Martin Ottesen

Characterization of two forms of glucoamylase from aspergillus niger

Aspergillus niger glucoamylases GI and GII (E.C. 3.2.1.3) were isolated from a commercial enzyme preparation by ammonium sulfate precipitation followed by DEAE-cellulose ion exchange chromatography. Both enzymes consist of a single glycosylated polypeptide chain. The molecular weights of GI and GII were determined by sedimentation equilibrium ultracentrifugation to 52,000 and 46,000, respectively, and by molecular sieving to 65,000 and 55,000. The amino acid compositions of GI and GII were very similar. Furthermore, the N-terminal amino acid sequence of the intact GI and GII as well as of their cyanogen fragments were identical, suggesting great homology in the primary structure of the two forms. In addition the digests of GI and GII produced respectively by Armillaria mellea protease, Staphylococcus aureus V8 protease, and submaxillary protease were analyzed by high pressure gel permeation chromatography. The elution profiles were also consistent with GI and GII having similar polypeptide chains. However, digestion with carboxypeptidase Y showed different C-terminal residues of the two forms.

Isolation of carboxypeptidase Y by affinity chromatography

Carboxypeptidase Y from bakers’ yeast has been purified in high yields by affinity chromatography. The affinity gel was prepared by coupling the specific inhibitor p-aminobenzylsuccinic acid via an azo linkage to Sepharoseglycyl-tyrosine. This affinity gel was able to bind carboxypeptidase Y specifically and quantitatively from a crude yeast autolysate.The isolated enzyme appeared homogeneous by gel electrophoresis and ultracentrifugation, while isoelectric focusing revealed the presence of two components with isoelectric points of pH 3.56 and 3.66, respectively. Small differences in amino acid composition and enzymatic properties between the enzyme from danish yeast and the corresponding enzyme isolated from Fleichmann yeast suggested the existence of more than one form of this enzyme.

Maturation of beer with α-acetolactate decarboxylase

Abstractα-Acetolactate decarboxylase isolated from Enterobacter aerogenes strain 1033 has been applied for maturation of beer. Addition of the enzyme to freshly fermented beer effected removal of α-acetolactate and α-aceto-α-hydroxybutyrate in 24 hours at 10 °C to a level below the taste treshold of the corresponding volatile diketones, diacetyl and 2,3-pentanedione, without affecting other important properties of the beer. By comparison of organoleptic properties, the beer matured in the presence of α-acetolactate decarboxylase was judged to be of an equally satisfactory quality when compared with conventionally prepared beer. The evidence suggest that apart from removal of diacetyl, 2,3-pentanedione and the precursors of these compounds from fermented beer no other important events related to flavour development occur during maturation of the type of beer studied. Consequently, it is concluded, that application of enzymes may allow flavour maturation of beer to be carried out in less than 24 hours.

Isolation of carboxypeptidase II from malted barley by affinity chromatography

A serine carboxypeptidase isolated from malted barley by affinity chromatography was termed malt carboxypeptidase II to distinguish it from another malt carboxypeptidase previously described (Carlsberg Res. Commun. 48, 217–230 (1983)), henceforth called malt carboxypeptidase I. Our nomenclature is in agreement with the nomenclature formerly suggested byMikola. Malt carboxypeptidase II has a molecular weight of 110,000–120,000. It appears to be a dimer where each monomer is composed of two peptide chains linked by disulfide bridges: one monomer contains an A-chain (34,000) and a B-chain (27,000), the other an A-chain and a C-chain (24,000). The enzyme contains 28 residues of glucosamine and 15% neutral sugar. The N-terminal sequence of the A-chain was NH2-Ala-Gly-Gly-His-Ala-Ala-Asp-Arg-Ile-Val- while the B- and C-chains appeared to be N-terminally blocked. The amino acid compositions of the B- and C-chains were identical suggesting that their different molecular weights are due to different contents of carbohydrate.Malt carboxypeptidase II is inhibited by diisopropyl phosphorofluoridate and by Hg++. It exhibits a strong preference for substrates containing Lys and Arg as C-terminal amino acid residues but it also hydrolyses substrates with hydrophobic amino acid residues in this position.

Primary structure of the aspartic proteinase A from Saccharomyces cerevisiae

Proteinase A was purified by an improved large scale procedure and split into fragments by means of trypsin, cyanogen bromide, hydroxylamine, and o-iodosobenzoic acid. On the basis of high degrees of homology with cathepsin D and pepsin its amino acid sequence was determined. Proteinase. A contains 329 amino acid residues, and in addition 8.5% neutral sugar and 1% glucosamine, attached to asparagines in positions 67 and 267. Proteinase A contains two disulfide bonds, as opposed to three in mammalian aspartic proteinases. Comparison with the tertiary structure of pepsin indicates, that the two catalytically essential aspartic acid residues, and the residues corresponding to their surroundings, are conserved. The sequence shows 46% identity with porcine cathepsin D and 40% with porcine pepsin. An aspartic proteinase from Saccharomyces carlsbergensis had the same N-terminal 40 amino acid sequence as proteinase A. Immunological cross-reactivity between proteinase A and calf chymosin was demonstrated by immune blotting assay.

Use of carboxypeptidase Y for carboxy-terminal sequence determination in proteins

Treatment of several proteins-native pancreatic ribonuclease, human carbonic anhydrase B and human carbonic anhydrase C- with carboxypeptidase Y in the presence of 0.5% sodium dodecyl sulphate resulted in the sequential release of amino acid residues from the carboxyl terminus of the polypeptides. This provides a convenient method to study the carboxy-terminal sequence of proteins whose carboxyl termini are inaccessible in the native state to cleavage by carboxypeptidase.

Fractionation and characterization of beer proteins

A combination of large scale gel filtration with preparative isoelectric focusing and ion exchange chromatography has been used for fractionation of beer proteins. From gel filtration on Sephadex G-150, a high molecular weight fraction, eluting close to the void volume of the column, a fraction appearing corresponding to a molecular weight of 44,000 and a fraction containing rather low-molecular weight (about 10,000) components were obtained and used for preparative isoelectric focusing in the pH-range 3.5–10. The high-molecular weight fraction was rich in carbohydrate and cross-reacted with yeast antibodies. After preparative isoelectric focusing the amino acid composition of the isolated subfractions resembled that of yeast cell wall components. Preparative isoelectric focusing of the two fractions with molecular weight about 44,000 and 10,000 revealed the presence of two classes of components based on their amino acid composition. One class, which seemed to be present in low concentration in nearly all isolated subfraction, had an amino acid composition resembling that of barley glutelins. In the fraction with molecular weight about 44,000 another class of components—having an amino acid composition resembling barley albumins and globulins—were observed in the region with isoelectric points of about pH 4–5. They cross-reacted immunologically with antibodies against soluble barley proteins. This class of components could also be separated from the glutelin-like constituents by ion exchange chromatography. However, they could not be completely separated from carbohydrate by the fractionation procedures employed. Carbamylation expents demonstrated that approx, half of the ε-amino groups of lysine residues of these proteins were blocked. Furthermore, partial amino acid sequence determination by the Edman procedure revealed a strong heterogeneity with respect to N-terminal amino acid residues. These observations are consistent with a suggestion that a large part of the beer proteins might be polypeptide chains crosslinked by carbohydrates.

Enzymes immobilized as crystals. Hydrogen isotope exchange of crystalline lysozyme

Immobilization of enzymes by crystallization and subsequent cross-linking provide structures characterized by a regular three-dimensional molecular arrangement and a high packing density. Compared to randomly immobilized enzymes, such structures permit more detailed analysis of the variations in kinetic properties arising from the three-dimensional network or perturbations of the molecular conformations. To obtain information on the effect of crystallization on the dynamic properties of the folded peptide chain in an enzyme molecule, hydrogen exchange rates were measured for both dissolved and crystalline lysozyme over a wide range of pH. Using this method, which reflects molecular oscillations between closely related conformations, no differences were detected between lysozyme in crystalline and dissolved state.

Determination of C-terminal sequences by digestion with serine carboxypeptidases: The influence of enzyme specificity

It is demonstrated that C-terminal sequence determinations of a number of proteins and peptides by digestion with serine carboxypeptidases often require use of different reaction conditions and/or use of carboxypeptidases with different specificities. The commonly utilized carboxypeptidase Y from yeast with preference for hydrophobic amino acid residues is conveniently supplemented by carboxypeptidase II from amlt which includes Lys and Arg in its specificity range. This renders it particularly suitable for C-terminal digestion of tryptic peptides. In some cases a phenylmercuric chloride modified derivative of carboxypeptidase Y which has a decreased preference for hydrophobic amino acid residues can be used with advantage.

Occurrence of ?-acetolactate decarboxylases among lactic acid bacteria and their utilization for maturation of beer

SummaryAcetolactate decarboxylase activity has been detected among three genera, nine species and 263 strains of lactic acid bacteria tested in the course of a screening for acetolactate decarboxylases amenable for use in brewing as maturation aid. Streptococcus diacetylactis strain FD-64-D was found to generate a decarboxylase exhibiting a satisfactory activity and an excellent stability at the pH prevailing in beer and wort. This decarboxylase could not be solubilized but enzymatically active, freeze-dried cells were effective for satisfactory flavour maturation of beer although difficulties were encountered during attempts to remove the applied cell material by filtration of the beer. Lactobacillus casei DSM 2547 was likewise found to produce a decarboxylase exhibiting a satisfactory activity and stability at the low pH of beer and which, in addition, was readily solubilized. A method has been developed for pilot scale production of preparations of this decarboxylase suitable for use in brewing.