Characterization of a purified beta-fructofuranosidase from Bifidobacterium infantis ATCC 15697

AIMS: To characterize the beta-fructofuranosidase of Bifidobacterium infantis ATCC 15697 and to compare it with other bacterial beta-fructofuranosidases. METHODS AND RESULTS: The beta-fructofuranosidase of B. infantis ATCC 15697 was purified 46.8 times over the crude extract by anion exchange chromatography, ultrafiltration and gel filtration. The sequence of 15 amino acid residues of the NH2 terminal was determined. This enzyme was a monomeric protein (Mr 70 kDa) with beta-fructofuranosidase and invertase activities. The isoelectric point was pH 4.3, the optimum pH 6.0 and pKas (4.5 and 7.2) of two active groups were obtained. The activities were inhibited by Hg2+ and p-chloromercuribenzoic acid (pCMB). The optimal temperature was 37 degrees C and activities were unstable at 55 degrees C. beta-fructofuranosidase activity was more efficient than that of invertase with Vm/Km ratios of 0.65 and 0.025 x 10-3 l min(-1) mg(-1), respectively. The enzyme catalyses the hydrolysis of fructo-oligosaccharides, sucrose and inulin at relative velocities of 100, 10 and 6, respectively. CONCLUSIONS: The enzyme of B. infantis ATCC 15697 is an exo-inulinase which has beta-fructofuranosidase and invertase activities. This protein was different from the beta-fructofuranosidase of another strain of B. infantis (B. infantis JCM no. 7007). SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of bacterial beta-fructofuranosidases, especially from bifidobacteria, has been gained.     

On the DNA binding protein II from Bacillus stearothermophilus. II. The amino acid sequence and its relation to those of homologous proteins from other prokaryotes

The complete amino acid sequence of DNA binding protein II from Bacillus stearothermophilus has been determined. The protein contains 90 amino acid residues and has a calculated Mr of 9716. The sequence is compared to homologous molecules from Escherichia coli, Thermoplasma acidophilum, and Pseudomonas aeruginosa (where only a partial sequence is available). The B. stearothermophilus molecule has 58% and 59% residues identical with the two forms of the E. coli protein and 32% with the T. acidophilum protein. There are totally conserved residues at positions 46-48 and 61-65 with an intervening cluster of basic amino acids in all four proteins.     

Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis.

The structural gene for a subtilisin J from Bacillus stearothermophilus NCIMB10278 was cloned in Bacillus subtilis using pZ124 as a vector, and its nucleotide sequence was determined. The nucleotide sequence revealed only one large open reading frame, composed of 1,143 base pairs and 381 amino acid residues. A Shine-Dalgarno sequence was found 8 bp upstream from the translation start site (GTG). The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprised of 275 residues. The productivity of subtilisin in the culture broth of the Bacillus subtilis was about 46-fold higher than that of the Bacillus stearothermophilus. The amino acid sequence of the extracellular alkaline protease subtilisin J is highly homologous to that of subtilisin E and it shows 69% identity with subtilisin Carlsberg, 89% with subtilisin BPN' and 70% with subtilisin DY. Some properties of the subtilisin J that had been purified from the Bacillus subtilis were examined. The subtilisin J has alkaline pH characteristics and a molecular weight of 27,500. It retains about 50% of its activity even after treatment at 60 degrees C for 30 min in the presence of 2 mM calcium chloride.     

Molecular cloning and characterization of the fructooligosaccharide-producing beta-fructofuranosidase gene from Aspergillus niger ATCC 20611

The fopA gene encoding a fructooligosaccharide-producing beta-fructofuranosidase was isolated from Aspergillus niger ATCC 20611. The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other beta-fructofuranosidases from A. niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others. We could successfully express the fopA gene in S. cerevisiae and the fopA gene product obtained from the culture supernatant of the S. cerevisiae transformant had similar characteristics to the beta-fructofuranosidase purified from A. niger ATCC 20611. However, we could not detect any beta-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S. cerevisiae. In western analysis of those samples, there was no protein product that is cross-reacted with anti-beta-fructofuranosidase antibody. These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.     

Analysis of the essential cell division gene ftsL of Bacillus subtilis by mutagenesis and heterologous complementation

The ftsL gene is required for the initiation of cell division in a broad range of bacteria. Bacillus subtilis ftsL encodes a 13-kDa protein with a membrane-spanning domain near its N terminus. The external C-terminal domain has features of an alpha-helical leucine zipper, which is likely to be involved in the heterodimerization with another division protein, DivIC. To determine what residues are important for FtsL function, we used both random and site-directed mutagenesis. Unexpectedly, all chemically induced mutations fell into two clear classes, those either weakening the ribosome-binding site or producing a stop codon. It appears that the random mutagenesis was efficient, so many missense mutations must have been generated but with no phenotypic effect. Substitutions affecting hydrophobic residues in the putative coiled-coil domain, introduced by site-directed mutagenesis, also gave no observable phenotype except for insertion of a helix-breaking proline residue, which destroyed FtsL function. ftsL homologues cloned from three diverse Bacillus species, Bacillus licheniformis, Bacillus badius, and Bacillus circulans, could complement an ftsL null mutation in B. subtilis, even though up to 66% of the amino acid residues of the predicted proteins were different from B. subtilis FtsL. However, the ftsL gene from Staphylococcus aureus (whose product has 73% of its amino acids different from those of the B. subtilis ftsL product) was not functional. We conclude that FtsL is a highly malleable protein that can accommodate a large number of sequence changes without loss of function.     

Hydrolysis of oligofructoses by the recombinant beta-fructofuranosidase from Bifidobacterium lactis

The ability of the beta-fructofuranosidase (EC 3.2.1.26) from Bifidobacterium lactis DSM 10140T to cleave a variety of fructooligosaccharides was characterised. We identified its gene on a cloned chromosomal DNA fragment by sequence similarity (69% identity) to the putative CscA protein encoded in the Bifidobacterium longum genome. The deduced amino acid sequence of 532 residues (59.4 kDa) appeared to be identical to the beta-fructofuranosidase from the same strain recently described by Ehrmann et al. (Curr. Microbiol. 2003, 46, 391-397). However, the characterisation of the heterologously expressed enzyme showed several discrepancies to the referred study. First, the B. lactis beta-fructofuranosidase gene was found to have 41% identity with CscA from E. coli in contrast to the 16% reported, therefore it was assigned to as CscA protein instead of BfrA. Second, we observed only low activity of the enzyme towards sucrose (6%) instead of the 100% previously reported. Instead, we measured highest activity (100%) of the enzyme with the oligofructose Raftilose as a substrate compared with the inulin of low degree of polymerisation Raftiline LS (29%) and the highly polymerised Raftiline HP (10%). Altogether, the enzyme showed high affinity to terminal beta(2-1) glycosyl linkages between fructose moieties. The Km values obtained for Raftilose, Raftiline LS and sucrose were 0.12, 7.08 and 8.37 mM, respectively, and V(max) values for the conversion to fructose were calculated to be 5, 21 and 17 micromol/min per mg of protein, respectively. Growth of B. lactis was supported by fructans of low degree of polymerisation (Raftilose and Raftiline LS), whereas we observed no growth with highly polymerised inulin (Raftiline HP).     

Molecular cloning and characterization of an alkalophilic Bacillus sp. C125 gene homologous to Bacillus subtilis sec Y.

A 1.8 kb HindIII DNA fragment containing the secY gene of alkalophilic Bacillus sp. C125 has been cloned into plasmid pUC119 using the B. subtilis secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs. The similarity of these ORFs to the sequences of the B. subtilis proteins indicated that they were the genes for ribosomal protein L15-SecY-adenylate kinase, in that order. The gene product of the alkalophilic Bacillus sp. C125 secY homologue was composed of 431 amino acids and its M(r) value has been calculated to be 47,100. The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments. The total amino acid sequence of alkalophilic Bacillus sp. C125 secY homologue showed 69.7% homology with that of B. subtilis secY. Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmic domains (73% identity) with less homologous regions present in extracellular domains (43% identity).     

The primary structure of ribosomal protein L2 from Bacillus stearothermophilus

The complete amino acid sequence of ribosomal protein L2 from the moderate thermophile Bacillus stearothermophilus has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with Staphylococcus aureus protease, trypsin and chymotrypsin, as well as by chemical cleavage with o-iodosobenzoic acid. The protein contains 275 amino acid residues and has a calculated molecular mass of 30201 Da. Comparison of this sequence with sequences of the corresponding proteins from Escherichia coli and from spinach and tobacco chloroplasts reveals that 60% of the residues of protein L2 from B. stearothermophilus are identical to those of the protein from E. coli and 45% are identical to those found in the two chloroplast proteins. There are extended regions of totally conserved sequence at positions 54-58 (GGGHK), 81-86 (EYDPNR), and 224-230 (MNPVDHP) in all four proteins.     

Cloning of the beta-amylase gene from Bacillus cereus and characteristics of the primary structure of the enzyme.

The gene encoding the beta-amylase of Bacillus cereus BQ10-S1 (SpoII) was cloned into Escherichia coli JM 109. A sequenced DNA fragment of 2,001 bp contains the beta-amylase gene. The N-terminal sequences (AVNGKG MNPDYKAYLMAPLKKI), the C-terminal sequences (SHTSSW), and the amino acid sequences of the five regions in the beta-amylase molecules were determined. The mature beta-amylase contains 514 amino acid residues with a molecular mass of 57,885 Da. The amino acid sequence homology with those of known beta-amylases was 52.7% for Bacillus polymyxa, 52.0% for Bacillus circulans, 43.4% for Clostridium thermosulfurogenes, 31.8% for Arabidopsis thaliana, 31.5% for barley, 29.9% for sweet potato, and 28.9% for soybean. Ten well-conserved regions were found between the N terminus and the area around residue 430, but the C-terminal region of 90 residues has no similarity with those of the plant beta-amylases. The homology search revealed that this C-terminal region has homology with C-terminal regions of the beta-amylase from C. thermosulfurogenes, some bacterial alpha-amylases, cyclodextrin glucanotransferase, and glucoamylase. Some of these sequences are known as the raw-starch-binding domain. These results suggest that B. cereus beta-amylase has an extra domain which has raw-starch-binding ability and that the domain has considerable sequence homology with those of other amylases or related enzymes from a wide variety of microorganisms.     

The partial amino acid sequence of the extracellular beta-lactamase I of Bacillus cereus 569/H.

The chemical structure of the extracellular beta-lactamase I of Bacillus cereus 569/H was investigated. Three electrophoretically homogenous charge variants of this enzyme were isolated and amino acid analysis of each revealed no significant differences. However, a degree of N-terminal heterogeneity was found by direct end-group modification of the protein and also on alignment of peptides from tryptic and chymotryptic digestion. The N-terminal heterogeneity observed was great enough to explain the production of the beta-lactamase I isoenzymes which are probably produced by postsynthesis modification of a single gene product. Over 80% of the amino acid sequence of beta-lactamase I was determined by the detailed analysis of peptides derived from tryptic, chymotryptic and thermolytic digests. Five polypeptide fragments were constructed from these data and aligned by comparison with the known amino acid sequences of the penicillinases produced by Bacillus licheniformis and Staphylococcus aureus (Ambler & Meadway, 1969). About 60% of the proposed sequence was identical with that of B. licheniformis penicillinase, whereas the S. aureus enzyme had only about 40% of its residues in common with beta-lactamase I. These results are discussed with reference to the possible evolutionary relationships existing between known beta-lactamases. Detailed evidence for the amino acid sequence proposed has been deposited as Supplementary Publication SUP 50044 (27 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5.     

Gene encoding a novel extracellular metalloprotease in Bacillus subtilis.

The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation.     

Isolation and characterization of cDNA clones for rat liver 10-formyltetrahydrofolate dehydrogenase.

We have isolated and characterized cDNA clones encoding rat liver cytosol 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6). An open reading frame of 2706 base pairs encodes for 902 amino acids of Mr 99,015. The deduced amino acid sequence contains exact matches to the NH2-terminal sequence (28 residues) and the sequences of five peptides derived from cyanogen bromide cleavage of the purified protein. The amino acid sequence of 10-formyltetrahydrofolate dehydrogenase has three putative domains. The NH2-terminal sequence (residues 1-203) is 24-30% identical to phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) from Bacillus subtilis (30%), Escherichia coli (24%), Drosophila melanogaster (24%), and human hepatoma HepG2 (27%). Residues 204-416 show no extensive homology to any known protein sequence. Sequence 417-900 is 46% (mean) identical to the sequences of a series of aldehyde dehydrogenase (NADP+) (EC 1.2.1.3). Intact 10-formyltetrahydrofolate dehydrogenase exhibits NADP-dependent aldehyde dehydrogenase activity. The sequence identity to phosphoribosylglycinamide formyltransferase is discussed, and a binding region for 10-formyltetrahydrofolate is proposed.     

Gene encoding a minor extracellular protease in Bacillus subtilis.

The gene for a minor, extracellular protease has been identified in Bacillus subtilis. The gene (epr) encoded a primary product of 645 amino acids that was partially homologous to both subtilisin (Apr) and the major internal serine protease (ISP-1) of B. subtilis. Deletion analysis indicated that the C-terminal 240 amino acids of Epr were not necessary for activity. This C-terminal region exhibited several unusual features, including a high abundance of lysine residues and the presence of a partially homologous sequence of 44 amino acids that was directly repeated five times. The epr gene mapped near sacA and was not required for growth or sporulation.     

The hypervariable region in the genes coding for entomopathogenic crystal proteins of Bacillus thuringiensis: nucleotide sequence of the kurhd1 gene of subsp. kurstaki HD1

One of the genes for the entomophatogenic crystal protein of Bacillus thuringiensis (subsp. kurstaki strain HD1) has been cloned in Escherichia coli, and its nucleotide sequence determined completely. The gene is contained within a 4360-bp-long HpaI-PstI DNA restriction fragment and codes for a polypeptide of 1,155 amino acid residues. The protoxin protein has a predicted Mr of 130,625. The E. coli-derived protoxin gene product is biologically active against Heliothis virescens larvae in a biotest assay. Extensive computer comparisons with other published B. thuringiensis subsp. kurstaki strains HD1, HD73, and B. thuringiensis subsp. sotto gene sequences reveal hypervariable regions in the first half of the protoxin coding sequence. These regions are responsible for the biological activity of the protein product of the cloned gene, and may explain the different biological activities of these different protoxins.     

Molecular cloning, nucleotide sequence, and expression of the structural gene for alkaline serine protease from alkaliphilic Bacillus sp. 221.

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus subtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6-61.7%). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

The complete covalent structure of protein B. The third major protein degraded during germination of Bacillus megaterium spores

The complete covalent structure of Protein B, the third major protein degraded during germination of Bacillus megaterium spores, has been determined. The intact protein was cleaved with the specific B. megaterium spore protease into three peptides, residues 1 to 31 (B-III), 32 to 66 (B-I), and 67 to 96 (B-II). Cleavage of the intact protein with trypsin allowed isolation of the peptide encompassing residues 61 to 77 (T-11) as well as the COOH-terminal peptide, residues 94 to 96 (T-4). Cleavage of Peptide B-I with trypsin or chymotrypsin allowed isolation of peptides encompassing residues 53 to 60 (B-I-T-2) and residues 52 to 66 (B-I-C-4), respectively. Subtractive Edman degradation of Peptide T-4, automated sequenator analysis of Peptides B-I, B-II, T-11, B-I-T-2, and B-I-C-4, previously published partial sequence data on the intact B-protein and carboxypeptidase V digestion of the intact protein provided the data from which the following unique sequence was deduced: NH2-Ala-Lys-Gln-Thr-Asn-Lys-Thr-Ala-Ser-Gly-Thr-Ser-Thr-Gln-His-15 Val-Lys-Gln-Gln-Asp-Ala-Gln-Ala-Ser-Lys-Asn-Asn-Phe-Gly-Thr-30 Glu-Phe-Gly-Ser-Glu-Thr-Asn-Val-Gln-Glu-Val-Lys-Gln-Gln-Asn-45 Ala-Gln-Ala-Ala-Asn-Lys-Ser-Gln-Asn-Ala-Gln-Ala-Ser-Lys-60 Asn-Asn-Phe-Gly-Thr-Glu-Phe-Ala-Ser-Glu-Thr-Ser-Ala-Gln-Glu-75 Val-Arg-Gln-Gln-Asn-Ala-Gln-Ala-Gln-Lys-Lys-Asn-Gln-Asn-90 Ser-Gly-Lys-Tyr-Gln-Gly-COOH. The primary sequence of the B-protein contains a large internal duplication (residues 17 to 50 and 52 to 85), and shows significant sequence homology with the A- and C-proteins, the other major proteins degraded during B. megaterium spore germination.