Molecular characterization, expression and antimicrobial activities of a c-type lysozyme from the mud crab, Scylla paramamosain.

Lysozyme is an important immune protein involved in the first line of defense for crustaceans. In the present study, a c-type lysozyme gene (SpLyzC) was cloned and characterized from the mud crab, Scylla paramamosain. The full-length cDNA was 849 bp with an open reading frame of 669 bp, and encoded a polypeptide of 223 amino acids with a calculated molecular mass of 23.7 kDa and an isoelectric point of 8.90. SpLyzC shared conserved active sites with c-type lysozymes from other species, detected in all tested tissues and had higher expression levels in hepatopancreas and gill tissues. The expression of SpLyzC was up-regulated in hepatopancreas and gill after infection with Vibrio parahaemolyticus and Staphylococcus aureus. The density of bacteria in the hemolymph and the mortality of crabs increased following infection with V. parahaemolyticus after SpLyzC expression was silenced by injecting double-strand RNA of SpLyzC. The recombinant protein of the S. paramamosain c-type lysozyme (rSpLyzC) exhibited antibacterial activities against Micrococcus lysodeikticus, S. aureus, Vibrio harveyi and V. parahaemolyticus. These results indicate that SpLyzC could help eliminate bacteria in S. paramamosain and may play an important role in resistance to bacterial infection.

[Progress of researches on lysozyme and its expression in Oncomelania hupensis].

Lysozyme generally exists in animals, plants and microorganisms, and it is used as a natural anti-infection material and one of the important non-specific immune factors in organisms. This paper reviews the progress of researches on its classification, gene structure and function, and expression regulation in Oncomelania hupensis, and on the factors affecting its activities in recent years, in order to further discuss its distribution in O. hupensis.

Molecular characterization, gene structure and antibacterial activity of a g-type lysozyme from the European sea bass (Dicentrarchus labrax L.).

In fish, the first line of defense is represented by the innate immune system and the lysozyme is one of the molecules involved in this mechanism of protection. Three types of lysozymes have been identified in metazoan, the c-type (chicken or conventional), the g-type (goose-type) and the i-type (invertebrate type). They are all involved in the hydrolysation of the bacterial cell wall. Our work has been focused on the molecular characterization, expression analysis by real-time PCR, both at basal condition and after in vivo challenges, and 3D structural studies on the g-type lysozyme from sea bass (Dicentrarchus labrax L.). Moreover, a recombinant sea bass lysozyme has been produced in Escherichia coli and used to investigate the activity of the enzyme at different pH and temperatures and to perform antibacterial assays against typical fish pathogens. The cloned sea bass cDNA for g-type lysozyme (accession number FN667957) consists of 742 bp and translates for a putative protein of 188 amino acids. The molecular weight is 20.251, 41Da with a theoretical pI of 8.53, two cysteine residues along the sequence and no putative signal peptide. These features of the enzyme are in agreement with the expected characteristics of a proper g-type lysozyme, except for the cysteine residues that in fish are quite variable in number. An alignment between known g-type lysozyme sequences evidences that the amino acid residues thought to be involved in the enzyme catalysis (Glu(71), Asp(84) and Asp(95) in sea bass) are quite well conserved between mammalian, avian and fish sequences. The sea bass g-type lysozyme gene is composed of four exons and three introns and this gene structure is more compact compared to other known fish lysozyme homologues. Modeling of 3D structure has been performed on the template structure of g-type lysozyme from Atlantic cod. The catalytic site appears well conserved when compared with known structures of fish g-type lysozymes (cod and salmon). The basal expression of lysozyme transcripts is highest in gills, followed by head kidney and peripheral blood leukocytes. The lysozyme expression is up regulated in head kidney leukocytes both after challenge with the fish bacterial pathogen Photobacterium damselae subsp. piscicida. The lytic activity, determined using as substrate Micrococcus lysodeikticus, was optimal at pH 5.5 and at a temperature of 30°C. In conclusion, these results suggest that the identified g-type lysozyme should be involved in the innate immune responses of sea bass.

Putative apolipoprotein A-I, natural killer cell enhancement factor and lysozyme g are involved in the early immune response of brown-marbled grouper, Epinephelus fuscoguttatus, Forskal, to Vibrio alginolyticus.

The gram-negative bacterium, Vibrio alginolyticus, has frequently been identified as the pathogen responsible for the infectious disease called vibriosis. This disease is one of the major challenges facing brown-marbled grouper aquaculture, causing fish farmers globally to suffer substantial economic losses. The objective of this study was to investigate the proteins involved in the immune response of brown-marbled grouper fingerlings during their initial encounter with pathogenic organisms. To achieve this objective, a challenge experiment was performed, in which healthy brown-marbled grouper fingerlings were divided into two groups. Fish in the treated group were subjected to intraperitoneal injection with an infectious dose of V. alginolyticus suspended in phosphate-buffered saline (PBS), and those in the control group were injected with an equal volume of PBS. Blood samples were collected from a replicate number of fish from both groups at 4 h post-challenge and analysed for immune response-related serum proteins via two-dimensional gel electrophoresis. The results showed that 14 protein spots were altered between the treated and control groups; these protein spots were further analysed to determine the identity of each protein via MALDI-TOF/TOF. Among the altered proteins, three were clearly overexpressed in the treated group compared with the control; these were identified as putative apolipoprotein A-I, natural killer cell enhancement factor and lysozyme g. Based on these results, these three highly expressed proteins participate in immune response-related reactions during the initial exposure (4 h) of brown-marbled grouper fingerling to V. alginolyticus infection.

Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

The complete amino acid sequence and enzymatic properties of an i-type lysozyme isolated from the common orient clam (Meretrix lusoria).

To determine the structure and functional relationships of invertebrate lysozymes, we isolated a new invertebrate (i)-type lysozyme from the common orient clam (Meretrix lusoria) and determined the complete amino acid sequence of two isozymes that differed by one amino acid. The determined sequence showed 65% similarity to a lysozyme from Venerupis philippinarum (Tapes japonica), and it was therefore classified as an i-type lysozyme. The lytic activities of this lysozyme were similar to those of previously reported bivalve i-type lysozymes, but unlike the V. philippinarum lysozyme, it did not exhibit an increase in activity in high ionic strength. Our data suggest that this lysozyme does not have a dimeric structure, due to the replacement of Lys108 which contributes to dimer formation in the V. philippinarum lysozyme. GlcNAc oligomer activities suggested an absence of transglycosylation activity and a higher number of subsites on this enzyme compared with hen egg lysozyme.

A bifunctional invertebrate-type lysozyme from the disk abalone, Haliotis discus discus: genome organization, transcriptional profiling and biological activities of recombinant protein.

Lysozyme is an important enzyme in the innate immune system that plays a vital role in fighting microbial infections. In the current study, we identified, cloned, and characterized a gene that encodes an invertebrate-type lysozyme from the disk abalone, Haliotis discus discus (abLysI). The full-length cDNA of abLysI consisted of 545 bp with an open reading frame of 393 bp that encodes 131 amino acids. The theoretical molecular mass of mature abLysI was 12.3 kDa with an isoelectric point of 8.03. Conserved features in other homologs, such as catalytic sites for lytic activity (Glu(30) and Asp(41)), isopeptidase activity (His(107)), and ten cysteine residues were identified in abLysI. Genomic sequence analysis with respect to its cDNA showed that abLysI was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative promoter region. Homology and phylogeny analysis of abLysI depicted high identity and closer proximity, respectively, with an annelid i-type lysozyme from Hirudo medicinalis, and indicated that abLysI is a novel molluscan i-type lysozyme. Tissue-specific expressional studies revealed that abLysI is mainly transcribed in hepatopancreas followed by mantle. In addition, abLysI mRNA expression was induced following bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and viral (viral hemorrhagic septicemia virus) challenges. Recombinantly expressed abLysI [(r)abLysI] demonstrated strong lytic activity against Micrococcus lysodeikticus, isopeptidase activity, and antibacterial activity against several Gram-positive and Gram-negative bacteria. Moreover, (r)abLysI showed optimum lytic activity at pH 4.0 and 60 °C, while exhibiting optimum isopeptidase activity at pH 7.0. Taken together, these results indicate that abLysI is potentially involved in immune responses of the disk abalone to protect it from invaders.

Two duplicated chicken-type lysozyme genes in disc abalone Haliotis discus discus: molecular aspects in relevance to structure, genomic organization, mRNA expression and bacteriolytic function.

Lysozymes are crucial antibacterial proteins that are associated with catalytic cleavage of peptidoglycan and subsequent bacteriolysis. The present study describes the identification of two lysozyme genes from disc abalone Haliotis discus discus and their characterization at sequence-, genomic-, transcriptional- and functional-levels. Two cDNAs and BAC clones bearing lysozyme genes were isolated from abalone transcriptome and BAC genomic libraries, respectively and sequences were determined. Corresponding deduced amino acid sequences harbored a chicken-type lysozyme (LysC) family profile and exhibited conserved characteristics of LysC family members including active residues (Glu and Asp) and GS(S/T)DYGIFQINS motif suggested that they are LysC counterparts in disc abalone and designated as abLysC1 and abLysC2. While abLysC1 represented the homolog recently reported in Ezo abalone [1], abLysC2 shared significant identity with LysC homologs. Unlike other vertebrate LysCs, coding sequence of abLysCs were distributed within five exons interrupted by four introns. Both abLysCs revealed a broader mRNA distribution with highest levels in mantle (abLysC1) and hepatopancreas (abLysC2) suggesting their likely main role in defense and digestion, respectively. Investigation of temporal transcriptional profiles post-LPS and -pathogen challenges revealed induced-responses of abLysCs in gills and hemocytes. The in vitro muramidase activity of purified recombinant (r) abLysCs proteins was evaluated, and findings indicated that they are active in acidic pH range (3.5-6.5) and over a broad temperature range (20-60 °C) and influenced by ionic strength. When the antibacterial spectra of (r)abLysCs were examined, they displayed differential activities against both Gram positive and Gram negative strains providing evidence for their involvement in bacteriolytic function in abalone physiology.

A lysozyme-like protein in Brucella abortus is involved in the early stages of intracellular replication.

Secretion of proteins in Gram-negative bacteria is a high-energy-consuming process that requires translocation across two membranes and a periplasmic space composed of a mesh-like layer, the peptidoglycan. To achieve this, bacteria have evolved complex secretion systems that cross these barriers, and in many cases there are specific peptidoglycanases that degrade the peptidoglycan to allow the proper assembly of the secretion machinery. We describe here the identification and characterization of a muramidase in Brucella abortus that participates in the intracellular multiplication in professional and nonprofessional phagocytes. We demonstrated that this protein has peptidoglycanase activity, that a strain with a clean deletion of the gene displayed a defect in the early stages of the intracellular multiplication curve, and that this is dependent on the lytic activity. While neither the attachment nor the invasion of the strain was affected, we demonstrated that it had a defect in excluding the lysosomal marker LAMP-1 but not in acquiring the reticulum endoplasmic marker calnexin, indicating that the gene participates in the early stages of the intracellular trafficking but not in the establishment of the replicative niche. Analysis of the assembly status and functionality of the VirB secretion apparatus indicated that the mutant has affected the proper function of this central virulence factor.

Transcriptional response of lysozyme, metallothionein, and superoxide dismutase to combined exposure to heavy metals and bacteria in Mactra veneriformis.

The response of the defense components lysozyme (LYZ), metallothionein (MT), and superoxide dismutase (SOD) to combined exposure to heavy metals and bacteria was assessed at transcriptional level in the surf clam Mactra veneriformis. First, the full-length LYZ cDNA containing 808 nucleotides and encoding 194 deduced amino acids was identified from the clam. Multiple alignments revealed that MvLYZ had a high identity with invertebrate-type LYZs from other mollusks. Next, clams were exposed to Vibrio parahaemolyticus and a mixture of cadmium and mercury, alone or in combination, for 7 days. Cumulative mortality of clams and mRNA expressions of the three defense components were analyzed. The highest cumulative mortality took place in the combined treatment on day 7. The expression of the three genes was up-regulated in response to treatments compared to the control with different response times and transcriptional levels; the response to combined exposure occurred earlier than to single exposure. Among the experimental groups, MvLYZ expression and MvSOD expression peaked in the combined treatment on day 3, whereas MvMT expression peaked in heavy metals treatment on day 5. Furthermore, interactive effects of heavy metals and Vibrio on transcriptional response changed over the exposure time. Therefore, transcriptional regulation of the three genes under combined exposure was more complex than under single exposure.

Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs.

BACKGROUND: Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily. RESULTS: Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a ß-hairpin pointed towards the substrate binding cleft. The invariant ß-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism. CONCLUSIONS: The seven families and subfamilies of the lysozyme superfamily all have in common a ß-hairpin structure which displays a family-specific sequence motif. These GH ß-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily.

Thermodynamics and structure of a salmon cold active goose-type lysozyme.

Atlantic salmon goose-type lysozyme (SalG) was previously shown to display features of cold-adaptation as well as renaturation following heat treatment. In this study differential scanning calorimetry (DSC) was carried out to investigate unfolding and potential refolding, while X-ray crystallography was used to study structural factors contributing to the temperature-related characteristics. The recombinant SalG has a melting temperature (T(m)) of 36.8 degrees C under thermal denaturation conditions and regains activity after returning to permissive (low) temperature. Furthermore, refolding is dramatically reduced in solutions with high SalG concentrations, coupled with significant protein precipitation. The structural features of SalG closely resemble those of other g-type lysozymes. However, the N-terminal region of SalG is less anchored to the rest of the molecule due to the absence of disulphide bonds, thus, contributing significantly to the low T(m) of SalG. The absence of disulphide bonds and the distribution of salt bridges may at the same time ease refolding leading to renaturation.

Accessory active site residues of Streptomyces sp. N174 chitosanase: variations on a common theme in the lysozyme superfamily.

The chitosanase from Streptomyces sp. N174 (CsnN174) is an inverting glycoside hydrolase belonging to family 46. Previous studies identified Asp40 as the general base residue. Mutation of Asp40 into glycine revealed an unexpectedly high residual activity. D40G mutation did not affect the stereochemical mechanism of catalysis or the mode of interaction with substrate. To explain the D40G residual activity, putative accessory catalytic residues were examined. Mutation of Glu36 was highly deleterious in a D40G background. Possibly, the D40G mutation reconfigured the catalytic center in a way that allowed Glu36 to be positioned favorably to perform catalysis. Thr45 was also found to be essential. Thr45 is thought to orientate the nucleophilic water molecule in a position to attack the glycosidic link. The finding that expression of heterologous CsnN174 in Escherichia coli protects cells against the antimicrobial effect of chitosan, allowed the selection of active chitosanase variants after saturation mutagenesis. Thr45 could be replaced only by serine, indicating the importance of the hydroxyl group. The newly identified accessory catalytic residues, Glu36 and Thr45 are located on a three-strand beta sheet highly conserved in GH19, 22, 23, 24 and 46, all members of the 'lysozyme superfamily'. Structural comparisons reveal that each family has its catalytic residues located among a small number of critical positions in this beta sheet. The position of Glu36 in CsnN174 is equivalent to general base residue in GH19 chitinases, whereas Thr45 is located similarly to the catalytic residue Asp52 of GH22 lysozyme. These examples reinforce the evolutionary link among these five GH families.

Molecular characterization, phylogeny, and expression of c-type and g-type lysozymes in brill (Scophthalmus rhombus).

Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill (Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25- and 23-fold at 12 and 24h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion.

Structural evidence for lack of inhibition of fish goose-type lysozymes by a bacterial inhibitor of lysozyme.

It is known that bacteria contain inhibitors of lysozyme activity. The recently discovered Escherichia coli inhibitor of vertebrate lysozyme (Ivy) and its potential interactions with several goose-type (g-type) lysozymes from fish were studied using functional enzyme assays, comparative homology modelling, protein-protein docking, and molecular dynamics simulations. Enzyme assays carried out on salmon g-type lysozyme revealed a lack of inhibition by Ivy. Detailed analysis of the complexes formed between Ivy and both hen egg white lysozyme (HEWL) and goose egg white lysozyme (GEWL) suggests that electrostatic interactions make a dominant contribution to inhibition. Comparison of three dimensional models of aquatic g-type lysozymes revealed important insertions in the beta domain, and specific sequence substitutions yielding altered electrostatic surface properties and surface curvature at the protein-protein interface. Thus, based on structural homology models, we propose that Ivy is not effective against any of the known fish g-type lysozymes. Docking studies suggest a weaker binding mode between Ivy and GEWL compared to that with HEWL, and our models explain the mechanistic necessity for conservation of a set of residues in g-type lysozymes as a prerequisite for inhibition by Ivy.

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts.

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.

Characterization, organization and expression of AmphiLysC, an acidic c-type lysozyme gene in amphioxus Branchiostoma belcheri tsingtauense.

The study on lysozymes remains open in amphioxus, a cephalochordate. Here we show the existence of c-type lysozyme gene (AmphiLysC) in amphioxus, first such data in the basal chordates including urochordate and cephalochordate. This is in contrast to the absence of c-type lysozyme genes in urochordate. It is found that there exist two copies of c-type lysozyme genes in amphioxus genome, and their gene organization is similar to vertebrate c-type lysozyme genes with respect to the number and the size of both exons and introns. AmphiLysC possesses main features characteristic of the digestive c-type lysozyme such as lower number of basic amino acids (low pI values) and pH-optimum in acidic range. Moreover, AmphiLysC is predominantly expressed in the gut. These indicate that AmphiLysC is possibly a digestive c-type enzyme. However, the ubiquitous expression of AmphiLysC in non-digestive tissues such as ovaries, testes, notochord, gill and muscle suggests that it may also play a non-digestive role like antibacterial activity. It is highly likely that AmphiLysC is an enzyme with a combined function of digestion and bacteriolysis.

Using 3D Hidden Markov Models that explicitly represent spatial coordinates to model and compare protein structures.

BACKGROUND: Hidden Markov Models (HMMs) have proven very useful in computational biology for such applications as sequence pattern matching, gene-finding, and structure prediction. Thus far, however, they have been confined to representing 1D sequence (or the aspects of structure that could be represented by character strings). RESULTS: We develop an HMM formalism that explicitly uses 3D coordinates in its match states. The match states are modeled by 3D Gaussian distributions centered on the mean coordinate position of each alpha carbon in a large structural alignment. The transition probabilities depend on the spread of the neighboring match states and on the number of gaps found in the structural alignment. We also develop methods for aligning query structures against 3D HMMs and scoring the result probabilistically. For 1D HMMs these tasks are accomplished by the Viterbi and forward algorithms. However, these will not work in unmodified form for the 3D problem, due to non-local quality of structural alignment, so we develop extensions of these algorithms for the 3D case. Several applications of 3D HMMs for protein structure classification are reported. A good separation of scores for different fold families suggests that the described construct is quite useful for protein structure analysis. CONCLUSION: We have created a rigorous 3D HMM representation for protein structures and implemented a complete set of routines for building 3D HMMs in C and Perl. The code is freely available from http://www.molmovdb.org/geometry/3dHMM, and at this site we also have a simple prototype server to demonstrate the features of the described approach.

The primary structure of a novel goose-type lysozyme from rhea egg white.

G-type lysozyme is a hydrolytic enzyme sharing a similar tertiary structure with plant chitinase. To discover the relation of function and structure, we analyzed the primary structure of new G-type lysozyme. The complete 185 amino acid residues of lysozyme from rhea egg white were sequenced using the peptides hydrolyzed by trypsin, V8 protease, and cyanogen bromide. Rhea lysozyme had sequence similarity to ostrich, cassowary, goose, and black swan, with 93%, 90%, 83%, and 82%, respectively. The six substituted positions were newly found at positions 3 (Asn), 9 (Ser), 43 (Arg), 114 (Ile), 127 (Met), and 129 (Arg) when compared with ostrich, cassowary, goose, and black swan lysozymes. The amino acid substitutions of rhea lysozyme at subsite B were the same as ostrich and cassowary lysozymes (Ser122 and Met123). This study was also constructed in a phylogenetic tree of G-type lysozyme that can be classified into at least three groups of this enzyme, namely, group 1; rhea, ostrich, and cassowary, group 2; goose, black swan, and chicken, and group 3; Japanese flounder. The amino acid sequences in assembled three alpha-helices found in this enzyme group (Thammasirirak, S., Torikata, T., Takami, K., Murata, K., and Araki, T., Biosci. Biotechnol. Biochem., 66, 147-156 (2002)) were also highly conserved, so that they were considered to be important for the formation of the hydrophobic core structure of the catalytic site and for maintaining a similar three-dimensional structure in this enzyme group.

Virtual enantiomers as the solution of optical activity's deterministic offset problem.

Deterministic offsets have remained one of optical activity's most intractable problems. To the extent that the mechanisms by which they are produced do not depend on the chiroptical properties of the sample, they can be eliminated by the subtraction of measurements done on both enantiomers. We show that it is possible to create, by purely optical means, by the sole use of half-wave retarders, the optical antipode of a chiral molecule, to measure the chiroptical properties of the molecule and of its optically generated antipode, and to recover, by subtracting the measurements, the offset-free data of the enantiomer which is physically present. We moreover show that it is possible to do the measurements in a way that eliminates offsets that might occur through the influence of the differing chiroptical properties of the two antipodes. The procedure can be repeated, and by doing so, an almost arbitrarily high precision can be reached. The method is demonstrated by offset-free Raman optical activity back-scattering spectra measured in the so-called scattered circular polarization mode, one of optical activity's so far largely unsolved measurement problems. Such measurements can now be done with 2 mg of substance, in throw-away capillary cells, and on compounds sealed in cylindrical vials.