Cold adaptation of a psychrophilic chitinase: a mutagenesis study.

The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.

Engineering the properties of a cold active enzyme through rational redesign of the active site.

In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 degrees C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 degrees C and a significantly increased activity at 20-25 degrees C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135-->Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints.

Modular structure, local flexibility and cold-activity of a novel chitobiase from a psychrophilic Antarctic bacterium.

The gene archb encoding for the cell-bound chitobiase from the Antarctic Gram-positive bacterium Arthrobacter sp. TAD20 was cloned and expressed in Escherichia coli in a soluble form. The mature chitobiase ArChb possesses four functionally independent domains: a catalytic domain stabilized by Ca(2+), a galactose-binding domain and an immunoglobulin-like domain followed by a cell-wall anchorage signal, typical of cell-surface proteins from Gram-positive bacteria. Binding of saccharides was analyzed by differential scanning calorimetry, allowing to distinguish unequivocally the catalytic domain from the galactose-binding domain and to study binding specificities. The results suggest that ArChb could play a role in bacterium attachment to natural hosts. Kinetic parameters of ArChb demonstrate perfect adaptation to catalysis at low temperatures, as shown by a low activation energy associated with unusually low K(m) and high k(cat) values. Thermodependence of these parameters indicates that discrete amino acid substitutions in the catalytic center have optimized the thermodynamic properties of weak interactions involved in substrate binding at low temperatures. Microcalorimetry also reveals that heat-lability, a general trait of psychrophilic enzymes, only affects the active site domain of ArChb.

Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.

Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.

Cloning, sequences, and characterization of two chitinase genes from the Antarctic Arthrobacter sp. strain TAD20: isolation and partial characterization of the enzymes.

Arthrobacter sp. strain TAD20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the Antarctic ice shell. Arthrobacter sp. strain TAD20 secretes two major chitinases, ChiA and ChiB (ArChiA and ArChiB), in response to chitin induction. A single chromosomal DNA fragment containing the genes coding for both chitinases was cloned in Escherichia coli. DNA sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of ArChiA (881 amino acids [aa]) and ArChiB (578 aa). ArChiA and ArChiB are modular enzymes consisting of a glycosyl-hydrolase family 18 catalytic domain as well as two and one chitin-binding domains, respectively. The catalytic domain of ArChiA exhibits 55% identity with a chitodextrinase from Vibrio furnissii. The ArChiB catalytic domain exhibits 33% identity with chitinase A of Bacillus circulans. The ArChiA chitin-binding domains are homologous to the chitin-binding domain of ArChiB. ArChiA and ArChiB were purified to homogeneity from the native Arthrobacter strain and partially characterized. Thermal unfolding of ArChiA, ArChiB, and chitinase A of Serratia marcescens was studied using differential scanning calorimetry. ArChiA and ArChiB, compared to their mesophilic counterpart, exhibited increased heat lability, similar to other cold-adapted enzymes.

Chitin deacetylases: new, versatile tools in biotechnology.

Chitin deacetylases have been identified in several fungi and insects. They catalyse the hydrolysis of N-acetamido bonds of chitin, converting it to chitosan. Chitosans, which are produced by a harsh thermochemical procedure, have several applications in areas such as biomedicine, food ingredients, cosmetics and pharmaceuticals. The use of chitin deacetylases for the conversion of chitin to chitosan, in contrast to the presently used chemical procedure, offers the possibility of a controlled, non-degradable process, resulting in the production of novel, well-defined chitosan oligomers and polymers.

Alkaline phosphatase from the Antarctic strain TAB5. Properties and psychrophilic adaptations.

The gene encoding alkaline phosphatase (AP) from the psychrophilic strain TAB5 was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1125 base pairs which encodes a polypeptide consisting of signal peptide of 22 amino acids and a mature protein of 353 amino acids was identified. The deduced protein sequence of AP exhibits a 38% identity to the AP III and AP IV sequences of Bacillus subtilis and conserves the typical sequence motifs of the core structure and active sites of APs from various sources. Based on the crystal structure of the mutated Escerichia coli AP D153H, a homology-based 3D model of the TAB5 AP was constructed on the basis of which various features of the enzyme amino-acid sequence can be interpreted in terms of potential psychrophilic adaptations. The AP gene was expressed in E. coli BL21(DE3) cells, the recombinant protein was isolated to homogeneity from the membrane fraction of the cells and its properties were examined. The purified TAB5 AP shows typical features of a cold enzyme: high catalytic activity at low temperature and a remarkable thermosensitivity. The use of this heat-labile enzyme, for dephosphorylation of nucleic acids, simplifies dephosphorylation protocols.

Yeast ascospore wall assembly requires two chitin deacetylase isozymes.

Chitin deacetylases are required for spore wall rigidity in Saccharomyces cerevisiae. Two chitin deacetylase genes (CDA1 and CDA2) have been identified in yeast. In this report we studied the biochemical properties of the chitin deacetylases encoded by CDA1 and CDA2 and we show how their elimination directly affects the ascospore wall assembly.

Mode of action of chitin deacetylase from Mucor rouxii on N-acetylchitooligosaccharides.

The mode of action of chitin deacetylase from the fungus Mucor rouxii on N-acetylchitooligosaccharides with a degree of polymerization 1-7 has been elucidated. Identification of the sequence of chitin oligomers following enzymatic deacetylation was verified by the alternative use of two specific exo-glycosidases in conjunction with HPLC. The results were further verified by 1H-NMR spectroscopy. It was observed that the length of the oligomer is important for enzyme action. The enzyme cannot effectively deacetylate chitin oligomers with a degree of polymerization lower than three. Tetra-N-acetylchitotetraose and penta-N-acetylchitopentaose are fully deacetylated by the enzyme, while in the case of tri-N-acetylchitotriose, hexa-N-acetylchitohexaose and hepta-N-acetylchitoheptaose the reducing-end residue always remains intact. Furthermore, the enzyme initially removes an acetyl group from the nonreducing-end residue of all chitin oligomers with a degree of polymerization higher than 2, and further catalyses the hydrolysis of the following acetamido groups in a processive fashion. The results are in agreement with the mode of action that the same enzyme exhibits on partially deacetylated water soluble chitosan polymers.

Stereospecificity of hydrogen transfer by the NAD(+)-linked alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123.

Investigation of the stereochemistry of the hydride transfer in reactions catalyzed by the recently isolated NAD(+)-linked alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123 was accomplished by using 1H NMR spectroscopy of the deuterated coenzyme. It was found that this new psychrophilic enzyme is a type A dehydrogenase. Moraxella sp. ADH reduces stereospecifically 2-butanone to produce (S)-2-butanol.

Purification and characterization of an alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123.

An NAD+-dependent alcohol dehydrogenase (ADH) of the Antarctic psychrophile Moraxella sp. TAE123 was purified to homogeneity with an overall yield of 16.7% and further characterized. The native enzyme had an apparent molecular mass of 240 kDa and consisted of four identical 52-kDa subunits. The pI of the enzyme was determined to be 5.5, while its optimum pH is 7.5. The enzyme contained 1 zinc atom/subunit and exhibited a remarkable thermal lability. Moraxella sp. TAE123 ADH exhibited a wide range of substrate specificity similar to its mammalian counterparts and in contrast to other microbial ADHs. It oxidized mainly primary and secondary aliphatic alcohols. The highest reaction rate was observed when ethanol was used as substrate. A gradual decrease in rate was observed by increasing the length and branching of the carbon chain of the alcohol. This enzyme oxidized effectively large bulky alcohols, such as diphenylmethanol. Reduction of aldehydes and ketones was also observed. N-terminal amino acid sequence analysis of the enzyme did not reveal any similarity with the amino termini of all other ADHs, while an unexpected significant similarity was observed with the amino terminal sequence of four prokaryotic aldehyde dehydrogenases.

[Isolation and specificity of novel restriction endonucleases Bsp40091 and AsiI, isoschizomers of BamHI]. / Vydelenie i opredelenie spetsifichnosti novykh éndonukleaz restriktsii Bsp40091 i AsiI izoshizomerov BamHI.

New type II restriction endonucleases AsiI and Bsp40091 are detected in Azotobacter species N55 and Bacillus species 4009, respectively. Purified preparations of the restriction enzymes free from interfering nucleases and phosphatases were obtained by column chromatography on phosphocellulose and heparin-sepharose (Asil) and phosphocellulose and DEAE-cellulose (Bsp40091). The yield of purified AsiI and Bsp40091 was 16 x 10(3) and 8 x 10(3) units per g of wet cells, respectively. The above restriction endonucleases recognize the 5'-G decreases GATCC-3' sequence on double-stranded DNA and cleave it as shown, thus being true isoschizomers of BamHI restriction endonuclease.

[Novel site-specific endonucleases from Brevibacterium species]. / Novye sait-spetsificheskie éndonukleazy Brevibacterium species.

New site-specific endonucleases BecAI and BecAII have been detected in Brevibacterium species A. Endonuclease BecAII free from contaminating nonspecific endonucleases, exonucleases, and phosphatases was isolated by column chromatography on phosphocellulose, heparin sepharose, and DNA cellulose. It recognizes and cleaves the 5'-GG decreases CC-3' sequence and is a true isoschizomer of HaeIII restriction enzyme. The other restriction endonuclease, BecAI, cleaves Ad2 DNA at least by 2 sites but not the DNA of phage lambda, T7, SV40, phiX174, and plasmides pBR322 and pUC19. The substrate specificity of BecAI indicates its appurtenance to the super rare restriction endonucleases.

Isolation and characterization of CspBI, a novel NotI isoschizomer from Corynebacterium species B recognizing 5'-GC/GGCCGC-3'.

Sixty-seven bacterial strains were surveyed for the presence of type II restriction endonucleases, especially concerning super-rare-cutting enzymes. Fourteen strains were found to contain specific enzymes. One of them CspBI from Corynebacterium species B was purified and characterized as an isoschizomer of NotI, which recognizes the palindromic octanucleotide sequence 5'-GC/GGCCGC-3' and cleaves at the position shown by the arrow. A comparison between the cleavage patterns on different DNAs, obtained with partially purified endonucleases from other detected producents including some strains of Corynebacterium, Cellulomonas and Rhizobium has shown that these enzymes do not belong to super-rare-cutting restriction endonucleases.

Cloning and characterization of the gene encoding PspPI methyltransferase from the Antarctic psychrotroph Psychrobacter sp. strain TA137. Predicted interactions with DNA and organization of the variable region.

The gene (pspPIM) encoding the PspPI DNA methyltransferase (MTase) associated with the PspPI restriction-modification (R-M) system (5'-GGNCC-3') of Psychrobacter species TA137 has been cloned and expressed in E. coli, and its nucleotide (nt) sequence has been determined. The coding region was 1248 nt in length and capable of specifying a 46826-Da protein of 416 amino acids (aa). The predicted sequence of the MTase protein displays ten sequence motifs characteristic of all prokaryotic m5C-MTases and shows the highest similarity to other MTases that methylate the GGNCC sequence, namely M . Eco47II and M . Sau96I. All three MTases methylate the internal cytosine within their recognition sequence. Sequence similarities between M . PspPI and its isospecific M . Eco47II and M . Sau96I as well as with four other m5C-MTases that methylate the related GGWCC sequence, namely M . SinI, M . HgiCII, M . HgiBI, M . HgiEI have been also found within the variable region of these proteins. On the basis of structural information from M . HhaI and M . HaeIII, several M . PspPI residues that are expected to interact with DNA can be predicted. Furthermore, an organization of the variable region of m5C-MTases into two segments exhibiting a pattern of conserved residues and a considerable degree of structural homologies is described.

Purification, crystallization and preliminary X-ray analysis of the M.BseCI DNA methyltransferase from Bacillus stearothermophilus.

The DNA methyltransferase M.BseC1 from B. stearothermophilus methylates the N6 atom of the 3' adenine in the sequence 5'-ATCGAT-3'. The 579-residue protein has been isolated and crystallized using seeding and microdialysis techniques. The crystals are monoclinic, space group P2(1) with cell dimensions a = 53.7, b = 85.7, c = 151.8 A and beta = 95.1 degrees, two molecules in the asymmetric unit and diffract to at least 2.5 A resolution.

Two sporulation-specific chitin deacetylase-encoding genes are required for the ascospore wall rigidity of Saccharomyces cerevisiae.

The formation of the ascospore wall of Saccharomyces cerevisiae requires the coordinate activity of enzymes involved in the biosynthesis of its components such as chitosan, the deacetylated form of chitin. We have cloned the CDA1 and CDA2 genes which together account for the total chitin deacetylase activity of the organism. We have shown that expression of these genes is restricted to a distinct time period during sporulation. The two genes are functionally redundant, each contributing equally to the total chitin deacetylase activity. Diploids disrupted for both genes sporulate as efficiently as wild type cells, and the resulting mutant spores are viable under standard laboratory conditions. However, they fail to emit the natural fluorescence of yeast spores imparted by the dityrosine residues of the outermost ascospore wall layer. Moreover, mutant spores are relatively sensitive to hydrolytic enzymes, ether, and heat shock, a fact that underscores the importance of the CDA genes for the proper formation of the ascospore wall.

[Bsp153AI and BspM39I--new isoschizomers of PvuII restriction enzyme]. / Bsp153AI i BspM39I--novye izoshizomery restriktazy PvuII.

New restriction endonucleases, Bsp153AI and BspM39I, were isolated from Bacillus species strains 153A and M39, respectively. The enzymes recognize and cleave the nucleotide sequence [sequence: see text] and are true isoschizomers of restriction endonuclease PvuII.

Purification and characterization of chitin deacetylase from Colletotrichum lindemuthianum.

Chitin deacetylase (EC 3.5.1.41), the enzyme that catalyzes the hydrolysis of acetamido groups of N-acetyl-D-glucosamine in chitin, has been purified to homogeneity from the culture filtrate of the fungus Colletotrichum lindemuthianum and further characterized. The enzyme is a glycoprotein, and its apparent molecular mass was determined to be approximately 150 kDa. The glycosylation pattern of the enzyme is consistent with a mixture of N-linked glycans including oligomannosidic hybrid and/or complex type, and its carbohydrate content is approximately 67% by weight. Chitin deacetylase is active on several chitinous substrates and chitin derivatives, is not considerably inhibited by carboxylic acids, especially acetic acid, and exhibits a remarkable thermostability. The enzyme requires at least two N-acetyl-D-glucosamine residues (chitobiose) for catalysis. When glycol chitin (a water-soluble chitin derivative) was used as substrate, the optimum temperature for enzyme activity was determined to be 50 degrees C, and the optimum pH was approximately 8.5.

Preparation of high purity alkaline phosphatase from calf intestine using dye-ligand chromatography.

For the effective application of alkaline phosphatase from calf intestine in Molecular Biology research highly purified enzyme and free from contaminating DNases, DNA nicking, ribonuclease and phosphodiesterase activities is required. We now report the use of a two-step procedure which involves chromatography on a Mimetic Blue AP-Agarose, a commercially available adsorbent and Heparin Sepharose to purify calf intestinal alkaline phosphatase from a crude commercial preparation to homogeneity. Purified enzyme preparations were free from contaminating DNases, DNA nicking, ribonuclease and phosphodiesterase activities and exhibited a specific activity (3.800 units/mg) which is one of the highest reported among the existing high purity commercial preparations. It is therefore concluded that the reported purification protocol can be used routinely to prepare high purity alkaline phosphatase suitable for use in Molecular Biology research.