Mannitol Dehydrogenase Research Articles

Generation and analysis of expressed sequence tags from the mangrove plant, Acanthus ebracteatus Vahl

Salinity is a major abiotic stress that greatly affects plant growth and crop production. Sodium ions in saline soil are toxic to plants because of their adverse effects on potassium nutrition, cytosolic enzyme activities, photosynthesis, and metabolism. It is important to identify genes involved in salinity tolerance from mangrove plants that survive under saline conditions. In this study, a total of 864 randomly selected cDNA clones were isolated and sequenced from the primary cDNA library of Acanthus ebracteatus. Among the 521 readable sequences, 138 of them were assembled into 43 contigs, whereas 383 were singletons. Sequence analyses demonstrated that 349 of these expressed sequence tags showed significant homology to functional proteins, of which 18% are particularly interesting as they correspond to genes involved in stress response. Some of these clones, including putative mannitol dehydrogenase, plastidic aldolase, secretory peroxidase, ascorbate peroxidase, and vacuolar H+-ATPase, may be related to osmotic homeostasis, ionic homeostasis, and detoxification.

NADP-dependent Mannitol Dehydrogenase, a Major Allergen of Cladosporium herbarum

Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of approximately 57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and d-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a lambda-ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.

Overexpression of Saccharomyces cerevisiae mannitol dehydrogenase gene (YEL070w) in glycerol synthesis-deficient S. Cerevisiae mutant

Saccharomyces cerevisiae ORF YEL070w encodes mannitol dehydrogenase (MDH), as confirmed by the present results. Six highly homologous peptide motifs were identified in the amino acid sequence of the YEL070w product by comparison with those of microbial MDH proteins. In particular, a typical coenzyme-binding site and an active site that are specific to enzymes participating in the dehydrogenation of alcohols groups were identified in these homologous peptide motifs. S. cerevisiae cells overexpressing YEL070w exhibited MDH activity showing high substrate specificity for mannitol and a coenzyme requirement of NAD + in the oxidation reaction. The salt-sensitive phenotype of S. cerevisiae glycerol-synthesis-deficient mutant ( ΔGPD1 and ΔGPD2) was complemented and tolerance to several stressors (i.e., heat and sugar) was observed with YEL070w expression.

Determination of sugarcane deterioration at the factory: Development of a rapid, easy and inexpensive enzymatic method to measure mannitol

Mannitol, formed mainly by Leuconostoc mesenteroides bacteria, is a very sensitive indicator of sugarcane deterioration that directly affects processing and can predict problems from dextran and levan polysaccharides. An enzymatic method has been developed to measure mannitol in juice pressed from consignments of sugarcane delivered to the factory. This screening tool will allow factory staff to rapidly know which consignments of cane will affect processing negatively or reject consignments that will cause unacceptable processing problems. Mannitol is directly measured on a spectrophotometer using mannitol dehydrogenase as the enzyme catalyst. The stability of the reagents, limited cane juice preparation and linearity are described. This method is accurate, comparing favorably with an ion chromatography method, and can be easily performed using existing equipment in sugarcane factories. The coefficient of variation (CV) for cane juices ranged from 1.73% to 5.13% with the highest CVs occurring for low mannitol concentrations in undeteriorated cane. Mannitol can be measured after ∼7 min at room temperature and within 4 min if a 40 °C waterbath is used. The method is highly specific for mannitol and was not affected by the presence of sucrose, glucose, fructose, or dextran. The current cost is only ∼60 US cents per analysis. Further studies on the viability of the method at the factory, and as a screening tool for breeding programs for cane freeze tolerance, are being undertaken.

Glass-fiber disks provide suitable medium to study polyol production and gene expression in Eurotium rubrum

Eurotium species often dominate the fungal population in stored grain and are responsible for spoilage. In this study we tested the usefulness of glass fiber disks to aid the analysis of growth, polyol content and gene expression in E. rubrum in response to various water activities. Growth measurements based on ergosterol content and conidial production indicated that E. rubrum grew as well at 0.86 aw as 0.98 aw. The rate of growth was considerably reduced at 0.83 aw and 0.78 aw. In contrast, under our conditions, Aspergillus flavus and A. nidulans were able to grow only in the highest water activity (0.98 aw). Mannitol was the predominant polyol in all three fungal species grown at 0.98 aw. When E. rubrum was grown at 0.86 aw or lower, glycerol comprised greater than 90% of the total polyols. After a shift from 0.86 aw to 0.98 aw, mannitol levels in E. rubrum increased to 89% of the total polyols within 24 h. Of six genes whose expression was measured by quantitative real-time PCR, three were affected by water activity. Expression of putative hydrophobin and mannitol dehydrogenase genes was higher at 0.98 aw than at 0.86 aw. A putative triacylglycerol lipase gene was expressed at higher levels in 0.86 aw. The results of this study indicate that the disk method is suitable to study the effects of water activity on growth, polyol biosynthesis and gene expression in E. rubrum. The results also indicate the potential competitiveness of E. rubrum over A. flavus and A. nidulans in low water environments associated with stored grain.

Glass-fiber disks provide suitable medium to study polyol production and gene expression in Eurotium rubrum

Eurotium species often dominate the fungal population in stored grain and are responsible for spoilage. In this study we tested the usefulness of glass fiber disks to aid the analysis of growth, polyol content and gene expression in E. rubrum in response to various water activities. Growth measurements based on ergosterol content and conidial production indicated that E. rubrum grew as well at 0.86 aw as 0.98 aw. The rate of growth was considerably reduced at 0.83 aw and 0.78 aw. In contrast, under our conditions, Aspergillus flavus and A. nidulans were able to grow only in the highest water activity (0.98 aw). Mannitol was the predominant polyol in all three fungal species grown at 0.98 aw. When E. rubrum was grown at 0.86 aw or lower, glycerol comprised greater than 90% of the total polyols. After a shift from 0.86 aw to 0.98 aw, mannitol levels in E. rubrum increased to 89% of the total polyols within 24 h. Of six genes whose expression was measured by quantitative real-time PCR, three were affected by water activity. Expression of putative hydrophobin and mannitol dehydrogenase genes was higher at 0.98 aw than at 0.86 aw. A putative triacylglycerol lipase gene was expressed at higher levels in 0.86 aw.. The results of this study indicate that the disk method is suitable to study the effects of water activity on growth, polyol biosynthesis and gene expression in E. rubrum. The results also indicate the potential competitiveness of E. rubrum over A. flavus and A. nidulans in low water environments associated with stored grain.

Gluconobacter oxydans NAD-dependent, D-fructose reducing, polyol dehydrogenases activity: screening, medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l(-1), 7.2 g peptone l(-1) and 1.8 g yeast extract l(-1). Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5-10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2.

Enzymatic production of D-mannitol with the Leuconostoc pseudomesenteroides mannitol dehydrogenase coupled to a coenzyme regeneration system

The mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 was studied for its characteristics and capacity for enzymatic mannitol synthesis, coupled to a coenzyme regeneration system. The producing strain was cultivated for 6.5 hours on a medium containing both D-glucose and D-fructose as carbon sources. Efficient release of the intracellular enzyme was obtained by cell treatment with toluene, Triton permeabilisation, or sonication. The crude enzyme extract showed optimal D-fructose reduction at pH 5.5 and 50°C, whereas D-mannitol oxidation was best at pH 8.0 and 40°C. The polyol dehydrogenase was stable up to 40°C and within the pH-range of 6.5–8.5. The crude enzyme extract displayed high substrate specificity. With NADPH, enzyme activity was only 30% of that observed with NADH. The molecular weight of the native enzyme was found to be 136 kDa. The mannitol dehydrogenase preparation was tested for its capacity to produce D-mannitol from D-fructose. Since the mannitol dehydrogenase is NADH-dependent, efficient coenzyme regeneration was needed. Regeneration of NADH from NAD+ was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2. The best initial reaction rates were obtained with 0.5 mM NAD+ and 100 mM substrate.

Possible roles for mannitol and mannitol dehydrogenase in the biotrophic plant pathogen Uromyces fabae.

Levels of the C6-polyol mannitol were observed to rise dramatically in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant Vicia faba. Mannitol was found in millimolar concentrations in extracts and apoplastic fluids of infected leaves and also in extracts of spores. We suggest that this polyol might have at least a dual function: first, as a carbohydrate storage compound, and second, as a scavenger of reactive oxygen species. Mannitol accumulation is accompanied by high expression of a mannitol dehydrogenase (MAD1) in haustoria. While MAD1 transcripts were detected in haustoria only, immunolocalization studies show that the gene product is also present in spores. Kinetic and thermodynamic analyses of the MAD1p catalyzed reactions indicate that the enzyme might be responsible for the production of mannitol in haustoria and for the utilization of mannitol in spores. Since V. faba is normally unable to synthesize or utilize polyols, the multipurpose usage of mannitol seems an ideal strategy for the fungal pathogen.

Cloning, Expression, Purification, and Analysis of Mannitol Dehydrogenase Gene<I> mtlK</I> from <I>Lactobacillus brevis</I>

Cloning, Expression, Purification, and Analysis of Mannitol Dehydrogenase Gene<I> mtlK</I> from <I>Lactobacillus brevis</I>

The mannitol cycle in Pleurotus ostreatus (Florida)

In mushroom, presence of the mannitol cycle has not been reported so far although the polyol is supposed to be generated by the reduction of fructose by mannitol dehydrogenase. This study submits evidence for the presence of the mannitol cycle in Pleurotus ostreatus. The key enzyme of the cycle, mannitol-1-phosphate dehydrogenase (M1PDH), was present appreciably in all the developmental stages of the mushroom. However, the enzyme level dropped significantly at the onset of sporulation. The presence of M1DPH was confirmed by isozyme analysis and RT-PCR mediated amplification of a ∼400 bp DNA fragment.

The mannitol cycle in Pleurotus ostreatus (Florida)

In mushroom, presence of the mannitol cycle has not been reported so far although the polyol is supposed to be generated by the reduction of fructose by mannitol dehydrogenase. This study submits evidence for the presence of the mannitol cycle in Pleurotus ostreatus. The key enzyme of the cycle, mannitol-1-phosphate dehydrogenase (M1PDH), was present appreciably in all the developmental stages of the mushroom. However, the enzyme level dropped significantly at the onset of sporulation. The presence of M1DPH was confirmed by isozyme analysis and RT-PCR mediated amplification of a ∼400 bp DNA fragment.

Polymerase chain reaction (PCR) for the detection of celery ( Apium graveolens ) in food

A method based upon polymerase chain reaction (PCR) for the detection of celery (Apium graveolens) in food was developed. The method involves DNA isolation by chaotropic or non-chaotropic solid-phase extraction and PCR with primers oriented to the sequence of the nuclear gene encoding mannitol dehydrogenase. The PCR method was shown to be specific for celery, producing a 279 bp fragment with four celery varieties and negative results with other species commonly present together with celery in food products (16 samples). The detection limit of PCR was 490–1530 pg DNA, which corresponds to 102 genome copies. When evaluated with model samples of celery in meat pâtes, a detection limit of 0.1% (w/w) was determined. When used to analyse food products from the market (dried vegetable seasonings, dehydrated bouillons), all four products declared to contain celery were correctly identified as positive and all three products in which celery was not declared were identified as negative.

Alternaria alternata mannitol dehydrogenase, a fungal allergen

Rationale Mannitol dehydrogenase (MtDH) has previously been shown to be the major allergen of Cladosporium herbarum. Since cross-reactivity has been demonstrated for several fungal allergens we have cloned the homolog protein of Alternaria alternata and tested it in respect to its IgE-reactivity. Methods A. alternata MtDH was cloned by screening of an in vivo excised A. alternata cDNA library (pBluescript SK) using the C. herbarum homolog as a hybridization probe. In order to obtain a recombinant non fusion and a 6xHIS fusion protein, the coding sequence was subcloned into the pMW172 and the pHIS-parallel2 vector, respectively. Expression of the recombinant proteins was performed in the E. coli strain Bl21(DE3). The recombinant non fusion protein was purified by ammonium sulphate precipitation, hydrophobic interaction chromatography, anion exchange chromatography and Cibacron Blue F3G-A affinity chromatography. IgE immunoblots were performed utilizing the 6xHIS fusion protein purified by Ni 2+ complexed Chelating Sepharose affinity chromatography. Results A. alternata MtDH has been cloned. The coding sequence of the full-length cDNA clone comprises 798 bp and 266 amino acids, respectively. Protein sequence analysis revealed an identity of 74% and a homology of 80,5% between the MtDHs of A. alternata and C. herbarum. The purified recombinant protein was enzymatically active as shown by spectrophotometrical analysis. In IgE-immunoblots 9 out of 28 (34%) A. alternata patients reacted with the 6xHIS-MtDH. Conclusion Mannitol dehydrogenase was shown to be a new important allergen of A. alternata.

Indicators of freeze-damaged sugarcane varieties which can predict processing problems

In raw sugar manufacture, the quality of the sugarcane supply to the factory plays the most important role in production costs. Sugarcane can be very susceptible to damage by freezes, and freeze-deteriorated sugarcane can cause problems in processing which sometimes leads to a factory shut-down. A reliable indicator of whether a certain shipment of sugarcane can be processed economically is still needed. This study was undertaken during the 2002/2003 harvest season to measure the cold-tolerance performance of eight commercial sugarcane varieties, and to establish indicators which can predict processing quality and problems. Varieties studied included CP 70-321, CP 79-318, LHo 83-153, LCP 85-384, HoCP 85-845, HoCP 91-555, HoCP 96-540 a newly released Louisiana variety, and TucCP 77-42, developed in Argentina. Freezing temperatures occurred between January 18–19 and 24–25, 2003. The minimum field temperature recorded was −5.1 °C on January 25. Freezing conditions prevailed for 10–14 h during each freeze. Serious tissue damage occurred within the stalks of all varieties following the January 24–25 freeze, including freeze cracks. Samples were taken one day before the first freeze (pre-freeze control) on January 17, and subsequently again 12, 18 and 26 days post-freeze. Marked changes for most indicators of freeze-deterioration, for all varieties, were observed, particularly 18 and 26 days post-freeze, and viscosity increased and percentage pol-filterability decreased on freeze-deterioration. Variety TucCP 77-42 had significantly ( P<0.05) the worst cold-tolerance, even after 12 days. Strong polynomial trends or quadratic fits existed between ASI-II dextran and titratable acidity ( r 2=0.916) and pH ( r 2=−0.883). Deterioration effects became greater than varietal effects at threshold levels of ∼2500 and ∼2800 ppm/Brix dextran for titratable acidity and pH, respectively. Titratable acidity and pH may, therefore, only be useful in predicting problems caused by severe dextran concentrations. Reactions of Leuconostoc mesenteroides of importance to sugarcane deterioration, including the production of dextran, levan, and alternan polysaccharides are fully described. Mannitol, produced by mannitol dehydrogenase from Leuconostoc, was a slightly better predictor of viscosity ( r 2=0.838) than both ASI-II ( r 2=0.802) and Haze ( r 2=0.814) dextran, because it can indicate all Leuconostoc polysaccharides. In comparison, ethanol ( r 2=0.676), leucrose ( r 2=0.711), and pH ( r 2=−0.711) were only moderately correlated with viscosity. Mannitol was also better than dextran at predicting percentage pol-filterability, although substances present in the undeteriorated cane juice and sucrose interfere with this processing parameter. Overall, mannitol was the best predictor of sugarcane deterioration which contributes to sucrose losses, dextran, viscosity, and to a lesser extent, filterability problems. Model mannitol degradation reactions, simulating industrial conditions, showed that no mannitol degradation occurred, even after 1 h at 99 °C, pH 5.4 and high or low Brix, which further supports the use of mannitol to indirectly measure dextran and/or deterioration in the factory.

Evidences of high carbon catabolic enzyme activities during sporulation of Pleurotus ostreatus (Florida).

Measurements of the specific activities of the representative enzymes of different pathways linked to carbohydrate metabolism indicate that glycolysis and TCA cycles are the major route of carbohydrate catabolism in the sporulating phase of fruiting body development in Pleurotus ostreatus. Enzymes of the pentose phosphate pathway always showed lower specific activities as compared to those of the enzymes of the glycolytic pathway. The activity of NADP linked glutamate dehydrogenase which is known to be an anabolic enzyme decreased drastically in sporulating fruiting bodies and in spore containing gill tissue (spore bearing structure). Mannitol dehydrogenase activity declined significantly in the sporulating phase of P. ostreatus. The high rate of metabolism during sporulation was further supported by a lower rate of gluconeogenesis at this stage. Concentrations of all the major sugars of the fruiting body (mannitol, glucose and trehalose) decreased in the mature fruiting body and gill tissue. This indicated high catabolic activities at this stage of development.

Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation.

A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle. First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant E. coli strain. By contrast whole cells of the strain were unable to produce D-mannitol from D-fructose. To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed. FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form D-mannitol from D-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up D-fructose, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM D-mannitol in 17 h. Due to equimolar formation of sodium hydroxide during NAD(+)-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h. Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h. The yield Y(D-mannitol/D-fructose) was 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for d-mannitol formation.

Purification and characterization of a novel mannitol dehydrogenase from a newly isolated strain of Candida magnoliae.

Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37 degrees C and 10.0 and 40 degrees C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k(cat) = 823 s(-1), K(m) = 28.0 mM, and k(cat)/K(m) = 29.4 mM(-1) s(-1)) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.

Purification and characterisation of mannitol dehydrogenase from Lactobacillus sanfranciscensis

Mannitol dehydrogenase (MDH) was purified and characterised from Lactobacillus sanfranciscensis. Two peptide fragments of MDH were N-terminally sequenced for the first time in the genus Lactobacillus. The purified enzyme had an apparent molecular mass of 44 kDa and catalysed both the reduction of fructose to mannitol and the oxidation of mannitol to fructose. The K m value for the reduction reaction was 24 mM fructose and that for the oxidation 78 mM mannitol. The optimum temperature was 35°C, the pH optima for the reduction or oxidation were 5.8 and 8, respectively.

Genetic Diversity Among Iranian Isolates of Rhizoctonia solani Kühn Anastomosis Group1 Subgroups Based on Isozyme Analysis and Total Soluble Protein Pattern

Abstract Rhizoctonia solani is a destructive fungal pathogen with a wide host range. The R. solani complex species includes several divergent groups delimited by affinities for hyphal anastomosis. In this study, genetic variation among 20 isolates of R. solani anastomosis group 1 (AG1) subgroups (AG1‐IA and AG1‐IB) collected from Mâzandaran province, Iran, and standard isolates of these subgroups, was determined by isozyme analysis and total soluble protein profile. Mycelial protein pattern and isozyme analysis were studied using denaturing and non‐denaturing polyacrylamide gel electrophoresis, respectively. A total of 15 enzyme systems were tested, among which six enzymes including esterase, alkaline phosphatase, superoxide dismutase, octanol dehydrogenase, lactate dehydrogenase and mannitol dehydrogenase generated distinct and reproducible results. The soluble protein patterns were similar among the R. solani isolates examined; however, minor differences in banding pattern were observed between the two subgroups. In isozyme analysis, a total of 64 electrophoretic phenotypes were detected for all six enzymes used. Based on cluster analysis and similarity matrix, the fungal isolates were divided into two genetically distinct groups of I and II consistent with the previously reported AG1‐IA and AG1‐IB subgroups in AG1. Group I represented all isolates belonging to AG1‐IA subgroup, whereas group II represented all isolates belonging to AG1‐IB subgroup. Results from isozyme analysis suggest that the subgrouping concept within AGs is genetically based.