Rhamnogalacturonan alpha-d-galactopyranosyluronohydrolase. An enzyme that specifically removes the terminal nonreducing galacturonosyl residue in rhamnogalacturonan regions of pectin

Abstract

A new enzyme, rhamnogalacturonan (RG) alpha-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing beta-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 &mgr;m and a maximum reaction rate of 160 units mg-1 was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50 degreesC. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40 degreesC) and up to 60 degreesC (for 3 h).

Figure 1

a, HPAEC of the purified hexasaccharide 1 fraction (peak a1; structures in Table I); b, this fraction after degalactosylation, which generates as major product tetrasaccharide 3 (peak b1); c, this fraction after degalactosylation and subsequent derhamnosylation, which generates as major product trisaccharide 5 (peak c1).

Figure 2

Detailed purification scheme of RG-galacturonohydrolase from Pectinex Ultra SP produced by A. aculeatus.

Figure 3

Chromatography of the protein fraction that was eluted from the Q-Sepharose column at 35 mm NaCl, on a chelating Sepharose Fast Flow column loaded with Cu²⁺ ions. For elution a pH gradient of pH 6.0 to 4.0 (buffer B) in 20 mm Bis-Tris containing 500 mm NaCl was used (Fig. 2). Solid line, A₂₈₀; dotted line, percent buffer B; ▪, RG-galacturonohydrolase activity; ○, RG-rhamnohydrolase activity (expressed as percentages of sugar released from the total amount present in the substrate).

Figure 4

a, Optimum pH of RG-galacturonohydrolase, 100% = enzyme activity at optimum pH; b, pH stability of RG-galacturonohydrolase, 100% = activity of untreated enzyme; c, optimum temperature of RG-galacturonohydrolase, 100% = enzyme activity at optimum temperature; and d, temperature stability of RG-galacturonohydrolase, 100% = activity of untreated enzyme.

Figure 5

a, HPAEC of hexasaccharide 1 fraction (peak a1; structures in Table I) before (bottom) and after (top) 45 h of incubation with RG-galacturonohydrolase; b, tetrasaccharide 3 (peak b1) before (bottom) and after (top) 45 h of incubation with RG-galacturonohydrolase; c, trisaccharide 5 (peak c1) before (bottom) and after (top) 45 h of incubation with RG-galacturonohydrolase.

Figure 6

Partial ¹H-NMR spectra showing the stereochemical course of hydrolysis of linear RG oligomers by the RG-galacturonohydrolase. H-1 resonances of the GalA released are indicated (α-GalA and β-GalA).