Isolation and Characterization of a Bacillus cereus Mutant Strain Hyperproductive of Exo-β-Amylase

Starting with a strain of Bacillus cereus excreting about 40-fold more β-amylase than does the original wild-type strain, we isolated, after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine, a strain designated BQ10-S1 SpoIII which showed under optimal conditions a further 5.5-fold increase in β-amylase activity. The amylase production of this strain was observed to increase in the presence of 0.5% glucose or 1% maltose and, more markedly, in the presence of 2% soluble starch in the culture medium. The enzyme produced by this strain was immunologically identical to the wild-type enzyme, suggesting that either the copy number of the gene or the efficiency of enzyme synthesis from it, or both, are altered in this strain.     

Regulation and genetic enhancement of beta-amylase production in Clostridium thermosulfurogenes.

We studied the general mechanism for regulation of beta-amylase synthesis in Clostridium thermosulfurogenes. beta-Amylase was expressed at high levels only when the organism was grown on maltose or other carbohydrates containing maltose units. Three kinds of mutants altered in beta-amylase production were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained starch-glucose agar plates. beta-Amylase was produced only when maltose was added to cells growing on sucrose in wild-type and catabolite repression-resistant mutant strains, but the differential rate of enzyme synthesis in constitutive mutants was constant regardless of the presence of maltose. In carbon-limited chemostats of wild-type and catabolite repression-resistant mutant stains, beta-amylase was expressed on maltose but not on glucose or sucrose. beta-Amylase synthesis was immediately repressed by the addition of glucose. Therefore, we concluded that beta-amylase synthesis in C. thermosulfurogenes was inducible and subject to catabolite repression. The addition of cAMP did not eliminate the repressive effect of glucose. The mutants were generally characterized in terms of beta-amylase production, growth properties, fermentation product formation, and alterations in glucose isomerase and glucoamylase activities. A hyperproductive mutant produced eightfold more beta-amylase on starch medium than the wild type and more rapidly fermented starch to ethanol.     

General Biochemical Characterization of Thermostable Extracellular beta-Amylase from Clostridium thermosulfurogenes

Clostridium thermosulfurogenes, an anaerobic bacterium which ferments starch into ethanol at 62 degrees C, produced an active extracellular amylase and contained intracellular glucoamylase but not pullulanase activity. The extracellular amylase was purified 2.4-fold, and its general physicochemical and catalytic properties were examined. The extracellular amylase was characterized as a beta-amylase (1,4-alpha-d-glucan maltohydrolase) based on demonstration of exocleavage activity and the production of maltose with a beta-anomeric configuration from starch. The beta-amylase activity was stable and optimally active at 80 and 75 degrees C, respectively. The pH optimum for activity and the pH stability range was 5.5 to 6 and 3.5 to 6.5, respectively. The apparent [S](0.5V) and V(max) for beta-amylase activity on starch was 1 mg/ml and 60 U/mg of protein. Similar to described beta-amylase, the enzyme was inhibited by p-chloromercuribenzoate, Cu, and Hg; however, alpha- and beta-cyclodextrins were not competitive inhibitors. The beta-amylase was active and stable in the presence of air or 10% (vol/vol) ethanol. The beta-amylase and glucoamylase activities enabled the organism to actively ferment raw starch in the absence of significant pullulanase or alpha-amylase activity.     

Identification of tms-26 as an allele of the gcaD gene, which encodes N-acetylglucosamine 1-phosphate uridyltransferase in Bacillus subtilis.

The temperature-sensitive Bacillus subtilis tms-26 mutant strain was characterized biochemically and shown to be defective in N-acetylglucosamine 1-phosphate uridyltransferase activity. At the permissive temperature (34 degrees C), the mutant strain contained about 15% of the wild-type activity of this enzyme, whereas at the nonpermissive temperature (48 degrees C), the mutant enzyme was barely detectable. Furthermore, the N-acetylglucosamine 1-phosphate uridyltransferase activity of the tms-26 mutant strain was much more heat labile in vitro than that of the wild-type strain. The level of N-acetylglucosamine 1-phosphate, the substrate of the uridyltransferase activity, was elevated more than 40-fold in the mutant strain at the permissive temperature compared with the level in the wild-type strain. During a temperature shift, the level of UDP-N-acetylglucosamine, the product of the uridyltransferase activity, decreased much more in the mutant strain than in the wild-type strain. An Escherichia coli strain harboring the wild-type version of the tms-26 allele on a plasmid contained increased N-acetylglucosamine 1-phosphate uridyltransferase activity compared with that in the haploid strain. It is suggested that the gene for N-acetylglucosamine 1-phosphate uridyltransferase in B. subtilis be designated gcaD.     

Comparison of active mutants and wild-type aspartate transcarbamoylase of Escherichia coli

Two active mutants of aspartate transcarbamoylase from Escherichia coli have been purified from strains which produce large quantities of enzyme. Each enzyme was isolated from a different spontaneous revertant of a pyrimidine auxotrophic strain produced by mutagenesis with nitrogen mustard. Both enzymes exhibit allosteric properties with one having significantly less and the other more cooperativity than wild-type enzyme. Isolated catalytic subunits had different values of Km and Vmax. Studies on hybrids constructed from mutant catalytic and wild-type regulatory subunits (and vice versa) indicate that catalytic chains encoded by pyrB and not the regulatory chains encoded by pyrI were affected by the mutations. Differential scanning calorimetry experiments support these conclusions. Both mutant enzymes undergo ligand-promoted conformational changes analogous to those exhibited by wild-type enzyme: a 3% decrease in the sedimentation coefficient and a 5-fold increase in the reactivity of the sulfhydryl groups of the regulatory chains. Interactions between catalytic and regulatory chains in the mutants are weaker than those in the wild-type enzyme. The gross conformational changes of the mutants upon adding the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, in the presence of the substrate, carbamoylphosphate, and the activator, ATP, correlate with differences in cooperativity. The mutant with lower cooperativity is more readily converted from the low-affinity, compact, T-state to the high-affinity, swollen, R-state than is wild-type enzyme; this conversion for the more cooperative enzyme is energetically less favorable.     

The reduction of starch accumulation in transgenic sugarcane cell suspension culture lines

Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product because it reduces the amount of sucrose that can be crystallized from molasses. In an attempt to reduce the starch content of sugarcane, the activities of ADP-glucose pyrophosphorylase (AGPase) and beta-amylase were manipulated using transgenic approaches. Transformation vectors to reduce AGPase activity and to increase plastidial beta-amylase activity were constructed and used for the transformation of sugarcane calli. The results of the manipulations were analyzed in suspension cultures. AGPase activity was reduced down to between 14 and 54% of the wild-type control. This led to a reduction in starch concentration down to 38% of the levels of the wild-type control. beta-Amylase activity was increased in the transgenic lines by 1.5-2 times that of the wild-type control. This increase in activity led to a reduction in starch amounts by 90% compared to wild-type control cells. In both experiments, the changes in starch concentrations could be correlated with the change in enzyme activity. There were no significant effects on sucrose concentrations in either experiment, indicating that these approaches might be useful to engineer regenerated sugarcane for optimized sucrose production.     

Water stress enhances beta-amylase activity in cucumber cotyledons

Cotyledons detached from 4-d-old cucumber (Cucumis sativus L.) seedlings were subjected to water stress (air-drying or PEG-treatment) to examine the effects of the stress on carbohydrate metabolism. Amylolytic activity in the cotyledon was increased about 6-fold by water stress within 1 d. The substrate specificity and the action pattern indicated that beta-amylase is responsible for the activity. Activities of azocaseinase, malate dehydrogenase and triose-phosphate isomerase were not affected by water stress, indicating that the effect of the stress on beta-amylase is rather specific. Cycloheximide-treatment strongly reduced the enhancement of beta-amylase activity. The hypocotyl of cucumber seedlings also exhibited an increase in the enzyme activity when subjected to water stress. The major free sugars in cucumber cotyledons were glucose, fructose, maltose, and sucrose; sucrose being the most abundant. Sucrose content in excised, unstressed cotyledons increased markedly during the incubation. Changes in other free sugars were small compared with that of sucrose. Starch also accumulated in unstressed cotyledons. In stressed cotyledons more sucrose and less starch accumulated than in unstressed ones. Such results were discussed in relation to the enhancement of beta-amylase activity.     

Negative regulation in the expression of a sugar-inducible gene in Arabidopsis thaliana. A recessive mutation causing enhanced expression of a gene for beta-amylase.

Expression of a beta-amylase gene of Arabidopsis thaliana (AT beta-Amy) is regulated by sugars. We identified a mutant, hba1, in which the level of expression of AT beta-Amy in leaves of plants that had been grown in a medium with 2% sucrose was significantly higher than that in wild-type plants. Higher that wild-type levels of beta-amylase in hba1 plants depended on the presence of 1 to 2% sucrose or 1% glucose in the medium, whereas leaves of mutant plants grown with higher levels of sugars had beta-amylase activities similar to those in leaves of wild-type plants. The hba1 phenotype was recessive and did not affect levels of sugars and starch in leaves. It is proposed that expression of AT beta-Amy is regulated by a combination of both positive and negative factors, dependent on the level of sugars, and that HBA1 might function to maintain low-level expression of AT beta-Amy until the level of sugars reaches some high level. Results of crosses of hba1 plants with transgenic plants that harbored an AT beta-Amy:GUS transgene with 1587 bp of the 5'-upstream region suggested that HBA1 affects expressions of AT beta-Amy in trans. The hba1 plants also had growth defects and elevated levels of anthocyanin in their petioles. However, sugar-related changes in levels of several mRNAs other than beta-amylase mRNA were unaffected in hba1 plants, suggesting that only a subset of sugar-regulated genes is under the control HBA1.     

The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae

The co-regulation of the main mevalonic acid pathway enzymes was investigated in the yeast Saccharomyces cerevisiae. It was found that a 6-fold increase in FPPS activity compared with that of the wild-type strain FL100 did not cause significant changes in HMG-CoA reductase activity, while the amounts of synthesized dolichols and ergosterol increased by 80 and 32%, respectively. The disruption of the SQS gene in the strain grown in the presence of ergosterol repressed the activities of both FPP synthase and HMG-CoA reductase to a comparable degree, whereas in the same strain starved for ergosterol the activity of FPPS was 10-fold higher and HMG-CoA reductase activity was practically unchanged. We show that FPPS is the enzyme that regulates the flow rate of synthesized mevalonic acid pathway products independent of HMG-CoA reductase and SQS.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: enhancement of PHA production and alteration of polymer molecular weight

In vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 61-3 (phaC1(Ps)) has been performed to generate highly active enzymes. In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro. Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) [P(3HB)] from glucose, compared to recombinant E. coli harboring the wild-type PHA synthase gene (0.1 wt %). Recombinant E. coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %). The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase. In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme. The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity. Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo.     

Selection and study of a Candida molischiana mutant derepressed for β-glucosidase production

Abstract A mutant strain of Candida molischiana was selected. Analysis of the exocellular activity of Candida molischiana 35M5N grown on different carbon sources revealed that the biosynthesis of β-glucosidase is derepressed in this yeast strain. The strain is not a hyper-producer mutant. There were no observed differences in the endocellular and parietal activities of the wild and mutant strains. However, the mutant strain produced 35-fold more enzyme than the wild-type in the culture medium with glucose as carbon source. When glucose was used as carbon source, the mutant strain produced 90% more exocellular enzyme than when cellobiose was used as the carbon source.     

Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production.

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.     

Purification from a yeast mutant of mitochondrial F1 with modified beta-subunit. High affinity for nucleotides and high negative cooperativity of ATPase activity

Mitochondrial F1 containing genetically modified beta-subunit was purified for the first time from a mutant of the yeast Schizosaccharomyces pombe. Precipitation by poly(ethylene glycol) allowed us to obtain a very stable and pure enzyme from either mutant or wild-type strain. In the presence of EDTA, purified F1 retained high amounts of endogenous nucleotides: 4.6 mol/mol and 3.7 mol/mol for mutant and wild-type F1, respectively. The additional nucleotide in mutant F1 was ATP; it was lost in the presence of Mg2+, which led to a total of 3.4 mol of nucleotides/mol whereas wild-type F1 retained all its nucleotides. Mutant F1 bound more exogenous ADP than wild-type F1 and the same total nucleotide amount was reached with both enzymes. Kinetics of ATPase activity revealed a much higher negative cooperativity for mutant than for wild-type F1. Bicarbonate abolished this negative cooperativity, but higher concentrations were required for mutant F1. The mutant enzyme was more sensitive than the wild-type one to azide inhibition and ADP competitive inhibition; this indicated stronger interactions between nucleotide and F1 in the mutant enzyme. The latter also showed increased sensitivity to N,N'-dicyclohexylcarbodiimide irreversible inhibition.     

Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum

Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity. The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1). A 3-fold reduction of the kcat and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed. Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues. In the presence of 50 mM methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue. Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis. Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser. Seven of the mutant enzymes had specific activities lower than 50% of the wild-type. Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate. These findings suggest an important functional or structural role for His-516 in the wild-type enzyme.     

Mutants of Streptococcus gordonii Challis over-producing glucosyltransferase

Two mutants of Streptococcus gordonii which over-produced extracellular polysaccharide when grown on sucrose-containing medium were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The mutants, designated strains OB20 and OB30, expressed 2.6-fold and 4.7-fold respectively more glucosyltransferase (GTF) activities than the wild-type strain. Transformation experiments suggested that the two mutants carried different mutations, denoted gtf-20 and gtf-30. A double mutant (gtf-20 gtf-30) was constructed and this strain produced 6.4-fold more GTF. Enzymes from wild-type and mutant strains were biochemically indistinguishable and they synthesized structurally identical glucans. Increasing the Na+ concentration of the bacterial growth medium reduced GTF production in all strains by about 60%. Tween 80 also inhibited enzyme production and more specifically reduced GTF synthesis by the mutants. The mutations gtf-20 and gtf-30 appear to define separate genetic loci involved in regulating expression of GTF activity in S. gordonii.     

Escherichia coli E-39 ADPglucose synthetase has different activation kinetics from the wild-type allosteric enzyme

Kinetic and binding studies have shown that Lys39 of Escherichia coli ADPglucose synthetase is involved in binding of the allosteric activator. In order to study structure-function relationships at the activator binding site, this lysine residue was substituted by glutamic acid (Lys39----Glu) by site-directed mutagenesis. The resultant mutant enzyme (E-39) showed activation kinetics different from those of the wild-type enzyme. The level of activation of the E-39 enzyme by the major activators of E. coli ADPglucose synthetase, 2-phosphoglycerate, pyridoxal phosphate, and fructose-1,6-phosphatase was only approximately 2-fold compared to activation of 15- to 28-fold respectively, for the wild-type enzyme. NADPH, an activator of the wild-type enzyme, was unable to activate the mutant enzyme. In addition, the concentrations of the above activators necessary to obtain 50% of the maximal stimulation of enzyme activity (A0.5) were 5-, 9-, and 23-fold higher, respectively, than those for the wild-type enzyme. The E-39 enzyme also had a lower apparent affinity (S0.5) for the substrates ATP and MgCl2 than the wild-type enzyme and the values obtained in the presence or absence of activator were similar. The concentration of inhibitor giving 50% of enzyme activity (I0.5) was also similar for the E-39 enzyme in the presence or absence of activator. These results indicate that the E-39 mutant enzyme is not effectively activated by the major activators of the E. coli ADPglucose synthetase wild-type enzyme, and that this amino acid substitution also prevents the allosteric effect that the activator has on the wild-type enzyme kinetics, either increasing its apparent affinity for the substrates or modulating the enzyme's sensitivity to inhibition.     

In vitro reconstitution of cdc25 regulated S. cerevisiae adenylyl cyclase and its kinetic properties.

The attenuated GTP regulation adenylyl cyclase (CDC35) lysates or membranes prepared from cells of a cdc25ts strain is enhanced 2.5- to 6-fold by mixing these lysates or membranes with lysates or membranes from a cdc35ts strain harboring wild-type CDC25. The kinetics of activation of the Saccharomyces cerevisiae adenylyl cyclase in vitro is first order, as is the activation of mammalian adenylyl cyclase. The rate of enzyme activation in the presence of non-hydrolysable analogs of GTP increases with the number of CDC25 gene copies present in the cell. When GppNHp was used the rate of activation of the cyclase in a strain harboring a multicopy plasmid of CDC25 was 7.0-fold higher than the rate in an isogenic strain with the cdc25-2 mutation. The rate of adenylyl cyclase activation from a strain with a disrupted CDC25 gene is 14.7-fold lower than the rate in an isogenic strain containing the CDC25 gene on a multicopy plasmid. The reconstitution experiments described provide direct biochemical evidence for the role of the CDC25 protein in regulating the RAS dependent adenylyl cyclase in S.cerevisiae. The reconstitution experiments and the kinetic experiments may also provide a biochemical assay for the CDC25 protein and can form the basis for its characterization. In this study we also show that adenylyl cyclase activity in ras1ras2byc1 cells is found in the soluble fraction, whereas in wild-type strain it is found in the membrane fraction. Overexpression of the gene CDC25 in the ras1ras2bcy1 strain relocalizes adenylyl cyclase activity to the membrane fraction. This finding suggests a biochemical link between CDC25 and CDC35 in the absence of RAS, in addition to its role in regulating RAS dependent adenylyl cyclase.     

Formation of Isoamylase by Pseudomonas

We have isolated a Pseudomonas sp. (strain SB15) which produces an isoamylase (EC 3.2.1.9). Highest yields of this enzyme were obtained when the bacterium was grown in shaken culture in a medium containing maltose, dextrin, starch, or isomaltose. Specific carbon and nitrogen sources were required for growth. The most satisfactory medium consisted of 2% maltose, 0.4% sodium glutamate, 0.3% diammonium hydrogen phosphate, and other inorganic salts. The optimal pH for enzyme production was 5 to 6. The enzyme is stable between pH 3 and 6 but is extremely labile above pH 7. It splits amylopectin completely by combined action with beta-amylase but does not attack pullulan.     

High-level expression of a novel FMN-dependent heme-containing lyase,phenylacetaldoxime dehydratase of Bacillus sp. strain OxB-1, in heterologous hosts

We examined the overexpression of a novel FMN-dependent heme-containing lyase, phenylacetaldoxime dehydratase (Oxd) of Bacillus sp. strain OxB-1, in Escherichia coli and Bacillus subtilis. Several plasmids were constructed to express the enzyme under the control of the lac promoter or its own promoter, together with or without nitrilase and a possible regulatory protein that is present in the wild-type genome. The enzyme was expressed using E. coli transfected with the plasmid pOxD-9OF. Expression was under the control of the lac promoter in the pUC18 vector and was much more effective when the start codon was changed from TTG to ATG. When the transfected cells were grown at 37 degrees C, the enzyme was produced mainly in inactive inclusion bodies, whereas the enzyme was largely soluble and active when the cells were grown at 30 degrees C. The production of active enzyme was markedly enhanced by increasing the volume of culture medium. This had the effect of slowing the rate of apoenzyme synthesis. A slow rate of synthesis allows for a more efficient incorporation of heme cofactor into the apoenzyme than a fast rate of synthesis. Under optimized conditions, the enzyme was produced in an active and soluble form at 15,000U/L of culture, which is about 1500-fold higher than the amount produced by the wild-type strain. Moreover, the enzyme comprised over 40% of total extractable cellular protein.