Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii.

The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

Optimization of the production of Chondrus crispus hexose oxidase in Pichia pastoris.

Hexose oxidase (D-hexose:O(2)-oxidoreductase, EC 1.1.3.5, HOX) normally found in the red alga Chondrus crispus was produced heterologously in different host systems. Full-length HOX polypeptide was produced in Escherichia coli, but no HOX activity could be detected. In contrast, active HOX could be produced in the methylotrophic yeast Pichia pastoris. Several growth physiological and genetic approaches for optimization of hexose oxidase production in P. pastoris were investigated. Our results indicate that specific growth conditions are essential in order to produce active HOX with the correct conformation. Furthermore, HOX seems to be activated by proteolytic cleavage of the full-length polypeptide chain into two fragments, which remain physically associated. Attempts to direct HOX to the extracellular compartment using the widely used secretion signals from Saccharomyces cerevisiae invertase or alpha-mating factor failed. However, we show in this study that HOX is transported out of P. pastoris via a hitherto unknown mechanism and that it is possible to enhance this secretion by mutagenesis from below the detection limit to at least 250 mg extracellular enzyme per liter.

Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization.

Three beta-galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 beta-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four beta-galactosidases were highly specific for hydrolysis of beta-D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.

High-efficiency synthesis of oligosaccharides with a truncated beta-galactosidase from Bifidobacterium bifidum.

An exceptionally large beta-galactosidase, BIF3, with a subunit molecular mass of 188 kDa (1,752 amino acid residues) was recently isolated from Bifidobacterium bifidum DSM20215 [Møller et al. (2001) Appl Environ Microbiol 67:2276-2283]. The BIF3 polypeptide comprises a signal peptide followed by an N-terminal beta-galactosidase region and a C-terminal galactose-binding motif. We have investigated the functional importance of the C-terminal part of the BIF3 sequence by deletion mutagenesis and expression of truncated enzyme variants in Escherichia coli. Deletion of approximately 580 amino acid residues from the C-terminal end converted the enzyme from a normal, hydrolytic beta-galactosidase into a highly efficient, transgalactosylating enzyme. Quantitative analysis showed that the truncated beta-galactosidase utilised approximately 90% of the reacted lactose for the production of galacto-oligosaccharides, while hydrolysis constituted a 10% side reaction. This 9:1 ratio of transgalactosylation to hydrolysis was maintained at lactose concentrations ranging from 10% to 40%, implying that the truncated beta-galactosidase behaved as a "true" transgalactosylase even at low lactose concentrations.

Hexose oxidase from the red alga Chondrus crispus. Purification, molecular cloning, and expression in Pichia pastoris.

Hexose oxidase from Chondrus crispus catalyzes the oxidation of a variety of mono- and disaccharides including D-glucose, D-galactose, maltose, and lactose. The enzyme has previously been partially purified and was reported to be a highly glycosylated, copper-containing protein with a relative molecular mass of approximately 130,000 (Sullivan, J. D., and Ikawa, M. (1973) Biochim. Biophys. Acta 309, 11-22). We report here the purification to homogeneity of hexose oxidase from C. crispus. The purified enzyme was cleaved with cyanogen bromide and endoproteinase Lys-C and the peptide fragments were subjected to amino acid sequence analysis. Oligonucleotides were designed on the basis of the peptide sequences and a cDNA clone encoding C. crispus hexose oxidase was obtained using polymerase chain reaction on reverse transcribed cDNA. The nucleotide sequence of the hexose oxidase cDNA contained an open reading frame of 546 amino acid residues with a predicted relative molecular mass of 61,898. No significant sequence similarity was found between hexose oxidase and other protein sequences available in data bases. Expression of the hexose oxidase cDNA in Pichia pastoris as an active enzyme confirmed the identity of the DNA sequence. Native hexose oxidase from C. crispus was characterized and compared with purified, recombinant enzyme.

A new glucose oxidase from Aspergillus niger: characterization and regulation studies of enzyme and gene.

A new glucose oxidase from Aspergillus niger was isolated and characterized. The enzyme showed different kinetic and stability characteristics when compared to a commercially available batch of A. niger glucose oxidase. The gene encoding the new glucose oxidase was isolated and DNA sequence analysis of the coding region showed 80% identity to the sequence of a glucose oxidase gene previously published. However, the similarity of the non-coding sequences up- and downstream of the open reading frame was much less, showing only 66% and 50% identity respectively. Despite the low degree of similarity between the promotor region of the new gene and the previously published one, the new glucose oxidase was likewise induced by calcium carbonate. In addition, we showed that this induction occurred on the transcriptional level. Observations concerning the effect of gluconolactone and the levels of glucose-6 phosphate isomerase upon calcium carbonate induction suggested that the enhancement of glucose oxidase biosynthesis by calcium carbonate was accompanied by a metabolic shift from glycolysis to the pentose phosphate pathway.

The NH2-terminal amino acid sequence of the immunochemically partial identical major allergens of Alder (Alnus glutinosa) Aln g I, birch (Betula verrucosa) Bet v I, hornbeam (Carpinus betulus) Car b I and oak (Quercus alba) Que a I pollens.

The tree pollen major allergens (Aln g I), Bet v I, Car b I and Que a I, were purified to near homogeneity. Rabbit antibodies raised towards the major allergen Bet v I react with the major allergens of alder, hazel, hornbeam and oak, although with distinct variation in affinity. Immunochemically the major allergens seem to resemble one another more within than between the botanically established families of the fagale order. The physico-chemical parameters (molecular weight, pI values, amino acid composition and NH2-terminal amino acid sequence) of the major allergens exhibit a higher degree of consistency than do the immunochemical parameters, indicating that nuances in the allergens' secondary and tertiary structures are likely to govern the immunochemical differences.

The cell adhesion molecule Cell-CAM 105 is an ecto-ATPase and a member of the immunoglobulin superfamily.

Cell-CAM 105 (C-CAM), a cell adhesion molecule in rat hepatocytes, was digested with trypsin, and peptides were isolated and sequenced by Edman degradation. The sequences of 4 peptides agreed with different regions of rat liver ecto-ATPase. Detailed biochemical analyses confirmed the identity between C-CAM and the ecto-ATPase. C-CAM/ecto-ATPase is a transmembrane protein having 4 immunoglobulin-like domains in the extracellular portion, demonstrating membership of the immunoglobulin superfamily. The ATPase activity suggests that ATP might influence cell adhesion, which would explain the inhibitory effect of exogenously added ATP on adhesion of several cell types.

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA.

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.

The binding of mouse hybridoma and human IgE antibodies to the major fecal allergen, Der p I, of Dermatophagoides pteronyssinus. Relative binding site location and species specificity studied by solid-phase inhibition assays with radiolabeled antigen.

The relative binding site location and species specificity of 19 mouse hybridoma antibodies, produced in four laboratories, to Dermatophagoides pteronyssinus major fecal allergen, Der p I, was studied by using immobilized mAb and inhibitions of radiolabeled Ag binding. Four mAb groups were defined, within which 4, 6, 8, and 5 mAb, respectively, cross-inhibited each other. Five mAb were members of both group 2 and 3, demonstrating a considerable overlap of epitopes between the corresponding antibody-binding regions. The degree of mAb species specificity, as assessed by inhibition with cold Der p I and Ag Der m I and Der f I from the related species, Dermatophagoides microceras and Dermatophagoides farinae, was highly variable even for mAb binding to the same region on the Ag. Five cases of cross-reactivity between Der p I and Der m I and one case of cross-reactivity between Der p I and Der f I were found. The N-terminal 30 amino acids of the three species showed 7 substitutions between Der p I and Der m I/Der f I and 2 between Der f I and Der p I/Der m I. Single mAb inhibited up to 65% of labeled Der p I binding to immobilized human IgE from allergic patients' sera and up to 24% of labeled Der p I binding to immobilized rabbit antibodies. The spectrum of species specificities in human IgE sera, as assessed by inhibitions with cold Ag, was similar to that of the mAb. No evidence for the presence of strictly sequential epitopes, reactive with either mAb or human IgE was found, as judged from the weak inhibitory activity of acid-denatured Der p I.

Localization and functional significance of a polymorphic determinant in the third component of human complement.

A polymorphic epitope in the third component of human complement was studied. This allotypic system is distinct from the electrophoretically determined C3 S/F polymorphism and is defined by the recognition of one allotype by a monoclonal antibody. Allotypic protein variants, C3F+ (reactive with this antibody) and C3S- (non-reactive with the antibody), were purified. Deglycosylation studies and N-terminal sequencing of CNBr fragments, reactive with the antibody, revealed that the polymorphic epitope was present in a beta chain fragment of mol. wt 20,000. In the intact C3 molecule, this fragment is situated with N-terminus at residue No. 202, using the numbering of the cDNA derived amino acid sequence of human prepro C3. Addition of Fab fragments from the alloselective antibody preferentially inhibited the activity of C3F+ in a haemolytic assay which is selective for the C3 activity in the alternative complement pathway.

Pig intestinal microvillar maltase-glucoamylase. Structure and membrane insertion.

The NH2-terminal sequence (25 residues) of amphiphilic single polypeptide chain maltase-glucoamylase (EC 3.2.1.20) was determined by gas-phase sequencing. The result indicates that the NH2-terminal segment anchors the enzyme to the microvillar membrane. The single-chain form and the proteolytically processed two-chain form have two distinct active sites differing in heat stability. However, both sites are sensitive to chonduritol B-epoxide and have similar substrate specificity. The amphiphilic single-chain maltase-glucoamylase and the amphiphilic proteolytically processed form were inserted into liposomes and studied by electron microscopy. The results showed that the enzyme is predominantly present as a homodimeric complex in the membrane.

Cell-free synthesis of the D2-cell adhesion molecule: evidence for three primary translation products.

The D2-cell adhesion molecule (D2-CAM) is a membrane glycoprotein that is involved in cell-cell adhesion in the nervous system. To study the biosynthesis of D2-CAM we have translated free and membrane-bound polysomes from rat brain in vitro in the rabbit reticulocyte lysate system. D2-CAM was exclusively synthesized on membrane-bound polysomes. The primary translation products of D2-CAM were three polypeptides of apparent molecular weights 187,000, 134,000, and 112,000. No interconversion between these polypeptides was detected. In contrast to previous suggestions, we conclude that all three D2-CAM polypeptides are primary translation products. When translating polysomes from embryonic and postnatal rat brain, we found that the relative amounts of the three polypeptides synthesized varied with age. Their molecular weights, however, were not age-dependent.

Purification of gamma-enolase messenger ribonucleic acid from rat brain by an immunoadsorption method.

Messenger ribonucleic acid (mRNA) coding for the brain-specific protein gamma-enolase was isolated by an immunopurification procedure. Rat brain polysomes including nascent polypeptide chains were reacted with specific gamma-enolase antibody. The polysome-antibody complexes were subsequently adsorbed to protein A-Sepharose. After extensive washing, RNA was eluted and applied to an oligo(dT)-cellulose column. Purified mRNA was translated in vitro in a mRNA-dependent rabbit reticulocyte lysate system. The synthesized product was identical to gamma-enolase synthesized by free polysomes from rat brain. Immunoisolated gamma-enolase mRNA was enriched 380-fold compared to total mRNA extracted from free polysomes. This result indicates that low-abundance mRNAs may conveniently be isolated from brain tissue by immunoadsorption of polysomes.