ABSTRACT
Insoluble potato dietary fibre, isolated from potato pulp, can be enzymatically hydrolysed with the pectolytic enzyme preparation Pectinex Ultra SP from Novo Nordisk A/S, in order to produce soluble fibre. The soluble fibre has valuable functional properties for the food industry. Cloned monocomponent enzymes from Pectinex Ultra SP (arabinofuranosidase, endoglucanase II, pectin lyase, polygalacturonase I, rhamnogalacturonan acetyl esterase, rhamnogalacturonase a, rhamnogalacturonase b and xylanase I) were added in order to increase the yield. Surprisingly, however, the yield is not increased when any of the monocomponent enzymes are added. To describe the results a new model designated 'the competitive activity adsorption model' is proposed. The model is based on the fact that the enzymes are adsorbed to the substrate before action. A combination of the Langmuir adsorption isotherm and basic enzyme kinetics shows that different enzymes that adsorb competitively will have an inhibitory effect on each other and consequently decrease the hydrolysis rate and thereby the yield. The model has been confirmed by an experiment in which the fibre has been pre-treated with rhamnogalacturonan acetyl esterase.
Subject(s)
Cell Wall/chemistry , Enzymes/chemistry , Polysaccharides/chemistry , Acetylesterase/chemistry , Adsorption , Algorithms , Dietary Fiber , Hydrolysis , Kinetics , Solanum tuberosumABSTRACT
Lanatoside 15'-O-acetylesterase (LAE) from in-vitro-cultivated cells of Digitalis lanata Ehrh. was isolated and partially sequenced. The enzyme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Sepharose hydrophobic interaction chromatography followed by CM Sepharose cation-exchange chromatography to more than 330 mumol.s-1.(g protein)-1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed a major band at 39 kDa. The protein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chromatography on Sephacryl 200 revealed a single activity peak with an apparent molecular mass of about 85 kDa. Electrophoresis under nondenaturating conditions of purified LAE showed only one band with esterase activity. The intensity of this band was correlated with that of the 39-kDa band after SDS-PAGE. About 30% of the protein, including the N-terminus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homology to other hydrolases and apoplasmic proteins. It included the probable location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activities in the chemical transformation of cardenolides, but rather low in the proembryogenic masses of the embryogenic cell strain VIII. It increased during the development of somatic embryos. The LAE activity in leaves of D. lanata plants was in the range 4-24 nmol.s-1.(g protein)-1.
Subject(s)
Acetylesterase/isolation & purification , Digitalis/enzymology , Plants, Medicinal , Plants, Toxic , Acetylesterase/chemistry , Acetylesterase/metabolism , Amino Acid Sequence , Carbohydrate Sequence , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Kinetics , Molecular Sequence DataABSTRACT
A rhamnogalacturonan acetylesterase (RGAE) was purified to homogeneity from the filamentous fungus Aspergillus aculeatus, and the NH2-terminal amino acid sequence was determined. Full-length cDNAs encoding the enzyme were isolated from an A. aculeatus cDNA library using a polymerase chain reaction-generated product as a probe. The 936-base pair rha1 cDNA encodes a 250-residue precursor protein of 26,350 Da, including a 17-amino acid signal peptide. The rha1 cDNA was overexpressed in Aspergillus oryzae, a filamentous fungus that does not possess RGAE activity, and the recombinant enzyme was purified and characterized. Mass spectrometry of the native and recombinant RGAE revealed that the enzymes are heterogeneously glycosylated. In addition, the observed differences in their molecular masses, lectin binding patterns, and monosaccharide compositions indicate that the glycan moieties on the two enzymes are structurally different. The RGAE was shown to act in synergy with rhamnogalacturonase A as well as rhamnogalacturonase B from A. aculeatus in the degradation of apple pectin rhamnogalacturonan. RNA gel blot analyses indicate that the expression of rhamnogalacturonan degrading enzymes by A. acculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.
Subject(s)
Acetylesterase/genetics , Acetylesterase/metabolism , Aspergillus/enzymology , Aspergillus/genetics , Pectins/metabolism , Acetylesterase/chemistry , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , DNA, Fungal/genetics , Gene Expression Regulation, Fungal , Genes, Fungal , Glucose/metabolism , Glycosylation , Mass Spectrometry , Molecular Sequence Data , Molecular Weight , Protein Sorting Signals/chemistry , Protein Sorting Signals/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
An acetyl esterase was purified from cell walls isolated from mung bean hypocotyls. The purified enzyme had an apparent Mr of 43,300 and an apparent pI > 9. It rapidly deesterified triacetin and p-nitrophenylacetate and slowly released acetate from beet and flax pectins, the deesterification rate being increased by previous demethylation of the pectins. No significant peptide sequence identity between the acetyl esterase and any known protein could be found in protein data bases.