THE ACTIVITY OF YEAST INVERTASE AS A FUNCTION OF OXIDATION-REDUCTION POTENTIAL

The activity of yeast invertase as a function of oxidation-reduction potential has been investigated using a large number of oxidants and reductants. The activity is constant over the range of E_h from –270 to +600 mv., but above E_h = +600 mv. there is a sharp decrease in activity reaching 0 at E_h = +1,000 mv. The inhibiting action of strong oxidants is upon the enzyme rather than on the substrate and appears to be essentially irreversible Experiments indicate that the inhibiting action of strong oxidants on invertase is primarily related to their high oxidation-reduction potential rather than to a specific toxic action unrelated to E_h. The effects of oxidation-reduction potential upon invertase activity are independent of the purity of the enzyme, since they are the same for commercial invertases, fresh bakers'' yeast, powdered bakers'' yeast, brewers'' yeast, and highly purified invertase. Possible mechanisms involved in the inactivation of invertase by oxidants are discussed.     

THE ROLE OF CARBONIC ANHYDRASE IN CERTAIN IONIC EXCHANGES INVOLVING THE ERYTHROCYTE

1. The acceleration by bicarbonates of the swelling and hemolysis of erythrocytes in solutions of ammonium salts, first reported by Ørskov, is strikingly dependent upon carbonic anhydrase, being almost abolished by inhibitors of this enzyme such as KCN and sulfanilamide, and under suitable conditions being enhanced by its addition to the external solution. This behavior gives support to the theory of "catalyzed diffusion" as an explanation of the Ørskov effect. 2. The inhibitory effects of both sulfanilamide and KCN seem to be capable of complete reversal on washing the erythrocytes in isotonic salt solutions. The full effect of KCN appears almost instantly; that of sulfanilamide requires a period measured in seconds, or possibly even in minutes, to reach its maximum, the delay presumably being due to the slower penetration of the erythrocyte by this substance. Under favorable conditions the effect of concentrations of sulfanilamide of a few hundredths of a milligram per cent can be demonstrated. No similar effects have been obtained with sulfapyridine. 3. Bicarbonates also have a "catalytic" effect on the response of the internal pH of erythrocytes to changes in that of their surroundings. The resulting volume changes of the cell, which otherwise frequently require many minutes for their completion, may take place within a few seconds in the presence of low concentrations of bicarbonates. At a given pH value the effect of the latter substances is chiefly on the rate of the change and only to a minor extent on its magnitude. It may be further accelerated under appropriate conditions by the addition to the cell suspension of carbonic anhydrase, and can be almost abolished by KCN and by sulfanilamide. 4. Volume changes of erythrocytes associated with exchanges of Cl'' for SO₄'''' ions are greatly accelerated by low concentrations of bicarbonates, this effect being likewise dependent upon carbonic anhydrase. There is some evidence that in this case the exchange takes place, at least in part, in two steps: Cl'' for HCO₃'' and HCO₃'' for SO₄''''.     

Multiresidue determination of sulfonamides in edible catfish, shrimp and salmon tissues by high-performance liquid chromatography with postcolumn derivatization and fluorescence detection

A liquid chromatographic (LC) method for determining 14 sulfonamide (SA) (sulfanilamide, sulfadiazine (SDZ), sulfathiazole, sulfapyridine, sulfamerazine (SMR), sulfamethazine (SMZ), sulfamethizole, sulfamethoxypyridazine, sulfachloropyridazine (SCP), sulfamonomethoxine, sulfadoxine, sulfamethoxazole, sulfadimethoxine (SDM), and sulfaquinoxaline (SQX)) residues in edible catfish, shrimp and salmon tissues was developed and validated at 5, 10 or 20 ng g(-1). The method was then used to determine residues in tissues of catfish, shrimp and salmon dosed with six selected sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfachloropyridazine, sulfadimethoxine and sulfaquinoxaline). All assays were within U.S. Food and Drug Administration guidelines for recovery and intra-assay variability. The method was developed to determine possible sulfonamide residues in aquacultured catfish, shrimp and salmon produced for food.     

THE INFLUENCE OF ELECTROLYTES UPON THE ELECTROPHORETIC MIGRATION OF BACTERIA AND OF YEAST CELLS

1. It seems first of all clear from our results that the effect of electrolytes upon electrophoretic charge is essentially the same, whether one is dealing with silica dust, bacteria, or yeast cells, although certain quantitative differences appear which will later be discussed. 2. The normal negative charge on the suspended particles appears to be slightly increased by very low concentrations of electrolytes, markedly so in the case of yeast cells. Increase in charge due to minimal concentrations of electrolytes has been recorded by Loeb (1922) for collodion particles. 3. Higher concentrations of electrolytes cause a marked and progressive decrease in negative charge, sometimes leading to an isopotential condition and sometimes to a complete reversal of charge with active migration toward the cathode. This effect is apparently due to the cation alone and increases with the valency of the cation, except that the H ion shows specially marked activity, between that of bivalent and trivalent ions. Since NaOH behaves like an ordinary univalent salt, increased alkalinity of a solution does not further depress the charge already depressed by salts; but, since the H ion is much more active than other univalent or bivalent ions, increased acidity does cause a further progressive depression of charge, even in salt solutions. Certain electrolytes appear to show individual peculiarities due to something else than their valency. Thus KCl for example is distinctly more effective than NaCl. Sodium chloride in general appears to exert less influence upon electrophoretic charge, either in low or high dilution, than do other compounds of univalent ions studied. This depressing effect of moderately high concentrations of electrolytes is much less marked with yeast cells than with Bacterium coli. Silica dust is still less affected by monovalent and bivalent ions than are the yeast cells but appears to be more affected than either yeast or Bacterium coli by AlCl₃. 4. Very high concentrations of AlCl₃ (above 10^–2 M) show a third effect, a decrease of the positive charge produced by concentrations of moderate molar strength. This is analogous to phenomena observed for trivalent salts by Northrop and De Kruif (1921–22) and for acid by Winslow, Falk, and Caulfield (1923–24). 5. Organic substances, such as glucose, glycerol, and saponin produce no effect on electrophoretic velocity until they reach a concentration at which viscosity changes are involved. 6. The first two results observed,—(a) the increase in charge as a result of slight additions of electrolytes, and (b) the marked decrease in charge with further concentration of electrolytes, depending on the valency of the cation, so far as vegetable cells are concerned, are entirely in accord with the theory of the Donnan equilibrium as worked out by Loeb (1922). We might assume in explaining such phenomena that the plant cell contains a certain proportion of unbound protein material and that the first modicum of cation which enters the cell is bound by the protein, leading to an increase in the relative negative charge of the cell as compared with its menstruum, while subsequent increments of cation remain unbound in the cell and thus lower its charge. When we find, however, that the same phenomena are apparent with collodion particles, as shown by Loeb, and with silica dust, it seems difficult to apply such a theory, involving the conceptions of a permeable membrane and unbound organic compounds. Loeb (1923–24) suggests that the primary increase may be due to an aggregation of anions in the part of the electrical double layer adjacent to the suspended particles; but why there should be first an aggregation of anions and later (with increasing concentration) an aggregation of cations, is not easy to conceive. The third result,—the reversion to a more negative charge in the presence of a marked excess of trivalent ions,—is again difficult to explain. Loeb, in this connection, postulates the existence of complex ion-protein compounds, which can scarcely be assumed in the case of the silica particles.     

Substrate recognition characteristics of human holocarboxylase synthetase for biotin ligation

Holocarboxylase synthetase (HCS) is an essential enzyme that catalyzes the incorporation of biotin into apo carboxylase and the biotinylation of the four biotin-dependent carboxylases in the human cell. Deficiency of HCS results in decreased activity of these carboxylases and affects various metabolic processes. Despite the importance of this enzyme, the recognition mechanism of the biotinoyl domain by human HCS (hHCS) has remained unclear. We have developed a method to express hHCS in the baculovirus system and used it to purify catalytically active, full-length hHCS. NMR experiments on the biotinoyl domains from acetyl-CoA carboxylase indicate that when hHCS is added, it recognizes the MKM motif in human and in Escherichia coli with a preference to the human biotinoyl domain. In addition, hHCS can biotinylate the biotinoyl domains from human and E. coli acetyl-CoA carboxylase at similar rates compared to the E. coli biotin protein ligase, BirA, which reacts very slowly with the human biotinoyl domain. We propose that the hHCS has greater substrate acceptability, while the BirA has higher substrate specificity. These results provide insights into substrate recognition by hHCS, which can be distinguished from BirA in this respect.     

A KINETIC ANALYSIS OF THE ENDOGENOUS RESPIRATION OF BAKERS' YEAST

The process of endogenous respiration of two strains of bakers'' yeast, Saccharomyces cerevisiae, was examined kinetically. The rate of respiration with respect to time in a non-nutrient medium was found to exhibit two phases: (a) a period of constant rate of O₂ consumption and CO₂ production (R.Q. = 1) characteristic of cells with ample concentrations of stored material; (b) a first order decline in rate of respiration with respect to time, where the rate was proportional to the concentration of some substrate, S. (R.Q. = 1 throughout second phase.) The nature of this substrate was reexamined and the evidence summarized confirms the notion that it is a carbohydrate, probably glycogen. These phases of endogenous respiration were shown to depend upon the age of the culture and the amount of substrate available.     

A microbiological assay for sterigmatocystin,T-2 toxin and zearalenone

A survey was done to find microorganisms useful for assaying sterigmatocystin; T-2 toxin and zearalenone.Staphylococcus aureus was found to be sensitive to T-2 toxin and zearalenone;Bacillus cereus was found to be sensitive to T-2 toxin only; andEscherichia coli was sensitive to sterigmatocystin. The response of the organisms to sterigmatocystin; T-2 toxin and zearalenone was found to be linear between 4 and 100 μg with sterigmatocystin toE. coli; between 2 and 25 μg with T-2 toxin toStaph, aureus andB. cereus; and between 4 and 100 μg with zearalenone toStaph, aureus. The lower limits of sensitivity of the test were 2 μg T-2 toxin and zearalenone, and 4 μg sterigmatocystin. The assay is rapid (15–17 hrs); simple and inexpensive; and can be used to verify the toxicity of samples and to confirm thin layer chromatographic results.     

THE EFFECTS OF SULFATHIAZOLE AND OF PROPYL CARBAMATE ON THE RATE OF OXYGEN CONSUMPTION AND GROWTH IN ESCHERICHIA COLI

1. The rates of growth and of oxygen consumption by cells of E. coli have been measured under identical conditions, and the effects of sulfathiazole (ST) and of n-propyl carbamate (PC) on these two processes have been compared. 2. The rate of growth was measured by (a) the increase in the viable cell count, (b) the increase in the optical density of the culture, (c) the increase in the rate of oxygen consumption, and (d) the decrease in the ammonia of the medium. The results as indicated by these several measures were identical under the conditions of these experiments. 3. Concentrations of ST or of PC which are just sufficient to stop growth completely, lower the rate of oxygen consumption per unit of bacterial protoplasm to a value approximately 50 per cent of that seen in the absence of the inhibitor. 4. It is shown that the rate of oxygen consumption in cells from old cultures is less affected by ST than is the rate of oxygen consumption by cells from young cultures. It is probable that the rate of oxygen consumption by "old" cells is lower than that of "young" cells. 5. The effects of ST and PC on both the rate of oxygen consumption and the rate of growth are very similar, indicating in a general way, that the mechanism of the actions of these two inhibitors is similar. Furthermore, since both of them produce appreciable inhibition of the rate of oxygen consumption while they are inhibiting growth, the possibility that the effect on oxygen consumption is the immediate cause of the effect on growth must be entertained.     

FORMATION OF AUXIN IN YEAST CULTURES

We have found far more auxin in the culture media of bakers'' yeast than was obtained by Kögl and Kostermans from the cells themselves. The production of auxin by yeast cells resembles the formation observed in other organisms such as Rhizopus and Rhizobium which also form auxins in their culture media. The auxin yield was found to increase with the concentration of sucrose and to decrease with the concentration of peptone. An inverse relation with the rate of cell multiplication was observed. Enlarged and elongated cells appeared only in those media which contained considerable amounts of auxin. The total auxin yield in the various cultures was found to be directly proportional, below pH 5, to the hydrogen ion concentration. Thus, it was proposed that certain growth conditions favor the breakage of the link between auxin and its protein carrier (Skoog and Thimann) 1940) and consequently accelerate the rate of excretion of auxin into the growth medium.     

Ribulose Diphosphate Carboxylase Synthesis in Euglena: III. Serological Relationships of the Intact Enzyme and its Subunits

Ribulose 1,5-diphosphate carboxylase was isolated from Euglena gracilis Klebs strain Z Pringsheim, Chlorella fusca var. vacuolata, and Chlamydobotrys stellata, and the subunits from each enzyme were separated and purified by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulfate. Rabbit antibody was elicited against purified Euglena ribulose 1,5-diphosphate carboxylase whole enzyme and the isolated large and small subunits. Euglena ribulose 1,5-diphosphate carboxylase showed partial immunological identity on Ouchterlony gels with the Chlorella and Chlamydobotrys carboxylases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates between antibody to the Euglena large subunit and the isolated large subunits of the Chlorella and Chlamydobotrys enzymes showed this was due to determinants on the large subunit. There was no serological affinity between the small subunits of the Euglena, Chlorella, and Chlamydobotrys carboxylases, and NH₂-terminal amino acid analyses provided further evidence of variability in the structure of the small subunits.     

Biochemical relationship of phosphoenolpyruvate carboxylases (PEPCs) from thermophilic archaea

An archaeal phosphoenolpyruvate carboxylase (PEPC) was purified from an acidophilic extreme thermophile, Sulfolobus acidocaldarius. The native enzyme was a homotetramer of 260±20 kDa molecular mass composed of 60±5 kDa subunits. The enzyme appeared to have a temperature optimum of 90°C and a pH optimum of 8.0. The activity of S. acidocaldarius phosphoenolpyruvate carboxylase was inhibited by l-aspartate and l-malate, but not enhanced by any metabolites. In comparison to the enzymatic and molecular properties of all other phosphoenolpyruvate carboxylases including another archaeal entity from the hyperthermophilic methanogen Methanothermus sociabilis, the archaeal phosphoenolpyruvate carboxylases were quite different from bacterial and eucaryal counterparts, and their small size and the lack of positively allosteric regulation were likely to be peculiar to the enzyme of the domain Archaea.     

SULFONAMIDE INHIBITION IN MONOCHRYSIS LUTHERP1

The effect of sulfonamides on growth of the chry-somonad, Monochrysis lutheri, in a synthetic sea-water medium was examined over a period of 14 days. The population increased at all sulfonamide concentrations during the first several days of incubation before inhibition became apparent. Inhibitory concentrations ranged from 0.01 to 1.0 mg%. Inhibition luas most pronounced in sulfathiazole; sulfamethazine, sulfapyridine, and sulfanilamide followed in decreasing order. p-Arninobenzoic acid (0.001-1.0 mg%) competitively reversed inhibition. Folic acid, thymine, adenine, and vitamin B₁₂ neither reversed the inhibition nor spared the requirement for p-aminobenzoic acid. The significance of the inhibition pattern and the potential use of antimetabolites in the marine environment were discussed.     

SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO

1. A method is given whereby the course of hydrolysis of sucrose by live yeast cells may be followed with precision equal to that found when invertase solutions prepared from autolyzed yeast are used to cause inversion. 2. The practical value of the equation of Nelson and Hitchcock as a means of following the course of enzymic hydrolysis of sucrose is hereby extended. 3. The inversion of sucrose by live yeast cells and by extracted invertase has been quantitatively compared. 4. The course of hydrolysis of sucrose by the invertase of Fleischmann''s yeast has been found to be identical in vivo and in vitro.     

EFFECTS OF YEAST,FERMENTATION TIME,AND PRESERVATION METHODS ON TARHANA

The physicochemical properties of tarhana soup produced with different dough treatments, fermentation times, and preservation methods were examined. Tarhana doughs were prepared with yogurt (control) or baker's yeast (Saccharomyces cerevisiae) and fermented for 3 days. Samples were taken at 24, 48, and 72 hr. Samples were then preserved via one of four methods: sun dried, dried in the shade, vacumn dried, and frozen. Frozen samples produced lower organic acid levels after 72 hr of fermentation in both control (0.68 g/100 g) and yeast (0.61 g/100 g) applications than samples that were dried (0.94 g/100 g control samples; 0.81 g/100 g samples with yeast). Increasing fermentation time resulted in a significant effect on the formation of organic acid in the tarhana (p < .01). At 72 hr of fermentation, total acidity increased 11%, 17%, and 23% for tarhana samples vacumn-dried, sun-dried, and dried in the shade, respectively. Preservation methods also affected the moisture, ash, crude protein, total acidity, pH, salt, fat, reducing sugar levels, and the sensory assestment of tarhana soup (p < .01). Sensory characteristics were not significantly affected by baker's yeast in any of the preservation methods used (p > .01). However, sensory scores for tarhana prepared from the samples dried in a sheltered area showed a reduction in color desireablilty as the fermentation time increased. The soup prepared from frozen tarhana (72 hr fermentation, with yeast) had the highest scores with respect to color, mouth feel, flavor, and overall acceptability. Vacuum-dried samples' scores in these areas were also high in comparison to the two other drying methods.     

Comparison of acetyl-CoA carboxylases from parsley cell cultures and wheat germ

The activity of acetyl-CoA carboxylase of suspension-cultured cells of parsley (Petroselinum hortense Hoffm.) is greatly stimulated by light soon after transferring cells to new culture medium. Parsley acetyl-CoA carboxylase has been purified from frozen cells by treatment of the crude protein extract with Dowex 1 × 2 and polyethyleneimine, precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose and blue Sepharose CL-6B, and gel filtration on Sepharose 6B. A recovery of about 8% has been achieved with a 300-fold increase in specific activity. Wheat germ acetyl-CoA carboxylase has been purified 2180-fold by a similar procedure. The two carboxylases have the following characteristics: Molecular weights of 840,000 for the parsley carboxylase and 700,000 for the wheat germ carboxylase have been estimated from the elution volumes of a calibrated Sepharose 6B column. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the carboxylases from parsley and wheat each are composed of one large subunit (M_r = 210,000 and 240,000, respectively) and possibly one smaller polypeptide component (M_r = 105,000 and 98,000, respectively). Avidin-binding experiments demonstrated that the 240,000 — M_r component of wheat germ carboxylase is the biotin-containing subunit of this enzyme. No isoenzymes of the parsley carboxylase could be demonstrated.     

Characterization and bioactivity of hepcidin-2 in zebrafish: Dependence of antibacterial activity upon disulfide bridges

Hepcidin is an antimicrobial peptide and iron-regulatory molecule with highly conserved disulfide bridges among vertebrates, but structural insights into the function in fish remains largely missing. We demonstrate here that recombinant hepcidin-2 from zebrafish is capable of inhibiting the growth of the Gram-negative bacteria Escherichia coli and Vibrio anguillarum, and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentrations (MICs) of 18, 15, 13 and 9 μM, respectively. We also show by TEM examination that recombinant hepcidin-2 is directly cidal to the cells of E. coli and S. aureus. Moreover, we find that hepcidin-2 displays affinity to LPS, LTA and PGN. All these data indicate that hepcidin-2 is both a pattern recognition molecule, capable of identifying LPS, LTA and PGN, and an antibacterial effector, capable of inhibiting the growth of bacteria. The data also show that the antibacterial activity of hepcidin-2 depends upon the disulfide bridges.     

THE PREADAPTIVE OXIDATION OF GALACTOSE BY YEAST

A preadaptive purely aerobic utilization of galactose by yeast cells has been demonstrated. Hence, the adaptation by yeast to galactose is not to its utilization per se, but specifically to its metabolism by a glycolytic mechanism. An examination of this preadaptive oxidation of galactose reveals that it has many characteristics in common with the endogenous metabolism of yeast. Included among these are the similarities of the R.Q. values and the response of the Q _OO2 and Q_COCO2 ^O₂ to KCN and iodoacetic acid. Further, a competitive interaction appears to exist between the endogenous respiration and the preadaptive oxidation of the galactose. The latter can replace the endogenous respiration as a source of energy for the adaptation to the fermentation of the galactose. Carbon balance studies of the galactose oxidation revealed that polysaccharide could be formed as a result of this metabolism during the preadaptive period. Non-adaptable cells were also found to possess the capacity to oxidize galactose in the complete absence of any ability to metabolize it anaerobically. The significance of these findings for the biochemistry and physiology of the adaptation is discussed.     

2-carboxy-d-hexitol 1,6-bisphosphate: An inhibitor of d-ribulose 1,5-bisphosphate carboxylase/oxygenase

2-Carboxy-d-hexitol 1,6-bisphosphate (CHBP) has been prepared from d-fructose 1,6-bisphosphate and cyanide. DEAE-Sephadex chromatography separated the reaction products into two fractions which were identified as CHBP and CHBP-lactone. CHBP is presumably a mixture of two diastereomers, 2-carboxy-d-glucitol 1,6-bisphosphate and 2-carboxy-d-mannitol 1,6-bisphosphate, but an attempt to separate these compounds was not successful. The material in the CHBP-lactone peak had no effect on d-ribulose 1,5-bisphosphate (RuBP) carboxylase. However, CHBP was a potent reversible inhibitor of RuBP carboxylases. This compound displayed an inhibition constant (K_i at pH 8.0 and 30 °C) of 1–2 μm with the enzymes from spinach and barley, while the K_i was 60–70 μm with bacterial RuBP carboxylases from Pseudomonas oxalaticus and Rhodospirillum rubrum. The mode of inhibition was competitive with respect to RuBP for all the carboxylases, and noncompetitive with respect to CO₂ for the enzymes from spinach, P. oxalaticus and R. rubrum. The results indicate that, in the binding of certain organic phosphates by RuBP carboxylases, there may be a fundamental difference between the enzymes isolated from microbial and from higher plant sources. RuBP oxygenase activities from spinach and P. oxalaticus were also inhibited by CHBP, with K_i values which were similar to those obtained with the carboxylase activity of the same enzymes. The mode of inhibition of the oxygenase activities was also competitive with respect to RuBP. Thus, it seems that the binding of CHBP is similar for the carboxylase and oxygenase reactions of the same enzyme.     

Antimicrobial resistance and toxin gene profiles of Staphylococcus aureus strains from Holstein milk

Isolation of Staphylococcus aureus (Staph. aureus) from Holstein milk samples with mastitis and nonmastitis was conducted to estimate its prevalence, antimicrobial resistance and toxin genes. A total of 353 milk samples were collected from three Chinese Holstein herds. Fifty‐three Staph. aureus isolates collected from 29 Staph. aureus‐positive samples were characterized via antimicrobial susceptibility, toxin genes and Pulsed‐field Gel Electrophoresis (PFGE) profiles. The prevalence of Staph. aureus was 4·0–9·5% in mastitic and 7·3–11·5% in nonmastitic samples in the analysed herds. Approximately 61·0% of Staph. aureus strains isolated from mastitis cows were resistant to ≥10 antimicrobials compared with 0% of isolates with nonmastitis. The most frequently observed super antigenic toxin gene was pvl (41·5%) followed by seh + pvl (13·2%). We did not find mecA‐positive methicillin‐resistant Staph. aureus (MRSA) strains, while mecA‐negative MRSA strains were identified in the three herds. PFGE results suggested potential transmission of Staph. aureus strains in different farms. These results open new insights into Staph. aureus transmission and antimicrobial resistance of Holstein dairy cows and into developing strategies for udder health improvement of dairy cattle.     

Expression of a thermostable cellulase gene from a thermophilic anaerobe in Saccharomyces cerevisiae

A cellulase gene from a thermophilic anaerobe was recloned in the yeast Saccharomyces cerevisiae. The maximum level of the gene expression in the recombinant yeast was 4.4 times higher than that in the Escherichia coli transformant harboring the same plasmid. Cellulase activity was observed only within the yeast cells. To compare the enzymatic properties of cellulase produced by the yeast and E. coli transformants, cellulases were purified to homogeneous state by only three purification steps of heat treatment, and cellulose affinity and ion exchange chromatographies. The molecular weights of the enzymes produced by the yeast and E. coli were 3.8 × 10⁴ and 4.0 × 10⁴, respectively by SDS-polyacrylamide gel electrophoresis. Neither of the enzymes was glycosylated. Although the molecular weights were slightly different, enzymatic properties and thermostability were almost indistinguishable between the enzymes produced by the yeast and E. coli transformants.