Search

The glycoprotein glucoamylase (EC 3.2.1.3) G1 from Aspergillus niger was digested with trypsin after 2-pyridylethylation and the resulting peptide fragments were separated by gel filtration followed by reverse phase HPLC. A different set of peptide fragments was obtained from the citraconylated, 2-pyridylethylated enzyme. These were separated by gel filtrations, affinity chromatography on Con A-Sepharose, and reverse phase HPLC. The amino acid sequence of the isolated peptide fragments was determined by automated Edman degradation and digestion with carboxypeptidases Y and B. The majority of the carbohydrate of glucoamylase G1 was located in a fragment which carried approximately 35 units of neutral sugar linked O-glycosidically to threonine and serine residues, while a minor fraction was located in a different tryptic fragment which contained a single N-glycosylated asparagine residue.

Enzymatically active, N-bromosuccinimide oxidized glucoamylase G2 (EC 3.2 1.3) was prepared in the presence of the inhibitor nearbose and inactive, oxidized G2 with capacity to bind substrate was prepared in the presence of maltose. Four out of 15 tryptophanyl residues were oxidized in the first derivative and five in the latter. In order to identify this fifth and essential residue, tryptic fragments of the two G2 derivatives were isolated. The peptide fragment from the inactive G2 derivative containing the additional oxindolealanine residue was located in HPLC chromatograms by UV-absorption measurements. Normal and second derivative UV-spectra, amino acid composition and NH2-terminal sequence of the isolated fragment led to identification of Trp(120) as a residue essential for activity. A short homologous amino acid sequence precedes both this Trp(120) and the Trp(83) which is involved in substrate binding in Taka-amylase A (J. Biochem. 95. 697–702 (1984)).

An endo-β-glucanase was purified from a commercial enzyme preparation of fungal origin by ammonium sulphate fractionation, ion exchange chromatography, and gel filtration followed by preparative isoelectric focusing. The enzyme was homogeneous in sedimentation equilibrium analysis from which the molecular weight was determined to be 23.500 in agreement with the value 23.653 calculated on the basis of the amino acid composition. The enzyme did not contain carbohydrate and a molecular weight of 24.000 estimated by polyacrylamide gel electrophoresis in dodecyl sulphate after 2-mercaptoethanol treatment, indicated that it consisted of a single polypeptide chain. It had an isoelectric point of 4.47. The enzyme rapidly decreased the specific viscosity of barley β-glucan with a small concomitant increase in reducing sugar. Its activity toward carboxymethyl-cellulose, acid-swollen cellulose, and a mixture of cellodextrins was low. Laminarin, carboxymethyl-pachyman, cellobiose, and p-nitrophenyl-β-D-glucoside were not hydrolyzed. The Km for hydrolysis of barley β-glucan and carboxymethyl-cellulose was 1.8 and 11 mg/ml, respectively, the corresponding molecular activities being 7750 and 750 equivalents of glucosidic bonds hydrolyzed per min per mole of enzyme. The products of exhaustive hydrolysis of barley β-glucan were 4.3% glucose, 4.2% disaccharide, 72.7% trisaccharide, and 18.8% tetrasaccharide, higher oligomers and polymers were absent. The enzymic activity was completely destroyed by treatment with N-bromosuccinimide but was insensitive to EDTA, sulfhydryl modifying reagents, and glucono-1,5-lactone.

The tryptophanyl residues of the Aspergillus niger glucoamylase G1 and G2 (EC 3.2.1.3) were oxidized by N-bromosuccinimide in both the presence and the absence of substrates and inhibitors of the enzyme. In the absence of protective ligands, 8 of 19 and 6 of 15 tryptophanyl residues in G1 and G2, respectively, were susceptible to modification with concomitant inactivation of the enzyme. The binding of acarbose, a potent inhibitor, prior to oxidation protected 2 tryptophanyl residues (Wa and Wb) in both G1 and G2 from the modification. After dissociation of acarbose-enzyme complexes with the aid of Tris, these derivatives retained about 80% of the initial enzymic activity. Further oxidation subsequently modified these 2 tryptophanyl residues resulting in total loss of activity. The substrates, maltose, maltotriose or soluble starch and the inhibitors, gluconolactone, maltitol or deoxynojirimycin protected one tryptophanyl residue (Wb) in both G1 and G2 from oxidation but did not prevent the inactivation. Characterization of the oxidized enzyme derivatives by difference UV absorption and by fluorescence spectroscopy indicated that 2 residues, Wa and Wb, are essential in the mechanism of glucoamylase action. One residue, Wb, is apparently involved in the binding of substrate, while a second, Wa, is an integral part of a catalytically capable active center.

In barley α-amylase 2, two and three tryptophans are protected against reaction with dimethyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide by β-cyclodextrin and the pseudooligosaccharide inhibitor aplanin, respectively. Fragments were generated from the enzyme derivatives by digestion with Armillaria mellea protease and trypsin, and isolated by RP-HPLC. The substituted tryptophans were identified by amino acid and sequence analyses of modified peptides. Aplanin and β-cyclodextrin both reduced the accessibility of Trp276 and −277. In addition, aplanin hindered modification of Trp206, and only this derivative retained activity. Trp206 probably belongs to the active site region, whereas Trp276 and −277 are located in a different binding site. This suggestion is supported by a comparison with the 3-D structure of Taka-amylase A guided by sequence homology between it and barley α-amylase.

Three inhibitors of animal cell-free protein synthesis were isolated from barley seeds by ammonium sulfate fractionation, CM-cellulose chromatography and gel filtration on Bio-Gel P-60. The three inhibitors were all single chain, basic proteins with molecular weights about 31,000 and with very similar amino acid compositions. In the rabbit reticulocyte lysate system, they inhibited in vitro translation to 50% at concentrations between 15 and 25 ng·ml−1, while in the wheat germ system, their effect was very weak. The three translation inhibitors showed no detectable ribonuclease activity neither on ribosomal RNA from rabbit liver nor on homopolynucleotides.

Barley malt α-amylases 1 and 2, the low and high pI isozymes, respectively, were isolated and characterized by partial amino acid sequencing. The NH2-terminal sequence of α-amylase 1 and the sequence of a peptide fragment of this protein obtained by cleavage with hydroxylamine matched the primary structure previously deduced from the nucleotide sequence of a barley α-amylase cDNA clone. In distinction to α-amylase 1, the homologous protein α-amylase 2 had a blocked NH2-terminus, presumably a pyroglutamic acid residue. Sequenced peptide fragments from α-amylase 2 matched the amino acid sequence previously derived from a cDNA clone corresponding to a barley α-amylase different from α-amylase 1.

Trypsin converts a purified β-amylase component from barley, β-amylase 1, to another catalytically active form of the enzyme characterized by a higher isoelectric point. The two main peptide fragments released during this limited proteolysis were purified by reverse-phase HPLC. Their complete sequences were determined to comprise 22 and 23 amino acid residues starting at the same NH2-terminal residue. Barley β-amylases are NH2-terminally blocked and the released peptides were confirmed to stem from the COOH-terminal end of the protein by comparison with the sequence deduced from a cDNA clone of barley β-amylase (Kreis. Williamson, Buxton, Pywell, Hejgaard andSvendsen, in preparation).

A small basic 10 kD abundant protein in barley seeds can convey up to 7% of the phosphatidylcholine in liposomes to potato mitochondria whereas cholesteryloleate is not transported. The demonstration of this activity combined with a somewhat more than 50% homology of its primary structure to that of other plant phospholipid transfer proteins are the bases for our naming it a barley lipid transfer protein (LTP).

Barley malt α-amylase 2 has been purified on a large scale and the resulting enzyme preparation used in various chemical modification experiments. Results from tryptophan and tyrosine specific modifying reagents, as well as difference and fluorescence spectroscopy techniques, which studied the interaction of a variety of ligands, indicate that tryptophanyl and tyrosyl residues are involved in enzyme activity. Of the 16 tryptophanyl residues present in the enzyme, 4 could be modified using dimethyl (2-hydroxy-5-nitrobenzyl)sulphonium bromide. This caused inactivation of the enzyme. Addition of the inhibitor aplanin, a pseudo-maltooligosaccharide, decreased the loss of enzyme activity and protected 2 tryptophanyl residues from modification. Aplanin also protected the enzyme from inactivation by tetranitromethane, a tyrosine specific reagent.

A commercial fungal β-glucanase preparation was immobilized by various methods including adsorption to DEAE-cellulose, cross-linking with glutaraldehyde, adsorption to a phenol-formaldehyde resin (Duolite) and to perlite followed by fixation with glutaraldehyde, covalent binding to glutaraldehyde-treated, partially hydrolyzed or partially aminolyzed nylon, and covalent binding in the Ugi-reaction to polyisonitrile-nylon. The recovery of enzymatic activity varied from 0.02 to 4.5% and the immobilized enzyme preparations showed specific activities from 1 to 25% of that of the starting material.

A basic protein was isolated from aqueous extracts of barley seeds. It contains 91 amino acid residues including 8 half-cystines, but no phenylalanine or tryptophan; the calculated molecular weight is 9.694. Amino acid composition and N-terminal sequence show that the protein corresponds to the probable amylase/protease inhibitor (PAPI) encoded by a formerly described cDNA. The protein is 50% homologous with an α-amylase inhibitor I-2 from seeds of Indian finger millet and it shows a distant homology with two Bowman Birk type protease inhibitors. However, the barley protein does not inhibit any of the enzymes tested. They include α-amylases, a β-amylase, β-glucanases, serine proteases, a sulfhydryl protease, aspartate proteases, and serine carboxypeptidases. Nevertheless, difference UV-spectroscopy indicated that the protein interacts with porcine pancreatic α-amylase. NMR-spectroscopy revealed that it possesses tertiary structure.

An inhibitor of animal cell-free protein synthesis from barley seeds (inhibitor II) has been chemically modified by means of group-specific reagents. Specific nitration of a single tyrosyl residue caused a substantial loss of the inhibitory activity. After cyanogen bromide cleavage and subfragmentation of the nitrated fragment by endoproteinase Lys-C, the essential residue was identified as Tyr(86) in the primary structure. Chemical modifications of carboxyl groups, lysyl residues, and arginyl residues also led to reduction of the activity, whereas modifications of tryptophanyl or methionyl residues had no significant effects.

Treatment of glucoamylase G2 with large excesses of different group specific reagents resulted in modification of 25% of the histidyl, 15% of the tyrosyl, 20-40% of the arginyl, 30-50% of the lysyl and none of the methionyl residues. The modified groups were not critical since the various derivatives possessed from 50% to 100% residual enzymatic activity and retained the thermostability. Carboxamidomethylation occurred specifically at His254 with essentially no change of the kinetic parameters for hydrolysis of maltose and starch. Removal of the two N-linked sugar units by endoglycosidase H was similarly without effect on activity, thermostability and chemical reactivity of the histidyl residues. H(+)-titration revealed that glucoamylase G2 carries a lower net charge throughout the pH-range 3-11 than predicted from its amino acid composition.

The primary structure of glucoamylase G1 (EC 3.2.1.3) from Aspergillus niger has been determined. Fragments of G1 were obtained by cleavage with either cyanogen bromide, hydroxylamine, or S. aureus V8 protease. The resulting peptides were separated using ion exchange chromatography on DEAE-Sephacel, gel filtration, and affinity chromatography on Con A-Sepharose. Secondary fragments were generated by cleavage with either o-iodosobenzoic acid or BNPS-skatole as well as by digestion with S. aureus V8 protease, trypsin, and endoproteinase Lys-C. These peptides were purified by the procedures mentioned above and by reverse phase HPLC. The present fragments were amino acid sequenced and this permitted, in combination with the tryptic peptides (Carlsberg Res. Commun. 48, 517–527 (1983)), identification of 574 of the 614 amino acid residues in G1. Sequencing of glucoamylase G1 cDNA, constructed from A. niger total mRNA, enabled deduction of the sequence of the remaining 40 amino acid residues localized to 6 short stretches. From the alignment of the fragments the complete primary structure of the enzyme was established. The amino acid sequence corresponds to a molecular weight of the polypeptide moiety of 65,424. Including both hexosamine and neutral carbohydrate contents the molecular weight of the present sample of G1 was calculated to be about 82,000. The majority of the carbohydrate of G1 is found in a highly glycosylated region of 70 amino acid residues which comprises about 35 O-glycosyl serine and threonine residues. This region ends approximately 100 residues from the C-terminus of the enzyme. Two N-glycosylated positions were found in the central part of the polypeptide chain. The molecule contains 9 half-cystine residues. No homology is apparent between the sequence of glycoamylase and various α-amylases.

Glucoamylases G1 and G2 from Aspergillus niger lost the catalytic activity by prolonged treatment with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide(EAC). A total of 23 and 16 carboxyl groups were substituted out of 71 and 54 present in the G1 and G2 enzyme, respectively. The kinetics and the pH-dependence of the inactivation were compatible with the existence of one essential carboxyl group with a pKa 5.5. The inhibitor acarbose (a pseudotetrasaccharide) and the substrate maltose prevented EAC-modification of 4 and 2 carboxyl groups, respectively, with considerable retention of enzyme activity. Following removal of these ligands, differential labelling of critical carboxyl residue(s) was achieved by means of [3H]EAC. Glucoamylase G2 with 12 EAC groups incorporated, prepared in the presence of acarbose, displayed a two-fold increase of Km and a slight reduction of Vmax for hydrolysis of starch relative to the unmodified enzyme. Both Km and Vmax for hydrolysis of maltose were almost unaffected. In addition, maltose and three inhibitors perturbed the UV absorbance spectrum of this derivative. An altered shape of the acarbose-induced difference spectrum implied substitution of a carboxyl group near a binding tryptophanyl residue at a distance from the catalytic site. Efficient protection by acarbose against only the second part of the biphasic course of inactivation similarly reflected that residues playing different roles in the mechanism of action reacted with EAC. In separate experiments, evidence was obtained for a preferred reaction of ethylenimine with enzymatically important carboxyl groups in glucoamylase.

Glucoamylase was retained in calcium alginate gels, when the molecular weight of the enzyme was increased, e.g., by attachment to polymeric supports or by intermolecular cross-linking. Alternatively, the enzyme could be retained by covalent attachment to the alginate matrix. In the majority of the glucoamylase derivatives more than 50% of the initial activity determined with maltose or partially hydrolyzed starch as substrate was recovered, while upon entrapment in gel particles, less than 40% and 6% of the initial activity towards maltose and partially hydrolyzed starch, respectively, was recovered. Thus, diffusional and steric limitations on substrate access seem to influence the apparent enzymic activity, in spite of the high permeability of calcium alginate gels. Dextran-coupled glucoamylase entrapped in calcium alginate gels has been used as a packed-bed reactor for continuous hydrolysis of oligodextrins during a period of two months with no change in activity.

A 39 kD protein has been extracted from barley flour with 0.1 M monothioglycerol at pH 5.0 and purified by (NH4)2SO4-precipitation, anion exchange and molecular sieve chromatography. It is an N-terminally blocked, non-glycosylated, single-chain protein present in at least two molecular forms of isoelectric points 5.18 and 5.22. The amino acid composition and partial sequence analysis reveal a relationship to barley endosperm Z protein which belongs to the serpin superfamily. The 39 kD protein inhibits alpha-chymotrypsin while little or no effect could be demonstrated on trypsin, subtilisin, proteinase K, S. aureus V8 protease, thermolysin or two malt thiol endoproteinases. The 39 kD protein is immunochemically related to the major protein component in beer.