Partial purification and properties of an endo-xylanase from cucumber seeds

Abstract

1991. Partial purification and properties of an endo-xylanase from cucumber seeds. -Physio!. Plant. 81: 327-334.An endo-xylanase. 1.4-^-D-xylan xylanohydrolase (EC 3.2.1.8) from immature cucumber (Cucumis sativus L. cv. Heinz 3534) seeds was partially purified using ammonium sulfate fractionation and chromatography on SP-Sephadex and Sephadex G-100 in order to determine its role in xylan metabolism during development. Attempts to further purify the enzyme using chromatography on DEAE-Sephadex, Bio-Gel HTP hydroxylapatite, Sephadex G-200 and con A-Sepharose 4B and native polyacrylamide ge! electrophoresis resulted in a significant decrease or complete loss of enzyme activity. Endo-xylanase had a native molecular weight of 96 kDa as determined by gel filtration, exhibited optimal activity ai pH 5.0 and 48°C, and was most stable from pH 4.0 to 5.0. Using beechwood 4-o-methyl-t)-glucurono-D-xylan dyed with Remazol Brilliant Blue R as substrate, the K^ was estimated to be 0.70 mg ml '. HgCI, at i mM inhibited endo-xylanase completely. Other metal ions inhibited the enzyme in the order Cu^* > Fe'* > Zn'+ > Ca-+ > Mn'*. The eihanol-soluble products of endo-xylanase action on beechwood xylan were isolated and characterized by consecutive chromatography on Bio-Gel PIO and P-2. The major reaction products were xylo-oligosaccharides [degree of polymerization (dp) > 10] but traces of xylobiose and free xylose were also isolated. The formation of xylo-oligosaccharides indicated that the reaction was catalyzed primarily by an endo-xylanase. The partially pure enzyme had no activity towards other cell wall polysaccharides such as cellulose, carboxymethyl cellulose, sodium carboxyi cellulose, potato starch, orange pectin, polygalacturonic acid, arabinogalactan and /J-giucan. However, it was able to hydrolyze larchwood and oat spelts xylan and a polysaccharide component from purified cucumber cell walls. The ability to utilize a substrate from cucumber cell walls supports the hypothesis that endo-xylanase is involved in the development of cucumber seeds.

Effect of nanocellulose/chitosan composite coatings on cucumber quality and shelf life

“…Softening is an important parameter to determine the quality loss of fleshy fruits (Macro et al 2010). Cucumber contains many enzymes that are associated with tissue softening (Rashmi and Roger 2009),which were previously described by several investigators (Bell, Etchells, and Jones 1951;Pressey and Avants 1975;McFeeters, Bell, and Fleming 1980;Miller, Dalmasso, and Kretchman 1989;Mujer, Kretchman, and Miller 1991;Omran et al 1991). Various investigators have made efforts to inhibit this tissue softening phenomenon in order to provide beneficial consequences to the consuming human (Fasina and Fleming 2001;Fasina, Fleming, and Thompson 2002;Hurr et al 2013;Marcin, Richard, and Deborah 2015).…”

Purification and properties of β‐xylosidase isozymes from cucumber seeds

“…Purified /3-xylosidase A was not able to hydrolyze any of the following p-nitrophenyl glycosides: a-L-arabinofuranoside, a-L-arabinopyranoside, a-and ^-o-gJueopyranosides, a-and ^-D-galactopyranosides, a-L-rhamnopyranoside, /3-D-mannopyranoside and /3-D-fucopyranoside as well as p-nitrophenyl ;S-D-galacturonide, p-nitrophenyl ^-D-glucuronide, /j-nitrophenyl ^-D-cellobioside and beeehwood xyian. However, it was able to hydrolyze xylo-oligosaccharides obtained after hydrolysis of beeehwood xyian by cucumber endo-xylanase (Mujer et al 1991). Incubation of these xylo-oligosaccharides (DP > 10) with /S-xylosidase A yielded free xylose as determined by thin-layer chromatography (Fig.…”

“…The use of native substrates from cucumber cell walls would help elucidate the action of /?-xylosidase during cell wall degradation in vivo. It has been demonstrated previously (Mujer et al 1991) that cucumber endo-xylanase could utilize a hemiceliuiose fraction from cucumber celi walls. It would be of interest, therefore, to examine the possible synergistic interaction of both enzymes in xyian metabolism during cucumber seed development.…”

“…This activity couid be due to the action of one or more enzymes such as endo-xylanase, exo-xylanase and/or ^-xyiosidase. In a previous study (Mujer et al 1991), we reported the partial purification and properties of an endo-xyianase from cucumber seeds. This enzyme cataiyzed the hydroiysis of beeehwood xyian and yielded xylo-oiigosaccharides as the major product and smail amounts of xyiobiose and free xyiose.…”