Increase In Specific Enzyme Activity Research Articles

Characterization of a novel cellobiose phosphorylase with broad optimal pH range from a tailings pond macrogenomic library

Glucose-1-phosphate, a crucial pharmaceutical intermediate, can be generated through the synergistic transformation of lignocellulose using cellulase and cellobiose phosphorylase (CBP), thus holding promise for a profitable biorefinery process. However, the reported optimal pH range of CBPs (6.0–7.5) does not align with that of cellulase (4.8), resulting in suboptimal efficiency in the one-pot conversion of cellulose to glucose-1-phosphate. In this study, we identified a novel cellobiose phosphorylase, CBP1942, comprising 811 amino acid residues (92.5 kDa), in tailing pond macrogenomes, exhibiting identity of 60% to 75% with reported CBPs. CBP1942 was efficiently expressed in E. coli BL21 and purified with His tagging. It maintains over 90% enzyme activity across pH 4–9. Compared to the benchmark CtCBP (GenBank ID: AAL67138.1), CBP1942 displays a 1.2-fold increase in specific enzyme activity at the optimal cellulase temperature (50 °C), indicating its superior suitability for cellulase compounding. CBP1942 exhibits Km and kcat values of 6.69 mM and 8.83 s−1, respectively, with a kcat/Km ratio 6.6 times higher than CtCBP, suggesting superior catalytic efficiency. Notably, CBP1942 shows remarkable thermal stability, retaining 94.67% enzyme activity after 3 h at 50 °C, while CtCBP retains only 78.33%. Additionally, CBP1942 complexed with cellulase enhances the conversion of corn stover, resulting in a 6.2-fold increase in glucose-1-phosphate yield compared to CtCBP. These findings indicate CBP1942’s potential as an industrial enzyme for corn stover conversion to glucose-1-phosphate.

Enhanced catalytic activity and stability of lactate dehydrogenase for cascade catalysis of D-PLA by rational design

Serving as a vital medical intermediate and an environmentally-friendly preservative, D-PLA exhibits substantial potential across various industries. In this report, the urgent need for efficient production motivated us to achieve the rational design of lactate dehydrogenase and enhance catalytic efficiency. Surprisingly, the enzymatic properties revealed that a mutant enzyme, LrLDHT247I/D249A/F306W/A214Y (LrLDH-M1), had a viable catalytic advantage. It demonstrated a 3.3-fold increase in specific enzyme activity and approximately a 2.08-fold improvement of Kcat. Correspondingly, molecular docking analysis provided a supporting explanation for the lower Km and higher Kcat/Km of the mutant enzyme. Thermostability analysis exhibited increased half-lives and the deactivation rate constants decreased at different temperatures (1.47–2.26-fold). In addition, the mutant showed excellent resistance abilities in harsh environments, particularly under acidic conditions. Then, a two-bacterium (E. coli/pET28a-lrldh-M1 and E. coli/pET28a-ladd) coupled catalytic system was developed and realized a significant conversion rate (77.7%) of D-phenyllactic acid, using 10 g/L L-phenylalanine as the substrate in a two-step cascade reaction.

Template-Independent Poly(A)-Tail Decay and RNASEL as Potential Cellular Biomarkers for Prostate Cancer Development.

Simple SummaryThe ultimate need in cancer tissue is to adapt translation machinery to accelerated protein synthesis in a rapidly proliferating environment. Our study was designed with the aim of integrating fundamental and clinical research to find new biomarkers for prostate cancer (PC) with clinical usefulness for the stratification prediction of healthy tissue transition into malignant phenotype. This study revealed: (i) an entirely novel mechanism of the regulatory influence of Poly(A) deadenylase in mRNAs translational activity and the 3′ mRNA untranslated region (3′UTR) length in cancer tissue and its regulation by the poly(A) decay; (ii) the RNASEL interrelationship with the inflammatory pattern of PC and corresponding tumor-adjacent and healthy tissue; and (iii) the sensitivity, specificity, and predictive value of these enzymes. The proposed manuscript is based on the use of specific biochemical and immunoassay methods with the principal research adapted for the use of tissue specimens.The post-transcriptional messenger RNA (mRNA) decay and turnover rate of the template-independent poly(A) tail, localized at the 3′-untranslated region (3′UTR) of mRNA, have been documented among subtle mechanisms of uncontrolled cancer tissue growth. The activity of Poly(A) deadenylase and the expression pattern of RNASEL have been examined. A total of 138 prostate tissue specimens from 46 PC patients (cancer specimens, corresponding adjacent surgically healthy tissues, and in their normal counterparts, at least 2 cm from carcinoma) were used. For the stratification prediction of healthy tissue transition into malignant phenotype, the enzyme activity of tumor-adjacent tissue was considered in relation to the presence of microfocal carcinoma. More than a four-times increase in specific enzyme activity (U/L g.prot) was registered in PC on account of both the dissociation of its inhibitor and genome reprogramming. The obtained ROC curve and Youden index showed that Poly(A) deadenylase identified PC with a sensitivity of 93.5% and a specificity of 94.6%. The RNASEL expression profile was raised significantly in PC, but the sensitivity was 40.5% and specificity was 86.9%. A significantly negative correlation between PC and control tissue counterparts with a higher expression pattern in lymphocyte-infiltrated samples were reported. In conclusion, significantly upregulated Poly(A) deadenylase activity may be a checkpoint for the transition of precancerous lesion to malignancy, while RNASEL may predict chronic inflammation.

Purification and characterization of a carboxymethyl cellulase from Artemia salina

Brine shrimp (Artemia salina) belong to a group of crustaceans that feed on microalgae and require a cellulase enzyme that can be used in ethanol production from marine algae. Protein with potential cellulase activity was purified and the activity analyzed under different conditions. After initial identification of cellulase activity by CMC cellulase, surface sterilization and PCR using 16s rRNA primers was conducted to confirm that the cellulase activity was not produced from contaminating bacteria. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. After the final purification, a 70-fold increase in specific enzyme activity was observed. SDS–PAGE results revealed that the cellulase enzyme had a molecular mass of 96kDa. Temperature, pH, and salinity values were found to be optimal at 55°C, pH 8.0, and 600mM NaCl, respectively. Specifically, the enzyme showed a fivefold increase in enzyme activity in seawater compared to 600mM NaCl in phosphate buffer. Further analysis of the purified enzyme by molecular spectrometry showed no match to known cellulases, indicating this enzyme could be a novel halophilic cellulase that can be used for the production of bioethanol from marine macroalgae.

Optimization of medium composition for extra cellular alginate lyases of a marine bacterium

nfactorial design was applied to elucidate media compositions that significantly affect alginate lyase formation. Sixteen trials were investigated and one with positive levels of yeast extract, peptone, sodium alginate and sodium chloride showed a 15-fold increase in specific enzyme activity. The F ratio revealed that two factors, sodium chloride and alginate, whether separately or combined are positively effective at a 1% level of significance.

Filarial glutathione S-transferase: its induction by xenobiotics and potential as drug target.

Glutathione-S-transferase (GST) a Phase-II drug detoxification enzyme, was detected in Setaria cervi, a bovine filarial parasite. In vitro effect of diethylcarbamazine, butylated hydroxyanisole and phenobarbitone on the GST of adult female S. cervi was assayed by the addition of these compounds in the maintenance medium. The specific activity of GST towards 1-chloro-2,4-dinitrobenzene was increased progressively 1.2-1.97, 1.3-2.4 and 1.2-2.7 times at 10-100 microM of diethylcarbamazine, butylated hydroxyanisole and phenobarbitone, respectively, after 5 h at 37 degrees C. Substrate specificity studies showed a higher increase in specific activity with ethacrynic acid and no change with cumene hydroperoxide. Although the intensity of GST activity band was more in extract from diethylcarbamazine or butylated hydroxyanisole treated worms extract, an extra band of activity appeared in those worm extracts compared to control worm extract. SDS/PAGE showed increased thickness of the band corresponding to purified GST in extracts from diethylcarbamazine/butylated hydroxyanisole/phenobarbitone treated worms. Purification and quantification of GST from diethylcarbamazine and butylated hydroxyanisole treated worms indicated an increase in enzyme specific activity. The increase in GST protein by these agents was blocked by prior treatment with actinomycin D, indicative of a transcription dependent response. The role of this enzyme in motility and viability of microfilariae and adult female was tested in vitro using a range of known GST inhibitors. Of those tested, ethacrynic acid, ellagic acid, 1-chloro-2,4-dinitrobenzene, cibacron blue and butylated hydroxyanisole reduced the viability and motility of microfilariae and adult female worms at micromolar concentrations. These results suggest that S. cervi GST is inducible in response to the antifilarial drug diethylcarbamazine and may play an important role in parasite's survival, thus could be a potential drug target.

Phosphorylation of eNOS initiates excessive NO production in early phases of portal hypertension.

Akt, also known as protein kinase B, is a serine/threonine kinase. Akt becomes active when phosphorylated by the activation of receptor tyrosine kinases, G protein-coupled receptors, and mechanical forces such as shear stress. Studies in vitro have shown that Akt can directly phosphorylate endothelial nitric oxide (NO) synthase (eNOS) and activate the enzyme, leading to NO production. The aim of this study was to test the hypothesis that the phosphorylation of eNOS plays a role in the enhanced NO production observed in early portal hypertension. Male Sprague-Dawley rats were subjected to either sham or portal vein ligation (PVL), and mesenteric arterial beds were used for ex vivo perfusion studies. Mesenteric arterial beds from PVL rats had an approximately 60-70% decrease in response to methoxamine (an alpha(1)-agonist and vasoconstrictor) compared with the sham group (P < 0.01). When N(G)-monomethyl-L-arginine (a NOS inhibitor) was added to the perfusion, the difference in perfusion pressure between the two groups was abolished, suggesting that enhanced NO production in the PVL group blunted the response to the vasoconstrictor. The reduced responsiveness in PVL was not due to changes in eNOS expression but was due to an increase in enzyme-specific activity, suggesting posttranslational modification of eNOS. The phosphorylation of eNOS at Ser(1176) was significantly increased by twofold (P < 0.05) in the PVL group. Furthermore, PVL significantly increased Akt phosphorylation (an active form of Akt) by threefold (P < 0.05). When vessels were treated with wortmannin (10 nM) to block the phosphatidylinositol-3-OH-kinase/Akt pathway, NO-induced vasodilatation was significantly reduced. These results suggest that the phosphorylation of eNOS by Akt activates the enzyme and may be the first step leading to an initial increase in NO production in portal hypertension.

Variable specific activity of Escherichia coli β‐galactosidase in bacterial cells

Escherichia coli lacZ is a frequently employed reporter gene for the monitoring of gene expression and recombinant protein production due the simple determination of beta-galactosidase activity in both qualitative and quantitative assays. In the absence of either total or recombinant protein synthesis, we observed a lack of correlation between protein amount and enzymatic activity in both engineered and native beta-galactosidases in Escherichia coli cells. A delayed fading of beta-galactosidase activity compared with the rapid degradation of intact protein suggests a progressive increase in enzyme-specific activity during the life of the protein. This intriguing event does not involve solubilization from major protein aggregates and it occurs both in vivo and in cell extracts, but not in solutions of purified protein. Possible explanations for this activation are examined in the context of the assisted protein folding network and proteolytic degradation of misfolded proteins.

Development of a mixed mode adsorption process for the direct product sequestration of an extracellular protease from microbial batch cultures

Direct product sequestration of extracellular proteins from microbial batch cultures can be achieved by continuous or intermittent broth recycle through an external extractive loop. Here, we describe the development of a fluidisable, mixed mode adsorbent, designed to tolerate increasing ionic strength (synonymous with extended productive batch cultures). This facilitated operations for the integrated recovery of an extracellular acid protease from cultures of Yarrowia lipolytica. Mixed mode adsorbents were prepared using chemistries containing hydrophobic and ionic groups. Matrix hydrophobicity and titration ranges were matched to the requirements of integrated protease adsorption. A single expanded bed was able to service the productive phase of growth without recourse to the pH adjustment of the broth previously required for ion exchange adsorption. This resulted in increased yields of product, accompanied by further increases in enzyme specific activity. A step change from pH 4.5 to 2.6, across the isoelectric point of the protease, enabled high resolution fixed bed elution induced by electrostatic repulsion. The generic application of mixed mode chemistries, which combine the physical robustness of ion-exchange ligands in sanitisation and sterilisation procedures with a selectivity, which approaches that of affinity interactions, is discussed.

Production of D-p-hydroxyphenylglycine by N-carbamoyl-D-amino acid amidohydrolase-overproducing Escherichia coli strains.

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene from Agrobacterium radiobacter NRRL B11291 has been successfully cloned and expressed in Escherichia coli. Subcloning of the D-carbamoylase gene into different types of vectors and backgrounds of E. coli strains showed that the optimal expression level of D-carbamoylase was achieved in a ColE1-derived plasmid with a 150-fold increase in specific enzyme activity compared to that in a pSC101-derived plasmid. In addition, the recombinant plasmids were very stable in the E. coli strain ATCC11303 but not in JCL1258 tested here. Employing the recombinant E. coli strain DH5alpha/pAH61 for D-p-hydroxyphenylglycine production showed that the cell was capable of transforming N-carbamoyl-D-hydroxylphenylglycine to D-p-hydroxyphenylglycine with a molar conversion yield of 100% and a production rate of 1.9 g/(L h). In comparison with A. radiobacter NRRL B11291, this productivity approximates a 55-fold increase in D-hydroxyphenylglycine production. This result suggests the potential application of recombinant E. coli strains for the transformation reaction.

Improved stearate phenotype in transgenic canola expressing a modified acyl-acyl carrier protein thioesterase.

The engineering of crops for selected fatty acid production is one of the major goals of plant biotechnology. The Garm FatA1, an acyl-acyl carrier protein (ACP) thioesterase isolated from Garcinia mangostana, generates an elevated stearate (18:0) phenotype in transgenic Brassica plants. By site-directed mutagenesis, we generated seven mutants that showed up to a 13-fold increase in specific enzyme activity toward 18:0-ACP in vitro. The seed-specific expression of mutant S111A/V193A in Brassica plants results in transgenic plants that accumulate 55-68% more stearate than plants expressing the wild-type enzyme. Our results demonstrate that a thioesterase can be engineered to increase specific activity and that its improved function demonstrated in vitro is retained in vivo.

Lichenase production by catabolite repression-resistant Bacillus subtilis mutants: optimization and formulation of an agro-industrial by-product medium

Among four isogenic catabolite repression-resistant Bacillus subtilis mutants grown on four different media, the strain crsA47 and the formula MII were chosen. Selection was supported by analyzing the results using the two-way Anova test. Supplementation of the medium with pectin resulted in a 1.33-fold increase in enzyme specific activity. A 2 n factorial experiment was then applied to elucidate medium components that significantly affect enzyme formation. Significant factors were simultaneously optimized using the steepest ascent method. Optimized medium (tryptone, 10; yeast extract, 10; pectin, 2; and NaCl, 2 g l −1) yielded a crude filtrate with lichenase activity of 114 U ml −1 and a specific activity of 20 U mg −1 dry protein within 24 h of cultivation. Catabolite repression of lichenase synthesis was further limited by growing the crsA47 mutant on complex raw materials. The enzyme was maximally produced (123 U ml −1, 38 U mg −1 protein) by the cofermentation of cotton seed meal (36 g l −1) and lemon peel (9 g l −1).

Osmoregulation and branchial Na +,K +-ATPase in the lobster Homarus gammarus acclimated to dilute seawater

Osmoregulatory ability and Na +,K +-ATPase activity in gills and epipodites were examined following transfer of the lobster Homarus gammarus from sea water (SW; 1292 mOsmol l −1; 38 ppt salinity) to dilute seawater (DSW; 680±5 mOsmol l −1; 20 ppt salinity). The lobster H. gammarus behaves as an osmoconformer in sea water and a poor hyperosmoregulator when equilibrated in dilute seawater, where haemolymph was maintained 154 mOsmol l −1 above that of the medium. Lobsters could withstand direct transfer from SW to DSW and, after about 12 h, the initial drop in blood osmoconcentration and principal inorganic osmolytes ceased and a gradual increase followed during the next 15 days. Chloride and sodium were the main osmotic effectors responsible for the slow gradual adjustment of the blood hyperosmoticity. In DSW-acclimated lobsters, the Na +,K +-ATPase specific activities of gills (arthrobranchia and podobranchia) homogenates and partially purified membrane vesicle fractions were, respectively, 2.0 and 3.5 to 3.9-fold higher that those of the SW-acclimated animals. Enzyme specific activities of homogenate and membrane vesicle fractions isolated from DSW pleurobranchia were 1.4-fold higher than those of the SW-acclimated animals. In trichobranchiate gills and epipodites, saponin treatment increased the Na +,K +-ATPase activity over the native enzyme activity of the respective tissues. The plasma membrane vesicles from trichobranchiate gills and epipodites were better purified when isolated from DSW- than from SW-acclimated lobsters. Homogenates and membrane vesicles (enrichment factors ranging from seven to eight) from epipodites allowed a several-fold increase in enzyme specific activity over trichobranchiate gills. When lobsters were acclimated to DSW, the Na +,K +-ATPase specific activity measured in saponin-treated homogenates of epipodites was positively correlated with the sodium concentration gradient between haemolymph and DSW. The results suggest that epipodites Na +,K +-ATPase activation in DSW should be correlated with the blood sodium gradient, i.e. osmoregulatory ability. Short-circuit current of the hemiepipodite isolated from lobsters acclimated to DSW and mounted in a micro-Ussing chamber was −232 μA cm −2 (negative charge flow driven from apical to basolateral side of preparation) and conductance was 65 mS cm −2, a value characteristic of a leaky epithelium. Half-maximal inhibition of current by the specific Na +,K +-ATPase inhibitor ouabain occurs at 0.78 mM. At 1 mM ouabain, more then 70% of the inhibited current is Na +,K +-ATPase-related ion transport in epipodites. These results provide, for the first time, physiological evidence that, besides trichobranchiate gills, epipodites have an osmoregulatory role in the lobster Homarus gammarus.

Production of Laccase by Immobilized Cells of Agaricus sp.: Induction Effect of Xylan and Lignin Derivatives

Laccase was produced in the supernatant of culture of a local isolate of Agaricus sp. obtained from decaying Ficus religiosa wood. The enzyme was produced at a constitutive level when growing the fungus in a nitrogen-limited medium supplemented with either glycerol, glucose, fructose, mannitol, arabinose, maltose, saccharose, cellulose, or cellobiose. A two- to sixfold increase in enzyme specific activity was observed when growing the strain in the presence of straw, xylan, xylose, lignosulfonate, veratryl alcohol, and ferulic and veratric acid. Experiments are consistent with the existence of an induction control on laccase and the absence of a form of carbon catabolite repression mediated by noninducing carbon sources. Immobilization of the Agaricus sp. on several supports, including polyurethane foam, textile strips, and straw, resulted in an increase of enzyme production as compared to cultivation in liquid medium.

Functional Domains of the α1 Catalytic Subunit of the AMP-activated Protein Kinase

The AMP-activated protein kinase is a heterotrimeric enzyme, important in cellular adaptation to the stress of nutrient starvation, hypoxia, increased ATP utilization, or heat shock. This mammalian enzyme is composed of a catalytic alpha subunit and noncatalytic beta and gamma subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of Saccharomyces cerevisiae. In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the alpha1 catalytic subunit, which modulate its activity, subunit association, and protein turnover. C-terminal truncation of the 548-amino acid (aa) wild-type alpha1 protein to aa 312 or 392 abolishes the binding of the beta/gamma subunits and dramatically increases protein expression. The full-length wild-type alpha1 subunit is only minimally active in the absence of co-expressed beta/gamma, and alpha1(1-392) likewise has little activity. Further truncation to aa 312, however, is associated with a large increase in enzyme specific activity, thus revealing an autoinhibitory sequence between aa 313 and 392. alpha-1(1-312) still requires the phosphorylation of the activation loop Thr-172 for enzyme activity, yet is now independent of the allosteric activator, AMP. The increased levels of protein expression on transient transfection of either truncated alpha subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis. Taken together, these data indicate that the alpha1 subunit of AMP-activated protein kinase contains several features that determine enzyme activity and stability. A constitutively active form of the kinase that does not require participation by the noncatalytic subunits provides a unique reagent for exploring the functions of AMP-activated protein kinase.

Multiple copies of PBS2, MHP1 or LRE1 produce glucanase resistance and other cell wall effects in Saccharomyces cerevisiae.

Five sequences were isolated by selection for multiple copy plasmids that conferred resistance to laminarinase, an enzyme that specifically degrades cell wall beta(1-3) glucan linkages. Strains carrying three of these plasmids showed alterations in cell wall glucan labelling. One of these plasmids carried PBS2, a previously identified, non-essential gene which produces a variety of phenotypes and encodes a mitogen-activated protein kinase kinase analogue (Boguslawski and Polazzi, 1987). Cells carrying PBS2 at multiple copy show a small decrease in cell wall beta(1-6) glucans. Measurements of beta(1-3) glucan synthase activity in multi-copy PBS2 cells showed an approximate 30-45% increase in enzyme specific activity while a pbs2 delta disruption strain showed a decrease in glucan synthase activity of approximately 45% relative to control. A pbs2 delta disruption strain was laminarinase super-sensitive and supersensitive to K1 killer toxin while a strain carrying PBS2 at multiple copy was resistant to killer toxin. A second plasmid carried a portion of the MHP1 gene which has been reported to encode a microtubule-interacting protein (Irminger-Finger et al., 1996). The MHP1 gene product is a predicted 1398 amino acid protein and only approximately 80% of the amino portion of this protein is required for laminarinase resistance. Cells carrying the amino portion of MHP1 at multiple copy show a decrease in high molecular weight cell wall beta(1-6) glucans and were killer toxin resistant while a disruption strain was viable and killer toxin super-sensitive. Cells carrying this plasmid showed decreased levels of high molecular weight beta(1-6) glucans and increased glucan synthase activity. The laminarinase resistance conferred by the third plasmid mapped to the previously uncharacterized YCL051W open reading frame and this gene was therefore named LRE1 (laminarinase resistance). The LRE1 gene encodes a non-essential 604 amino acid hydrophilic protein. Unexpectedly, cells carrying LRE1 at multiple copy show no alteration in cell wall glucans or glucan synthase activity. Subcloning experiments demonstrated that the production of these cell wall effects requires the presence of both LRE1 and YCL052C (PBN1), a second open reading frame present on the original plasmid. Cells carrying multiple copies of PBN1 alone show no significant alterations in cell wall glucans or glucan synthase activity, indicating that these effects require the presence of multiple copies of both genes.

Regulation of 5′-AMP-activated Protein Kinase Activity by the Noncatalytic β and γ Subunits

The mammalian 5'-AMP-activated protein kinase is a heterotrimer consisting of an alpha catalytic subunit and beta and gamma noncatalytic subunits, each of which is represented in a larger isoprotein family, related to the SNF1 kinase and its interacting proteins in yeast. In this study, we have used mammalian cell transfection to compare the activities of the two alpha subunit isoforms, alpha-1 and alpha-2, and to study the influence of the noncatalytic subunits on enzyme subunit association and activity. Expression of epitope-tagged protein subunits in COS7 cells indicates detectable but low level kinase activity for each of the two catalytic alpha subunits. Co-expression of alpha subunits with the beta or gamma subunits modestly increases kinase activity accompanied by the formation of alpha/beta or alpha/gamma heterodimers. Co-expression of all three subunits, however, is accompanied by a 50-110-fold increase in kinase activity with the formation of a heterotrimeric complex. In addition to binding of each noncatalytic subunit to the alpha subunit, the beta and gamma subunits bind to each other, likely resulting in a more stable heterotrimeric complex. The increase in kinase activity associated with expression of this heterotrimer is due both to an increase in enzyme-specific activity (units/enzyme mass) and to an apparent enhanced alpha subunit expression. Co-expression of a catalytically defective alpha subunit or the beta/gamma-binding COOH-terminal domain of the alpha subunit results in reduced heterotrimeric kinase activity. The synergistic positive regulatory roles for both the noncatalytic beta and gamma subunits of 5'-AMP-activated protein kinase contrasts with the Snf1p kinase, where only heterodimers of Snf1p and Snf4p seem to be required for maximum kinase activity.

Correlation of isocitrate lyase activity and riboflavin formation in the riboflavin overproducer Ashbya gossypii

Isocitrate lyase (ICL) was assayed during batch cultivations of Ashbya gossypii on soybean oil or glucose as carbon source. On soybean oil, a correlation between enzyme activity and riboflavin synthesis was observed. On glucose as carbon source, riboflavin overproduction started in the late growth phase when glucose was exhausted. ICL activity appeared in parallel and reached a maximum of 0.41 U (mg protein)-1. This suggested synthesis of vitamin B2 from the intracellular reserve fat. ICL specific activity correlated with the enzyme concentration detected by specific antibodies. Itaconate, an efficient inhibitor of ICL, was used as an antimetabolite to screen mutants with enhanced ICL activity. Cultivations of an itaconate-resistant mutant on soybean oil revealed a 15% increase in enzyme specific activity and a 25-fold increase in riboflavin yield compared to the wild-type. On the other hand, growth experiments on glucose resulted in an eightfold increase in riboflavin yield but showed a 33% reduction in ICL specific activity compared to the wild-type grown on the same medium. These results support the idea of an ICL bottleneck in the riboflavin overproducer A. gossypii when plant oil is used as the substrate.

Effects of Acids and Storage Temperatures on the Phospholipase A Activities in Oyster Digestive Glands.

Effects of the acids and storage temperatures on the phospholipase A (PLase A) activities in oyster digestive glands (ODG) were investigated. The results were summarized as follows ; 1) Specific activities of both PLase A1 and A2 in ODG treated with acetic acid and stood at 37°C were higher than those in control group. The increase of these enzyme activities in ODG treated with 4% acetic acid (pH 2.5) was larger than those with 1% and 16% acetic acid. The enhancement of the enzyme activities in ODG was induced within 1 hr after the treatment. 2) No significant change was observed in the specific activities of PLase A1 and A2 in ODG treated with 0.01 N HCl (pH 2.2) and stood at 37°C throughout experimental period (5 h). The values were not different from those in control group. 3) Protein contents in all ODG samples treated with acids were lower than those in control group. The ODG treated with 4% acetic acid at 37°C had the lowest value. 4) Specific activities of both PLase A1 and A2 increased 3 d after the storage at 5°C. The increase of these specific activities observed until 2 weeks after the storage at 5°C was considered to be due to the decrease ofprotein contents. The degree of increase in enzyme specific activity at 3 weeks, however, was proportionally larger than that of the decrease in protein contents. No significant change was observed in PLase A activity in ODG stored at both -20°C and -70°C. 5) The optimum temperatures of PLase A1 and A2 were 37°C. The results showed that an increase of PLase A activity was induced in the early stage of oyster marination stood at 37°C. It was suggested that hydrolysis in phospholipid and protein denaturation would precede the enhancement of PLase A, and temperature could be one of the important factors on the enzyme activation.

Effects of ultraviolet light irradiation in biotreatment of organophosphates.

Excess biomass accumulation in reactor biodegradation processes is undesirable: it increases the disposal cost and upsets the operation of biological reactors if not properly controlled. In this study, we investigated the feasibility of using ultraviolet (UV) light irradiation to reduce biomass accumulation and increase the specific biodegradation activity. UV irradiation has been widely used to introduce DNA damage in bacteria. Here we apply this technology to the biodegradation of organophosphates by recombinant Escherichia coli strains that contain a recA mutation and a cloned organophosphate hydrolase gene. We show that the recA negative strains after UV irradiation reduce the growth rate but increase the specific organophosphate hydrolase activity. This increase in specific enzyme activity is not owing to continued protein synthesis from the plasmid after the damage of chromosomal DNA by UV irradiation. Rather, it is likely to be caused by an increase in membrane permeability to the substrate. Kinetic analysis suggests that the membrane transport of paraoxon is the rate-limiting step in its biodegradation.