Cloning and Sequencing Analysis of Alginate Lyase Genes from the Marine Bacterium Vibrio sp. O2

We isolated a new marine bacteria, which displayed alginate-depolymerizing activity in plate assays, from seawater in Mihonoseki Harbor, Japan. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the genus Vibrio and it was named Vibrio sp. O2. The alginate lyase genes of Vibrio sp. O2 were cloned and expressed in Escherichia coli. Two alginate lyase-producing clones, pVOA-A4 and pVOA-B5, were obtained. The alginate lyase gene alyVOA from pVOA-A4 was composed of an 858-bp open reading frame (ORF) encoding 285 amino acid residues, while alyVOB from pVOA-B5 was composed of an 828-bp ORF encoding 275 amino acid residues. The degree of identity between the deduced amino acid sequences of AlyVOA or AlyVOB and Photobacterium sp. ATCC43367 alginate poly(ManA)lyase AlxM was 92.3% or 32.6%, respectively. Alginate lyase consensus regions corresponding to the sequences YFKAGXYXQ and RXELR were observed in all three of these sequences. AlyVOA and AlyVOB both degraded polymannuronate in plate assays and were therefore confirmed to be poly(β-D-mannuronate)lyases.     

Complete amino acid sequence of the thioesterase domain of chicken liver fatty acid synthase

The complete amino acid sequence of thioesterase domain of chicken liver fatty acid synthase has been determined by sequencing peptides produced by trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide cleavage. The thioesterase domain consists of 300 amino acid residues. All of the tryptic peptides of the thioesterase domain were isolated and sequenced, except the segment covered from position 109 to position 124. Peptides resulting from digestion by Staphylococcus aureus V8 protease and cyanogen bromide cleavage filled the missing part and overlapped the complete sequence of the entire thioesterase domain. The NH2 terminus of the thioesterase domain was determined to be lysine by sequencing the whole domain up to 20 residues while the COOH terminus was identified as serine through carboxyl peptidase Y cleavage. The active site of the thioesterase domain of chicken fatty acid synthase was suggested to be the serine on position 101 according to its homology with other serine-type esterases and proteases which have a common structure of -Gly-X-Ser-Y-Gly- with the variable amino acids X and Y disrupting the homology.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

Plant aldolase: cDNA and deduced amino-acid sequences of the chloroplast and cytosol enzyme from spinach

We report the sequences of full-length cDNAs for the nuclear genes encoding the chloroplastic and cytosolic fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) from spinach. A comparison of the deduced amino-acid sequences with one another and with published cytosolic aldolase sequences of other plants revealed that the two enzymes from spinach share only 54% homology on their amino acid level whereas the homology of the cytosolic enzyme of spinach with the known sequences of cytosolic aldolases of maize, rice and Arabidopsis range from 67 to 92%. The sequence of the chloroplastic enzyme includes a stroma-targeting N-terminal transit peptide of 46 amino acid residues for import into the chloroplast. The transit peptide exhibits essential features similar to other chloroplast transit peptides. Southern blot analysis implies that both spinach enzymes are encoded by single genes.     

The complete amino acid sequence of ribosomal protein S18 from the moderate thermophile Bacillus stearothermophilus

The amino acid sequence of ribosomal protein S18 from Bacillus stearothermophilus has been completely determined by automated sequence analysis of the intact protein as well as of peptides derived from digestion with Staphylococcus aureus protease at pH 4.0 and cleavage with cyanogen bromide. The carboxy-terminal region was verified by both amino acid analyses of chymotryptic peptides and by mass spectrometry from the terminal region. The protein contains 77 amino acid residues and has an Mr of 8838. Comparison of this sequence with the sequences of the S18 proteins from tobacco and liverwort chloroplasts and E. coli shows a relatively high similarity, ranging from 42 to 55% identical residues with the B. stearothermophilus S18 protein. The regions of homology common to all four proteins consist of several positively charged sections spanning the entire length of the protein.     

Cloning and Characterization of a Novel Oligoalginate Lyase from a Newly Isolated Bacterium Sphingomonas sp. MJ-3

A bacterium possessing alginate-degrading activity was isolated from marine brown seaweed soup liquefied by salted and fermented anchovy. The isolated strain was designated as Sphingomonas sp. MJ-3 based on the analyses of 16S ribosomal DNA sequences, 16S-23S internal transcribed spacer region sequences, biochemical characteristics, and cellular fatty acid composition. A novel alginate lyase gene was cloned from genomic DNA library and then expressed in Escherichia coli. When the deduced amino acid sequence was compared with the sequences on the databases, interestingly, the cloned gene product was predicted to consist of AlgL (alginate lyase L)-like and heparinase-like protein domain. The MJ-3 alginate lyase gene shared below 27.0% sequence identity with exolytic alginate lyase of Sphingomonas sp. A1. The optimal pH and temperature for the recombinant MJ-3 alginate lyase were 6.5 and 50°C, respectively. The final degradation products of alginate oligosaccharides were analyzed by electrospray ionization mass spectrometry and proved to be alginate monosaccharides. Based on the results, the recombinant alginate lyase from Sphingomonas sp. MJ-3 is regarded as an oligoalginate lyase that can degrade oligoalginate and alginate into alginate monosaccharides.     

Production and Isolation of Levan by Use of Levansucrase Immobilized on the Ceramic Support SM-10

The complete amino acid sequence of rye seed chitinase-a (RSC-a) has been analyzed. RSC-a was cleaved with cyanogen bromide and the resulting three fragments, CB1, CB2, and CB3, were separated by gel filtration. The amino acids of the N-terminal fragment CB1 were sequenced by analyzing the peptides produced by digestion with trypsin, lysylendopeptidase, or pepsin of reduced S-carboxymethyl ated or S-aminoethylated CB1. The sequences of fragments CB2 and CB3 were established by sequencing the tryptic peptides from reduced S-carboxymethylated CB2 and CB3, and by aligning them with the sequence of rye seed chitinase-c (RSC-c) to maximize sequence homology. The complete amino acid sequence of RSC-a was established by connecting these three fragments.

RSC-a consists of 302 amino acid residues including hydroxyproline residues, and has a molecular mass of 31,722 Da. RSC-a is basic protein with a cysteine-rich amino terminal domain, indicating that this enzyme belongs to class I chitinases. The amino acid sequence of RSC-a showed that the sequence from Gly60 to C-terminal Ala302 in this enzyme corresponds to that of RSC-c belonging to class II chitinases with 92% identity, and that RSC-a has high similarity to other plant class I chitinases but a longer hinge region and an extra disulfide bond.     

Structure of the beta-1,3-1,4-glucanase gene of Bacillus macerans: homologies to other beta-glucanases

Summary The nucleotide sequence of an 852 base pair (bp) DNA fragment containing the entire gene coding for thermostable beta- 1,3-1,4-glucanase ofBacillus macerans has been determined. ThebglM gene comprises an open reading frame (ORF) of 711 by (237 codons) starting with ATG at position 93 and extending to the translational stop codon TAA at position 804. The deduced amino acid sequence of the mature protein shows 70% homology to published sequences of mesophilic beta- 1,3-1,4-glucanases fromB. subtilis andB. amyloliquefaciens. The sequence coding for mature beta-glucanase is preceded by a putative signal peptide of 25 amino acid residues, and a sequence resembling a ribosome-binding site (GGAGG) before the initiation codon. By contrast with the processed protein, the N-terminal amino acid sequence constituting the putative leader peptide bears no or only weak homology to signal peptides of mesophilicBacillus endo-beta-glucanases. TheB. macerans signal peptide appears to be functional in exporting the enzyme to the periplasm inE. coli. More than 50% of the whole glucanase activity was localized in the periplasmic space and in the supernatant. Whereas homology to endo-1,4-beta-glucanases is completely lacking, a weak amino acid homology between the sequence surrounding the active site of phage T4 lysozyme and a sequence spanning residues 126 through 161 ofB. macerans endo-beta-glucanase could be identified.     

Complete Amino Acid Sequence of Neutral Protease from Bacillus subtilis var. amylosacchariticus

The neutral protease of Bacillus subtilis var. amylosacchariticus was cleaved chemically or digested with proteolytic enzymes, and the resultant peptides were separated and purified by high performance liquid chromatography. The sequence analyses of these peptides by the manual Edman procedure established the complete amino acid sequence of the enzyme. The neutral protease consisted of 300 amino acid residues with Ala and Leu as its amino- and carboxyl-termini, respectively, and the molecular weight was calculated to be 32,633. The sequence was found to be identical to that of B. subtilis 1A72 neutral protease, which was deduced from nucleotide sequencing. Comparison of the sequence with those of other Bacillus proteases revealed that the putative active site amino acid residues, Zn-binding ligands, and two Ca-binding sites were well conserved among them, as compared with those of thermolysin.     

cDNA cloning of an alginate lyase from a marine gastropod Aplysia kurodai and assessment of catalytically important residues of this enzyme

Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5′- and 3′-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed ∼40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg; however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

The primary structure of the alpha subunit of protocatechuate 3,4-dioxygenase. I. Isolation and sequence of the tryptic peptides.

The carboxymethylated alpha subunit of protocatechuate 3,4-dioxygenase was digested with trypsin. The 14 tryptic peptides were isolated by ion exchange chromatography on DEAE-Sephadex and by gel filtration chromatography. Automated Edman degradation and carboxypeptidase Y and B digestion were used to establish the sequence of these peptides. Further fragmentation of two tryptic peptides, T3 and T5, by Staphylococcus aureus protease and cyanogen bromide, respectively, was necessary to complete the sequences. The tryptic peptides accounted for a minimum of 199 residues out of a total of 202 residues predicted by amino acid analysis.     

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated M _r of 124000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.     

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 suhtilis. 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—61.70/0). 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.     

Amino acid sequence studies on sheep liver fructose-bisphosphatase. II. The complete sequence

The cyanogen bromide fragments of S-carboxymethylated fructose-bisphosphatase were purified. The amino acid sequences of the small fragments were determined by the dansyl-Edman method. The large fragments were subjected to proteolytic digestion to give smaller peptides more amenable for purification and sequencing by similar methods. Enzyme digests of the S-carboxymethylated enzyme gave overlap peptides containing the methionine residues. In conjunction with the amino acid sequence of the 60-residue N-terminal fragment previously determined on the S-peptide released by limited proteolysis with subtilisin the complete sequence of 336 residues was deduced. The sequence has been compared with the 335 residue sequence of pig kidney fructose-bisphosphatase and some areas of sequence for rabbit liver enzyme. The strong homology previously noted for the S-peptide sequence is maintained for the complete enzyme with only 34 changes in 336 residues when comparing the pig and sheep enzymes.     

Genetic characterization and expression of the novel fungal protease, EPg222 active in dry-cured meat products

EPg222 protease is a novel extracellular enzyme produced by Penicillium chrysogenum (Pg222) isolated from dry-cured hams that has the potential for use over a broad range of applications in industries that produce dry-cured meat products. The gene encoding EPg222 protease has been identified. Peptide sequences of EPg222 were obtained by de novo sequencing of tryptic peptides using mass spectrometry. The corresponding gene was amplified by PCR using degenerated primers based on a combination of conserved serine protease-encoding sequences and reverse translation of the peptide sequences. EPg222 is encoded as a gene of 1,361 bp interrupted by two introns. The deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a putative signal sequence of 19 amino acids (aa), a prosequence of 96 aa and a mature protein of 283 aa. A cDNA encoding EPg222 has been cloned and expressed as a functionally active enzyme in Pichia pastoris. The recombinant enzyme exhibits similar activities to the native enzyme against a wide range of protein substrates including muscle myofibrillar protein. The mature sequence contains conserved aa residues characteristic of those forming the catalytic triad of serine proteases (Asp42, His76 and Ser228) but notably the food enzyme exhibits specific aa substitutions in the immunoglobulin-E recognition regions that have been identified in protein homologues that are allergenic.     

Primary structure of a base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi

The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase M) from Aspergillus saitoi was determined. The sequence was determined by analysis of the peptides generated by digestion of heat-denatured RNase M with lysylendopeptidase, and the peptides generated from RCM RNase M by digestion with staphylococcal V8 protease or chemical cleavage with BrCN. It consisted of 238 amino acid residues and carbohydrate moiety attached to the 74th asparagine residue. The molecular weight of the protein moiety deduced from the sequence was 26,596. The locations of 10 half cystine residues are almost superimposable on those of RNase Rh from Rhizopus niveus and RNase T2 from Aspergillus oryzae which have similar base specificity. The homology between RNase M and RNase Rh and RNase T2 amounted to 97 and 160 amino acid residues, respectively. The amino acid sequences conserved in the three RNases are concentrated around the three histidine residues, which are supposed to form part of the active sites of these RNases.     

A Novel Alginate Lyase with High Activity on Acetylated Alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03

Summary To exploit alginate lyase which could degrade bacterial alginates, degenerate PCR and long range-inverse PCR (LR-IPCR) were used to isolate alginate lyase genes from soil bacteria. Gene algL, an alginate lyase-encoding gene from Pseudomonas sp. QD03 was cloned, and it was composed of a 1122 bp open reading frame (ORF) encoding 373 amino acid residues with the calculated molecular mass of 42.2 kDa. The deduced protein had a potential N-terminal signal peptide of 20 amino acid residues that was consistent with its proposed periplasmic location. Gene algL was expressed in pET24a (+)/E. coli BL21 (DE3) system. The recombinant AlgL was purified to electrophoretic homogeneity using affinity chromatography. The molecular weight of AlgL was estimated to be 42.8 kDa by SDS-PAGE. AlgL exhibited maximal activity at pH 7.5 and 37 °C. Na⁺, K⁺, Ca²⁺ and Ba²⁺ significantly enhanced the activity of AlgL. AlgL could degrade alginate and mannuronate blocks, but hardly degrade guluronate blocks. In particular, AlgL could degrade acetylated alginate of Pseudomonas aeruginosa FRD1 (approximately 0.54 mol of O-acetyl group per mol of alginate). It might be possible to use alginate lyase AlgL as an adjuvant therapeutic medicine for the treatment of disease associated with P. aeruginosa infection.     

Purification and Characterization of an Alginate Lyase from Marine Bacterium Vibrio sp. Mutant Strain 510-64

Marine Vibrio sp. 510 was chosen as a parent strain for screening high producers of alginate lyase using the complex mutagenesis of Ethyl Methanesulphonate and UV radiation treatments. The mutant strain Vibrio sp. 510-64 was selected and its alginate lyase activity was increased by 3.87-fold (reaching 46.12 EU/mg) over that of the parent strain. An extracellular alginate lyase was purified from Vibrio sp. 510-64 cultural supernatant by successive fractionation on DEAE Sepharose FF and two steps of Superdex 75. The purified enzyme yielded a single band on SDS-PAGE with the molecular weight of 34.6 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel alginate lyase. The substrate specificity results demonstrated that the alginate lyase had the specificity for poly G block.     

Cloning and Sequence Analysis of the Gene (alyII) Coding for an Alginate Lyase of Pseudomonas sp. OS-ALG-9

Pseudomonas sp. OS-ALG-9 produces several kinds of alginate-degrading enzymes both intra- and extracellularly. As a second alginate lyase of this bacterium, the gene encoding alyII has been cloned in Escherichia coli JM109 by shotgun techniques and then sequenced. The alyII gene has an open reading frame of 2141 bp encoding 713 amino acid residues with a calculated molecular mass of 79,803 Da. The deduced amino acid sequence did not show any extensive similarity with those of other known alginate lyases, however, hydrophobic cluster analysis showed that alyII belonged to class 3 of alginate lyases. The alginate lyase from E. coli harboring the alyII gene showed a single active band, which coincided with one of four major alginate lyases from the crude cell extracts of Pseudomonas sp. OS-ALG-9 on a zymogram.