Nucleotide sequence of the lipase gene lip2 from the antarctic psychrotroph Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine residues

The lip2 gene from the antarctic psychotroph Moraxella TA144 was sequenced. The primary structure of the Lip2 preprotein deduced from the nucleotide sequence is composed of 433 amino acids with a predicted Mr of 47,222. This enzyme contains a Ser-centered consensus sequence and a conserved His-Gly dipeptide found in most lipase amino-terminal domains. These sequences are involved in the lipase active site conformation since substitution of the conserved Ser or His residues by Ala and Gln, respectively, results in the loss of both lipase and esterase activities. Structural factors that would allow proper enzyme flexibility at low temperatures are discussed. It is suggested that only subtle changes in the primary structure of these psychrotrophic enzymes can account for their ability to catalyze lipolysis at temperatures close to 0 degrees C.     

Cloning and expression in Escherichia coli of three lipase-encoding genes from the psychrotrophic antarctic strain Moraxella TA144

The cloning and expression of genes from a psychrotrophic bacterium in a mesophilic host are described. Three lipase (Lip)-encoding genes (lip) from the antarctic psychrotroph, Moraxella TA144, were cloned by inserting Sau3AI-generated DNA fragments into the BamHI site of the pSP73 plasmid vector. To prevent heat denaturation of the gene product, the screening procedure on agar plates containing an emulsified lipid involved growing of Escherichia coli recombinant colonies at 25 degrees C followed by incubation at 0 degree C. The three recombinant (reLip) were cell-associated and differed by their respective specificity towards p-nitrophenyl esters of various aliphatic chain lengths. These cloned reLip conserved the main character of the wild-type enzymes, i.e. a dramatic shift of the optimal temperature of activity towards low temperatures and pronounced heat lability.     

Nucleotide and derived amino acid sequence of the subtilisin from the antarctic psychrotroph Bacillus TA39.

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph Bacillus TA39 was determined. The primary structure of the subtilisin precursor is composed of 420 amino acids giving rise to a mature enzyme of 309 amino acids. Asp-145, His-185 and Ser-361 are the proposed catalytic residues of the active site.     

Sequence of the subtilisin-encoding gene from an antarctic psychrotroph Bacillus TA41.

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph, Bacillus TA41, was determined. The primary structure of the subtilisin precursor corresponds to a preproenzyme of 419 amino acids. Asp144, His181 and Ser359 are the proposed catalytic residues of the protease active site.     

Nucleotide sequence of the maltotetraohydrolase gene from Pseudomonas saccharophila

The nucleotide sequence of the Pseudomonas saccharophila gene encoding maltotetraohydrolase (G4-forming amylase) has been determined. The coding region for the G4-forming amylase precursor contained 1653 nucleotides. The deduced precursor protein included an N-terminal 21-residue putative signal peptide; the deduced mature form of G4-forming amylase contains 530 amino acid residues with a calculated molecular mass of 57 740 Da. Sequence similarities between the G4-forming amylase and other amylolytic enzymes of species ranging from prokaryotes to eukaryotes are quite limited. However, three regions, which are involved in both the catalytic and substrate-binding sites of various amylolytic enzymes, are highly conserved in the G4-forming amylase of P. saccharophila.     

Nucleotide sequence of the alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum

The nucleotide sequence of the gene (apu) encoding the thermostable alpha-amylase-pullulanase of Clostridium thermohydrosulfuricum was determined. An open reading frame of 4425 bp was present. The deduced polypeptide (Mr 165,600), including a 31 amino acid putative signal sequence, comprised 1475 amino acids, with no cysteine residues. The structural gene was preceded by the consensus promoter sequence TTGACA TATAAT, a putative regulatory sequence and a putative ribosome-binding sequence AAAGGGGG. The codon usage resembled that of Bacillus genes. The deduced sequence of the mature apu product showed similarities to various amylolytic enzymes, especially the neopullulanase of Bacillus stearothermophilus, whereas the signal sequence showed similarity to those of the alpha-amylases of B. stearothermophilus and B. subtilis. Three regions thought to be highly conserved in the primary structure of alpha-amylases could also be distinguished in the apu product, two being partly 'duplicated' in this alpha-1,4/alpha-1,6-active enzyme.     

Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus

The gene (nplT) for a new type of pullulan-hydrolysing enzyme, neopullulanase, from Bacillus stearothermophilus TRS40 was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1764 bases and 588 amino acid residues (Mr 69144). Although the thermostable neopullulanase contained eight cysteine residues, they did not provide conformational stability by disulphide bonds. A comparison was made of the amino acid sequences of alpha-amylase, neopullulanase, isoamylase, pullulanase and cyclodextrin glucanotransferase. All the enzymes examined contained four highly conserved regions which probably constitute the active centres of the enzymes. The amino acid residues required for the specificity of neopullulanase are compared with those of alpha-amylase and other amylolytic enzymes.     

Nucleotide sequence of the ethidium efflux gene from Escherichia coli

The nucleotide sequence of the gene specifying the ethidium efflux system of Escherichia coli has been determined. The translated open reading frame has identified a membrane-bound polypeptide of 110 amino acids (11,960 Da) which shares 42% identity with a staphylococcal protein specifying resistance to ethidium.     

Nucleotide sequence of the tag gene from Escherichia coli.

We have determined the complete nucleotide sequence of the tag gene, encoding 3-methyladenine DNA glycosylase I from Escherichia coli. From the nucleotide sequence it is deduced that the tag enzyme consists of 187 amino-acids and has a calculated molecular weight of 21.1 kdaltons. The tag enzyme is unusually rich in cysteine (8 residues) with a cluster of three consecutive cysteines near the C-terminal end. The tag coded DNA glycosylase does not show significant sequence homology to the alkA coded glycosylase in spite of that both of these enzymes catalyze the release of free 3-methyladenine from alkylated DNA.