyl @-n-xylopyranosides was followed by measuring respectively phenol a t 280 nm [29] and liberated reducing sugar [30--311

Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

“…5). In fact the average B-factor of the active site acidic et al , 1969;Chen et al, 1987) including AHA with Ki = 10" M residues in the native structure is 15.3 A2, whereas the correspondat pH 7.2. Inhibition studies of a-glucosidase, trehalase, and ing value in the complex with Tris is 16.9 A2.…”

Xylan‐Degrading Enzymes of the Yeast Cryptococcus albidus

“…These observations effluent was assayed for activity of /I-xylosidase, pointed out that the soluble cell-bound P-xylosidase /?-xylanase and also P-glucosidase, since it has been reported that some microbial B-glucosidases are capis of a xylobiase type. able of hydrolynng P-xylosides [19].…”

Purification and Characterization of a Thermostable -Xylosidase from Thermomonospora fusca

“…The first bacterial /Ixylosidase isolated was from Bacillus pumilus (Kersters-Hilderson et al, 1969) and more recently the properties of a /I-xylosidase from Clostridium acetobutylicum have been reported (Lee & Forsberg, 1987). The /I-xylosidase activity of a thermophilic Thermomonospora strain has been described as notable in that it exhibits broad pH and temperature optima (Ristroph & Humphrey, 1985), although no attempt was made to isolate or purify the enzyme from crude cell extracts.…”