Soluble Isozymes Research Articles

MUSCLE PROTEIN AND LIVER ESTERASES BANDING PATTERNS AS BIOCHEMICAL MARKERS TO DETERMINE GENETIC DIVERSITY IN EGYPTIAN POPULATIONS OF Tilapia SPECIES

Genetic diversity across Tilapia species is important key fordevelopment aquaculture strains, protection of endangered populations and biogeographical inferences. Total soluble protein and esterases isozymes were extracted from flesh (muscles) and liver of all individuals from the three populations (Ryad, Bahr El-Baqar and Motobs) from each species under study to estimate the genetic diversity. With comparing the obtained bands from the three Oreochromis niloticus, it was found that 13 bands out of the 20 were common bands among the three populations. The three T. zilli populations exhibited 8 bands out of the 21 were common bands among those three populations. The three O. aurea populations exhibited 10 bands were common between three populations, while the rest of bands appeared in some of population and disappeared in the other. Ryad and Motobs individuals of O. niloticus showed four isozymes. Meanwhile, Bahr El-Baqar exhibited only three. Ryad and Bahr El-Baqar individuals of T. zilli showed three isozymes. Meanwhile, Motobs exhibited four ones. Maximum two isozymes were detected in population of O. aurea. Band 1 was dark in Ryad individuals and ranged from very faint, faint and dark in Bahr El-Baqar and Motobs. The phylogenetic relationship within the studied populations of the three locations concerning O. niloticus, T. zilli and O. aurea was conducted and different variations were detected. Population from Ryad was highly differentiated than other populations.

Structural differences between soluble and membrane bound cytochrome P450s

The superfamily of cytochrome P450s forms a large class of heme monooxygenases with more than 13,000 enzymes represented in organisms from all biological kingdoms. Despite impressive variability in sizes, sequences, location, and function, all cytochrome P450s from various organisms have very similar tertiary structures within the same fold. Here we show that systematic comparison of all available X-ray structures of cytochrome P450s reveals the presence of two distinct structural classes of cytochrome P450s. For all membrane bound enzymes, except the CYP51 family, the beta-domain and the A-propionate heme side chain are shifted towards the proximal side of the heme plane, which may result in an increase of the volume of the substrate binding pocket and an opening of a potential channel for the substrate access and/or product escape directly into the membrane. This structural feature is also observed in several soluble cytochrome P450s, such as CYP108, CYP151, and CYP158A2, which catalyze transformations of bulky substrates. Alternatively, both beta-domains and the A-propionate side chains in the soluble isozymes extend towards the distal site of the heme. This difference between the structures of soluble and membrane bound cytochrome P450s can be rationalized through the presence of several amino acid inserts in the latter class which are involved in direct interactions with the membrane, namely the F′- and G′-helices. Molecular dynamics using the most abundant human cytochrome P450, CYP3A4, incorporated into a model POPC bilayer reveals the facile conservation of a substrate access channel, directed into the membrane between the B–C loop and the beta domain, and the closure of the peripheral substrate access channel directed through the B–C loop. This is in contrast to the case when the same simulation is run in buffer, where no such channel closing occurs. Taken together, these results reveal a key structural difference between membrane bound and soluble cytochrome P450s with important functional implications induced by the lipid bilayer.

Construction of Streptomyces jumonjinensis isopenicillin N synthase double mutants, Ser119Ala/Glu120Gly and Thr308Ala/Thr309Val, overcomes insoluble expression in Escherichia coli

Of the isopenicillin N synthase isozymes, Streptomyces jumonjinensis isopenicillin N synthase (sjIPNS) is the only insoluble isozyme produced in Escherichia coli during heterologous expression. Hence, this study aims to optimise the soluble expression of sjIPNS in E. coli. By lowering the cultivation temperature from 37 to 25°C, previous studies have shown that the solubility of IPNS from S. clavuligerus (scIPNS), S. lipmanii (slIPNS) and Nocardia lactamdurans (nIPNS) in E. coli was much improved but the same could not be achieved for sjIPNS in this study. To decipher the uniqueness of the elusively insoluble sjIPNS, its amino acid sequence was compared to three other soluble isozymes, scIPNS, slIPNS and nIPNS. The computational analyses revealed two positions with adjacent sites at 119/120 and 308/309, where by sjIPNS differs significantly in terms of amino acid identities and properties. Site-directed mutagenesis was then used to alter these sites to investigate their influence on solubility. The two double mutants constructed, Ser119Ala/Glu120Gly and Thr308Ala/Thr309Val sjIPNS, were found to be soluble at 28 and 25°C, with optimal production up to 25.3 and 23.0%, respectively, of the total soluble proteins. Furthermore, both mutants were found to be catalytically active. The results of this study constitute the first report on the use of site-directed mutagenesis to successfully transform the solubility of sjIPNS to closely resemble that of other soluble bacterial IPNS isozymes.

Carbon isotopic fractionation in lipids from methanotrophic bacteria II: the effects of physiology and environmental parameters on the biosynthesis and isotopic signatures of biomarkers

Controls on the carbon isotopic signatures of methanotroph biomarkers have been further explored using cultured organisms. Growth under conditions which select for the membrane-bound particulate form of the methane monooxygenase enzyme (pMMO) leads to a significantly higher isotopic fractionation than does growth based on the soluble isozyme in both RuMP and serine pathway methanotrophs; in an RuMP type the Δδ 13C biomass equaled −23.9‰ for pMMO and −12.6‰ for sMMO. The distribution of biomarker lipids does not appear to be significantly affected by the dominance of one or the other MMO type and their isotopic compositions generally track those of the parent biomass. The 13C fractionation behaviour of serine pathway methanotrophs is very complex, reflecting the assimilation of both methane and carbon dioxide and concomitant dissimilation of methane-derived carbon. A limitation in CH 4 availability leads to the production of biomass which is 13C-enriched with respect to both carbon substrates and this occurs irrespective of MMO type. This startling result indicates that there must be an additional fractionation step downstream from the MMO reaction which leads to incorporation of 13C-enriched carbon at the expense of dissimilation of 13C-depleted CO 2. In these organisms, polyisoprenoid lipids are 13C-enriched compared to polymethylenic lipid which is the reverse of that found in the RuMP types. Serine cycle hopanoids, for example, can vary anywhere from 12‰ depleted to 10‰ enriched with respect to the CH 4 substrate depending on its concentration. Decrease in growth temperature caused an overall increase in isotopic fractionation. In the total biomass, this effect tended to be masked by physiological factors associated with the type of organism and variation in the bulk composition. The effect was, however, clearly evident when monitoring the 13C signature of total lipid and individual biomarkers. Our results demonstrate that extreme carbon isotopic depletion in field samples and fossil biomarker lipids can be indicative of methanotrophy but the converse is not always true. For example, the hopanoids of a serine cycle methanotroph may be isotopically enriched by more than 10‰ compared to the substrate methane when the latter is limiting. In other words, hopanoids from some methanotrophs such as M. trichosporium would be indistinguishable from those of cyanobacteria or heterotrophic bacteria on the basis of either chemical structure or carbon isotopic signature.

Hexokinases of tobacco leaves: influence of plant age on particulate and soluble isozyme composition

Changes in hexokinase particulate and soluble isozyme composition and activities in leaves of 65- and 115-d-old tobacco plants were determined by ion exchange chromatography on DEAE cellulose. During plant ageing, the activities of glucose and of fructose phosphorylating isozymes of particulate hexokinase decreased to 9.9 and 9.2 % of initial value, respectively. The activity of soluble hexokinase decreased to a lesser extent: that of glucose phosphorylating isozyme to 49.8 % and of fructose phosphorylating isozyme to 37.8 %. The activity of soluble fructokinase isozyme dropped to 34.8 %. Thus also the ratio of particulate and soluble isozymes was dependent on the age of leaf tissue.

Classification of Pythium ‘group F’ based on mycelial protein and isozyme patterns

Taxonomic characteristics were compared among 10 isolates ofPythium ‘group F’ in tems of the electrophoretic patterns of their mycelial proteins and isozymes. These isolates were obtained from water of three ponds in different seasons and have an identical morphology of zoosporangia. Attempts to cross the isolates with each other themselves and with other isolates from the same group failed.Pythium ‘group F’ is the most dominant of the pythia in the aquatic ecosystem and is difficult to identify because of the lack of sexual reproductive structures. Isozyme analysis proved useful in this respect. Comparisons of banding patterns of total soluble proteins and isozymes revealed five subgroups inPythium ‘group F’. Two isolates obtained from water of different ponds in different seasons showed the same protein and isozyme patterns. Our findings indicate that the use of total soluble protein and isozyme patterns for determining the variation withinPythium ‘group F’ could become a valuable adjunct to the morphological and physiological criteria.

Genomic Organization and Localization of Gene for Human Carbonic Anhydrase IV to Chromosome 17q

Carbonic anhydrase IV (CA IV) is a glycosylphosphatidylinositol-anchored membrane isozyme expressed on the luminal surfaces of pulmonary (and certain other) capillaries and on the luminal surface of proximal renal tubules. It is of interest for its functional importance in CO 2 and HCO 3 transport, its ancient evolutionary status Among CA isozymes, and its possible role in inherited renal abnormalities of HCO 3 transport. To determine the localization of the CA IV gene and define its genomic structure, we isolated and characterized a full-length genomic clone. The 9.5-kb gene contains eight exons and seven introns. The first exon (exon 1a) encodes the signal sequence. Exons 1b through 7 encode the remaining coding sequence. Exon 7 encodes the C terminus of the enzyme precursor, the C terminus of the mature protein and also contains the 120-bp sequence corresponding to the 3′ untranslated region of the cDNA. The positions of introns 3, 4, 5, and 6 are identical with the corresponding positions of introns in the genes for the soluble CA isozymes (CA I, II, III, and VII). However, intron 1a is not found in these genes, and the positions of introns lb and 2 in CA IV differ from the positions of the corresponding introns in genes for the soluble isozymes. The 5′ upstream region of the gene (-500 to - 1) is GC rich and contains 30 CpG dinucleotides. A TATA box sequence and a potential Sp1 binding site are identified upstream of the first exon. By using PCR on DNA from human/rodent somatic cell hybrids, the CA IV gene was assigned to chromosome 17. In situ hybridization confirmed this chromosome assignment and localized the gene to 17q23. CA IV is the first CA assigned to chromosome 17.

Localization of carbonic anhydrase IV in a specific capillary bed of the human eye.

Carbonic anhydrase (CA) activity plays an important role in controlling aqueous humor production in the eye and in regulating intraocular pressure. Prior studies identified the soluble isozymes CA II and CA I in the human eye and also suggested a distinct membrane-associated CA. We used an antibody to CA IV, the membrane-anchored isozyme from human lung, to study CA IV in eye tissues and to compare its distribution with that of CA II. We found intense immunostaining for CA IV associated with endothelial cells of one specific uveal capillary bed, the choriocapillaris. CA IV was not detected in endothelial cells of the contiguous capillaries of the iris or in endothelial cells of other vessels. Immunoreactivity for CA IV was also intense in epithelial and fiber cells of the lens but was not detectable in the neuroretina, the ciliary process (except for capillaries), and the cornea, all sites where immunostaining with anti-CA II antibody was intense. These studies indicate that the membrane-associated CA in human eye, which was suspected from histochemical studies, is CA IV. Defining the physiological role of this ocular isozyme remains a challenge.

Aspirin hydrolyzing esterases from rat liver cytosol

Unlike most esterases, which are predominantly bound to the microsomal fraction, the enzymes hydrolyzing acetylsalicylic acid are present in an equal amount in the cytosol. Two soluble isozymes were purified to homogeneity from rat liver and characterized as serine esterases with a M r , of 35,000. Both had the wide substrate spectrum characteristic of enzymes active in detoxication. Both had a very low K m for acetylsalicylate. Three other cytoplasmic enzymes active with aspirin were observed but these differed in their high M r (about 220,000) and their lack of reactivity with antibody to one of the homogeneous isozymes.

Analysis of Carbohydrate Properties Essential for Melanogenesis in Tyrosinases of Cultured Malignant Melanoma Cells by Differential Carbohydrate Processing Inhibition

In order to clarify the biologic significance of carbohydrate processing in tyrosinases for melanogenesis, we have studied the effect of differential carbohydrate processing inhibitors on the recovery process of interrupted melanization which occurs after exposure of cultured B-16 melanoma cells to the inhibitor of core carbohydrate synthesis, glucosamine (Glc). Treatment of this glycosylation-dependent repigmentation process with the early-stage carbohydrate processing inhibitors deoxynojirimycin (dNM), castanospermine (CS), and monensin (MS) at 0.8 mM, 40 micrograms/ml, and 30 nM, respectively, in the presence of 2 mM theophylline (Tp) almost completely inhibits the reappearance of the pigment 48-72 h after removal of Glc. In contrast, treatment with the later stage carbohydrate processing inhibitor swaisonine (SW) at 40-80 micrograms/ml does not interrupt the repigmentation process. Electrophoretic analysis of tyrosinases in the soluble fractions of these melanoma cells demonstrates that the alteration of soluble tyrosinase isozymes by all the processing inhibitors is associated with a dose-dependent loss of sialic acid-rich T1 tyrosinase and the concomitant appearance or increase of sialic acid-poor tyrosinases. In the large granule fraction, a recovery of membrane-bound tyrosinase (T3) is seen following both MS and SW treatments, whereas dNM treatment results in the substantial loss of T3 tyrosinase. At the electron microscopic level, a translocation of tyrosinase from GERL and coated vesicles to many unmelanized vacuolar premelanosomes occurs in MS-treated cells in contrast to its predominant distribution in the GERL-coated vesicle system of dNM-treated cells, which contain many unmelanized premelanosomes. The present evidence for differential effects on intracellular tyrosinase transfer and melanization by different stages of carbohydrate processing inhibition suggests that asparagine-linked oligosaccharides, relating to the first mannose-trimming stages, determine the function of tyrosinase transfer as well as melanization through a specific intracellular recognition process in pigment cells.

A Soluble Form of Bovine Rod Photoreceptor Phosphodiesterase Has a Novel 15-kDa Subunit

A substantial fraction (20-30%) of the bovine rod outer segment phosphodiesterase (PDE) activity is not associated with outer segment membranes prepared with buffers of moderate ionic strength; this PDE activity appears to represent a distinct, soluble isozyme. Although this PDE isozyme can be demonstrated to be present in sealed rod outer segments, it is discarded from most standard rod outer segment preparations. A method was developed that allowed the rapid purification of the soluble rod PDE by 2600-fold, to apparent homogeneity, using a monoclonal antibody column (ROS-1a). The soluble rod PDE isozyme has a novel Mr = 15,000 subunit (delta) in addition to subunits of Mr = 88,000 (alpha sol), 84,000 (beta sol), and 11,000 (gamma sol). The delta subunit comigrates with and may be identical to the cone PDE 15-kDa subunit. The small subunits of the soluble rod PDE and the membrane-associated rod PDE were isolated by reverse-phase chromatography. The gamma sol subunit was a potent inhibitor of trypsin-activated rod PDE, inhibiting 50% of 1 pM PDE activity at a concentration of 11 pM. This concentration was similar to that observed for the gamma subunit of the membrane-associated rod PDE. The purified delta subunit did not appear to affect PDE activity; this subunit was, however, unusually difficult to keep in solution. All of the kinetic and physical properties of the soluble rod PDE tested thus far are similar to those of the membrane-associated form, except for the presence of the delta subunit, suggesting that this unique subunit could mediate the solubility of the soluble rod PDE and the cone PDE in the intact photoreceptor.

The existence of apotyrosinase in the cytosol of Harding-Passey mouse melanoma melanocytes and characteristics of enzyme reconstitution by Cu(II)

This paper reports the effect of Cu(II) supplementation on the tyrosinase isozymes from Harding-Passey mouse melanoma. The dopa-oxidase activity of the microsomal and soluble isozymes is increased by incubation with Cu(II), whereas the activity of the unique ‘in vivo’ melanin-forming isozyme, bound to melanosomes, is not. Other divalent cations are ineffective in increasing the dopa-oxidase activity of tyrosinases. These results indicate the existence of a mixture of tyrosinase and apotyrosinase in the cytosol of melanocytes before reaching the melanosome. The paucity of Cu(II) in the cytosol could be one of the mechanisms of regulation contributing to avoid the formation of melanin outside the melanosome. Some kinetic characteristics of the enzymatic reconstitution of soluble and microsomal isozymes by Cu(II) are also studied, and the results suggest that the glycosylation of apotyrosinase during its maturation yields a conformational change favouring the binding of Cu(II) at the enzyme active site, by lowering the activation energy of the reconstitution reaction.

Elicitation of cotton isoperoxidases by Aspergillus flavus and other fungi pathogenic to cotton

The distribution of peroxidase(s) in non-inoculated cotton bolls and flowers was qualitatively determined. The soluble and bound forms of cotton peroxidase(s) were investigated for changes elicited by fungal infection. Inoculation of cotton bolls, 30 days post anthesis, with Aspergillus flavus, Fusarium equiseti, Fusarium moniliforme, Fusarium semitectum or Rhizoctonia solani followed by a 6-day incubation period stimulated soluble peroxidase activity by 2- to 6-fold. In addition, fungal stress elicited two to five distinct soluble peroxidase isozymes not observed or at very low levels in non-inoculated controls. The pl values by isoelectric focusing for these isoperoxidases were 4·25, 4·35, 4·50, 4·65, 4·75 and 4·83. The pI 4·50 form was produced in controls and was also the major isozyme in the stress profiles. Each cotton-pathogen combination produced a unique peroxidase profile. No differences between control and fungal-stressed bolls were observed with either the specific activity or the peroxidase profile in the case of ionically-bound or covalently-bound classes of the enzyme.

Positive renal response to intravenous acetazolamide in patients with carbonic anhydrase II deficiency.

Carbonic anhydrase II (CA II) is the only soluble isozyme of CA which is known to be expressed in kidney. We recently identified a deficiency of this enzyme as the basis for the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. In order to explore the physiological importance of CA II in the kidney, we studied the renal response to intravenously infused acetazolamide in two CA II-deficient patients and two control subjects. Following acetazolamide infusion, the CA II-deficient patients exhibited a prompt rise in urinary pH and HCO3- excretion similar to the response seen in control subjects. These findings indicate that CA II-deficient patients, who lack detectable CA II in their erythrocytes, still expressed an acetazolamide-inhibitable CA activity in their kidneys. These results can be explained in three ways: 1) the CA II deficiency which is profound in the erythrocytes of these patients may not be expressed in their kidney. 2) An acetazolamide-sensitive CA other than CA II, such as CA I and CA III, which is not normally expressed in kidney, is expressed in kidneys of CA II-deficient patients. 3) The CA II deficiency is expressed in kidney in these patients but the acetazolamide response is due to inhibition of the luminal, membrane-bound CA which is the product of a different gene and unaffected by the CA II deficiency mutation. We favor the third possibility.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification.

The clinical, radiological, and pathological findings in three siblings affected with the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification have been reported. In an effort to explain the pleiotropic effects of the mutation producing this disorder, we postulated a defect in carbonic anhydrase II (CA II), the only one of the three soluble isozymes of carbonic anhydrase that is known to be synthesized in kidney and brain. We report here biochemical and immunological evidence for the virtual absence of CA II in erythrocytes of patients affected with this condition, whereas CA I level is not reduced. Levels of CA II in erythrocyte hemolysates from asymptomatic obligate heterozygotes are about half of normal. These findings: (i) elucidate the basic defect in one form of inherited osteopetrosis; (ii) provide genetic evidence implicating CA II in osteoclast function and bone resorption; (iii) explain previous observations that carbonic anhydrase inhibitors block the normal parathyroid hormone-induced release of calcium from bone; (iv) clarify the role of renal CA II in urinary acidification and bicarbonate reabsorption; and (v) suggest a method to identify heterozygous carriers for the gene for this recessively inherited syndrome.

Isolation and Characterization of Infected and Uninfected Cells from Soybean Nodules

The distribution of organelles and associated enzymes between cells containing bacteroids and uninfected cells from nodules of Glycine max L. Merr. cv Amsoy 71 was investigated by separation of protoplasts on a sucrose step-gradient. Infected protoplasts were much larger, irregular in shape, and more dense than uninfected protoplasts. The peroxisomal enzymes, uricase and catalase, were present at much higher specific activity in the uninfected cell fraction. Allantoinase, an enzyme of the endoplasmic reticulum, had a greater specific activity in the uninfected cell fraction. Several enzymes whose products are required for purine biosynthesis, including phosphoglycerate dehydrogenase, aspartate aminotransferase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase, exhibited a higher specific activity in the uninfected cell fraction. Isozymes of aspartate aminotransferase were separated on native gels and located by an activity stain. The soluble isozyme was predominantly found in the uninfected cell fraction. These data suggest that peroxisomes, containing uricase and catalase for conversion of uric acid to allantoin, are present only in the uninfected cells of soybean nodules. The uninfected cells also appear to be the site of the allantoinase reaction.

Soluble peptidase isozymes of the Japanese Medaka (Oryzias latipes): tissue distributions and substrate specificities.

Peptidases catalyze the hydrolysis of di- and tripeptidases to their constituent amino acids. Five isozymes (PEP A, B, C, D, and S) were shown to be the products of independent genetic loci by several criteria including distinct adult tissue and substrate specificities, non-cross-reacting immunochemical properties, and independent genetic variation at three of the loci. Four of the peptidases had at least one substrate against which they contributed over 95% of the activity. These substrates were used for isozyme-specific assays. In adult tissues, three of the peptidases had higher activities in liver and intestine than in other tissues (PEP A, B, and S). PEP C had a 10-fold higher specific activity in brain than in other tissues.

New Regulators of Melanogenesis Are Associated with Purified Tyrosinase Isozymes

Three new regulatory factors in the melanogenesis pathway were recently described: dopachrome conversion factor accelerates the conversion of dopachrome to 5,6-dihydroxyindole; indole conversion factor accelerates the conversion of 5, 6-dihydroxyindole into melanin; and indole blocking factor retards the conversion of 5, 6-dihydroxyindole into melanin. Exposure of wild-type Cloudman melanoma cells in culture to melanotropin (MSH) removes blocking factor activity and increases indole conversion factor activity. The chemical nature of factors has not yet been determined. In this report we demonstrate that highly purified isozymes of tyrosinase from C57B1/6N murine hair bulbs and B16 murine melanoma are closely associated with conversion and blocking factor activities. The soluble isozymes. T1, T2, and T2 contain blocking factor activity, while isozyme T4, the major tyrosinase species found in melanosomes, contains activity that accelerates melanin formation from dopachrome. The results suggest that melanogenesis is regulated by the association of these different factors with the specific tyrosinase isozymes.

Characterization of esterase isozymes of Raillietina tetragona (Molin, 1858) (Cestoda).

Abstract Soluble esterase isozymes from Raillietina tetragona were separated in polyacrylamide gel electrophoresis, which revealed the presence of at least four fractions, possibly representing different esterases. The differences in the inhibitory effect of various chemicals on these fractions reveal them to be four types of esterases, namely: arylesterase, carboxylesterase, acetylesterase and cholinesterase.

A Comparative Study of Multiple: Acid Phosphatases in Cellular Fractions of Bean Hypocotyl

Six kinds of acid phosphatases were solubilized with Triton X-100 from the cell wall (W-I, W-II), mitochondrial (M-I, M-II, M-III) and microsomal (Ms) fractions of bean hypocotyl, and they were partially purified by using Sephadex gel filtration and DEAE-cellulose column chromatography. Acid phosphatases from the soluble fraction were also fractionated into 12 isozymes by electrophoresis, and the properties of the isozymes were compared. The soluble isozymes showed pH optima at 5 0, 5-3 and 5-6; the isozymes possessed high affinity to /7-nitrophenyl phosphate and ADP, and their mol. wts ranged from 30000 to 45000. Among the solubilized phosphatases, W-I, M-I and M-III showed maximum activity at pH 5 0 and their mol. wts were between 50000 and 110000. W-I proved to have high affinity to ATP and ??s-p-nitrophenyl phosphate as M-I did to /7-nitrophenyl phosphate and M-III to phenyl phosphate. The characteristics of these solubilized isozymes were different from those of the soluble isozymes. On the contrary, W-II, M-II and Ms were quite similar to those of the solubles in pH optima, substrate specificity, Km value, affinity to DEAE-cellulose and gel electrophoretic patterns. These results suggest that W-II, M-II and Ms were derived from the soluble isozymes.