M.J.F. Searle-van Leeuwen

The arabinanases of Aspergillus niger — Purification and characterisation of two α-l-arabinofuranosidases and an endo-1,5-α-l-arabinanase

Three arabinan-degrading enzymes were isolated from a preparation derived from Aspergillus niger. Steps used in the purification procedure included gel filtration on Bio-Gel, anion exchange on DEAE Bio-Gel A, adsorption on Bio-Gel HTP, treatment with crosslinked alginate and FPLC chromatography on Mono Q. α-l-Arabinofuranosidase A was active on p-nitrophenyl-α-l-arabinofuranoside (specific activity 67 U/mg protein) and on 1,5-α-l-arabinofuranose oligosaccharides, but not on 1,5-α-l-arabinan, beet arabinan, arabinan obtained from apple juice by ultrafiltration, or arabinoxylan. α-l-Arabinofuranosidase B was active on all these substrates, but the synthetic sugar derivative was its best substrate (specific activity 147 U/mg protein). Both enzymes released arabinose as the sole product. endo-1,5-α-l-Arabinanase was active towards UFR arabinan and beet arabinan, but 1,5-α-l-arabinan was its best substrate (specific activity 1·2 U/mg protein). During hydrolysis of this substrate the disaccharide and the trisaccharide accumulated as end-products. All three enzymes were glycoproteins with isoelectric points in the range of pH 4·5 to 6·5. Their optimum pH values were in the range of 3·7 to 5·0 and their optimum temperatures 50 to 60°C. Arabinofuranosidase A (Mfr 128000) had a Km value for p-nitrophenyl-α-l-arabinoside of 6 × 10−4m, and a Vmax value of 15·7 × 103 min−1. The corresponding kinetic parameters for arabinofuranosidase B (Mr 60 000) were 4·8 × 10−4m and 14·6 × 103 min−1. The Km and Vmax values for endo-arabinanase (Mr 35000) on 1,5-α-l-arabinan were 0·72 g/litre and 73 min−1, respectively.

Rhamnogalacturonan acetylesterase : a novel enzyme from Aspergillus aculeatus, specific for the deacetylation of hairy (ramified) regions of pectins

Rhamnogalacturonan acetylesterase, able to specifically hydrolyse the acetyl asters present in modified hairy (ramified) regions (MHR) of apple pectin, was identified. The enzyme removed about 70% of the total acetyl groups in MHR. This acetylesterase did not cause the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, including the acetylated smooth regions present in beet pectin. Pretreatment of pectic polysaccharides in order to remove arabinose side chains had no effect on the acetyl release, wor was an effect found on the rate or degree of acetyl release, when the purified acetylesterase was combined with pectolytic enzymes, pectin methylesterase or arabinanases.

Purification and characterization of a (1,4)-ß-D-arabinoxylan arabinofuranohydrolase from Aspergillus awamori.

SummaryAn enzyme able to split off arabinose sidechains from cereal arabinoxylans was isolated from a cell-free culture filtrate of Aspergillus awamori CMI 142717 containing milled oat straw as the carbon source. The enzyme was highly specific for arabinoxylans and, unlike other α-l-arabinofuranosidases reported in the literature, did not show any activity towards p-nitrophenyl α-l-arabinofuranoside, arabinans and arabinogalactans. This novel enzyme, which can be described as a (1,4)-β-d-arabinofuranohydrolase, had a molecular mass of 32 000 Da when determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and a specific activity of 22 units/mg on wheat arabinoxylan.

Effect of enzymatic deacetylation on gelation of sugar beet pectin in the presence of calcium

Abstract This study deals with the effect of methyl esters, acetyl groups, and neutral sugar side-chains on the gelation properties of sugar beet pectin with Ca 2+ . Sugar beet pectin was treated in the presence of Ca 2+ with the enzymes pectin methyl esterase (PE), pectin acetyl esterase (PAE), rhamnogalacturonan acetyl esterase (RGAE), arabinofuranosidase B (AF) and rhamnogalacturonase (RGase) in various combinations. Addition of RGAE plus PE or PAE plus PE to the pectin–Ca 2+ mixture significantly increased the release of acetyl groups and methyl esters, in comparison to the addition of only PE or PAE. This indicates that PE activity is hindered by the presence of acetyl groups both in the ‘smooth’ and in the `hairy’ regions. Also the PAE activity is hindered by the presence of methyl groups in the ‘smooth’ regions. Treatment with PAE plus PE led to a stiffer gel, as determined by the storage modulus ( G ′), than treatment with PE alone, while RGAE plus PE did not improve the gel forming properties. Addition of only PAE to the pectin–Ca 2+ mixture did not result in gel formation. A lower stiffness of the gel was found when RGase combined with RGAE and PE were added to the pectin–Ca 2+ mixture, in comparison to treatment with PE alone. Addition of AF plus PE to the pectin–Ca 2+ mixture gave similar rheological effects as treatment with only PE. A fraction representing the ‘smooth’ homogalacturonan regions, which was obtained after treatment of the beet pectin with RGase and subsequent size-exclusion chromatography, was also able to form a gel with Ca 2+ and PE. However, the gel formation was much slower, and the stiffness of the gel was lower than when the parental extract was used. Also with the modified pectin the treatment with PAE plus PE gave an increased stiffness of the gel in comparison to PE alone.

Purification and characterization of endo-1,4-β-D-galactanases from Aspergillus niger and Aspergillus aculeatus : use in combination with arabinanases from Aspergillus niger in enzymic conversion of potato arabinogalactan

Abstract Two galactanases, purified from experimental enzyme preparations derived from Aspergillus niger and Aspergillus aculeatus were found to be similar in a number of properties. They had similar molecular weights (Mr = 42–43 kD) and both showed highest activity on 1,4-β- D -galactan. Optimal activity was measured at 50–55°C and pH 4·00–4·25; optimal stability was observed in the pH range of 5–7 at 30°C. A sodium acetate buffer was found to be the best incubation buffer. Activity and stability were affected by Pb2+ (endo-galactanase from A. aculeatus was inhibited completely) and to a lesser extent by Ag+ and Zn2+ ions. Digestion of (arabino)-1,4-β- D -galactan resulted initially in a big shift in the Mw value of the bulk of (arabino)-1,4-β- D -galactan and formation of low galacto-oligomers, mainly tetra- and trimers of galactose. In the final stage of the reaction mono- and dimer accumulated as end products. Therefore a multiple attack mechanism was suggested for the endo-1,4-β- D -galactanases. The galactanases did not hydrolyse arabino-1,3/6-β- D -galactan. The effect of the temperature on their stability, their specific activities and their affinity for potato arabinogalactan differed; in the absence of substrate the A. niger and A. aculeatus endo-galactanase were stable up to 60°C and 35°C, respectively. The specific activities on potato arabinogalactan in a sodium acetate buffer pH 5·0, 30°C were found to be 158 and 244 u/mg, respectively. The Km values estimated for potato arabinogalactan were 0·77 and 0·31 g/litre, respectively. For an optimal breakdown of potato arabinogalactan combined action of arabinanases from A. niger and endo-galactanase was required. Arabinofuranosidase B did not stimulate the degradation of potato arabinogalactan by the endo-galactanases under the conditions used, whereas endo-1,5-α- L -arabinanase had an immediate synergistic effect with both endo-galactanases, indicating the presence of linear 1,5-α- L -arabinan side chains.

Degradation of arabinans by arabinanases from Aspergillus aculeatus and Aspergillus niger

Abstract An endo-arabinanase was purified from an enzyme preparation, derived from Aspergillus aculeatus. After SDS-gel electrophoresis a molecular weight of 45 kDa was estimated. The enzyme was reactive with antibodies raised against endo-arabinanase from Aspergillus niger, having the same molecular weight. Besides similarities, remarkable differences were observed for endo-arabinanases from A. niger and A. aculeatus. The enzyme from A. aculeatus was optimally active at a higher pH and produced a different spectrum of oligomers after incubation with linear arabinan. This was reflected in a relatively low concentration of oligomers with a degree of polymerization (DP) of 13 and a high concentration of oligomers with a DP of 6–7. Contrary to this, the concentration of oligomers produced by the A. niger endo-arabinanase gradually increased, going from DP 20 to DP 3. Both endo-arabinanases, as well as arabinofuranosidase B from A. niger, were studied with respect to the degradation of branched arabinans. Removal of arabinofuranosyl side chains by arabinofuranosidase B was essentially independent of the type of glycosidic linkage but had a tremendous effect on the digestibility by endo-arabinanase.

(1,4)-β-D-arabinoxylan arabinofuranohydrolase : a novel enzyme in the bioconversion of arabinoxylan

SummaryAn enzyme able to hydrolyse the terminal non-reducing α-l-arabinofuranoside residues from arabinoxylans only has been found. This enzyme was unable to split arabinofuranosyl linkages in a range of other arabinofuranosyl-containing substrates. Analysis of reaction mixtures of arabinoxylan with this enzyme did not show a shift in the molecular weight distribution of the arabinoxylan, even after 24 h of incubation. Only monomeric arabinose was released. 1H-Nuclear magnetic resonance studies of arabinoxylan treated with this enzyme, described as (1,4)-β-d-arabinoxylan arabinofuranohydrolase, indicated a specificity towards the single-substituted xylose in arabinoxylan.

Purification and characterization of β-amylase from Curculigo pilosa

Curculigo pilosa is traditionally used in the manufacture of sorghum beer in West Africa. β-Amylase was purified 100-fold with 38% yield from a crude extract, giving final specific activities of 4850 U/mg and 5650 U/mg using soluble starch and p-nitrophenyl maltopentaoside, respectively, as substrates. The molecular mass of the monomeric enzyme was 64 kDa and its pI 4.2. Both activity and thermostability are higher than reported for other plant β-amylases. The catalytic efficiency was lower for amylose than for starches and amylopectin. In contrast to other plant amylases, the β-amylase from C. pilosa is able to degrade raw starches from wheat, corn, potato and rice. In this respect, it resembles β-amylases from microbial origin. This property, and its high activity and stability, explain its traditional use in the manufacture of infant food and sorghum beer in Burkina Faso and could make it applicable for other biotechnological purposes.

Extraction, partial purification and characterization of β-amylase from the bulbs of G. klattianus.

Abstract The bulbs of Gladiolus klattianus are used in Burkina Faso in food processing. Activities of α-amylase and β-amylase were reported within those bulbs for the first time. The purification of the β-amylase involved buffer extraction, ammonium sulfate precipitation and gel filtration chromatography. The enzyme was purified 47 fold with 75% yield, giving a final specific activity of 2360 U/mg. The β-amylase from G. klattianus was shown to be a heterodimer protein of 60 and 12 kDa subunits. Optimum pH and temperature for the activity were 5.5°C and 55°C, respectively. The abundance of β-amylase in G. klattianus suggests its possible application for biotechnological purposes.