Unit Of Enzyme Activity Research Articles

ENZYMATIC ACIDOLYSIS OF EVENING PRIMROSE OIL WITH DOCOSAHEXAENOIC ACID USING RESPONSE SURFACE METHODOLOGY

ABSTRACT Lipase‐catalyzed acidolysis of evening primrose oil with docosahexaenoic acid (DHA) was carried out using Novozym‐435 from Candida antarctica as the biocatalyst. Several parameters, namely the amount of enzyme (X1; 100–200 enzyme activity units), incubation temperature (X2; 30–60C) and reaction time (X3; 18–30 h) were examined as independent variables of face‐centered cube design. Using response surface methodology (RSM), the optimum reaction conditions for DHA incorporation into evening primrose oil were determined and quadratic response surface was drawn from the mathematical model. The results demonstrated that the amount of enzyme, reaction temperature and reaction time significantly (P ≤ 0.05) affected the incorporation of DHA into evening primrose oil. Optimum reaction conditions for DHA incorporation were 162 units of enzyme at 43C in 27 h. The optimum value predicted by RSM for DHA incorporation was 31.9%. Analysis of variance showed that 95% (R2 = 0.95) of the observed variation was explained by the polynomial model. Close agreement existed between experimental and predicted values. The positional distribution of fatty acids in DHA‐enriched evening primrose indicated that DHA was randomly distributed among the sn‐2 and sn‐1 + sn‐3 positions of the DHA‐enriched evening primrose oil.

Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1.

α-Amylase from Thermococcus hydrothermalis: Re-cloning aimed at the improved expression and hydrolysis of corn starch

A gene encoding the thermostable α-amylase from hyperthermophilic archaeon Thermococcus hydrothermalis was cloned in Escherichia coli. In an effort to achieve the improved expression, four DNA primers (three reverse and one forward) were designed to yield three recombinant variants of α-amylase. The additional aim of the re-cloning was to verify how the changes at the C-teminal end of the recombinant α-amylase introduced by the pET vector and 6-His tag may affect the resulting specific activity of the variants. Biochemical characteristics of the recombinant α-amylases, i.e. molecular weight, temperature optimum, and pH optimum, were confirmed to be the same as those of the original α-amylase: 53.6 kDa, 85 °C, and 5.5, respectively. Concerning the specific activities, the addition of two residues succeeded by six histidines (LEHHHHHH) had in fact no influence, whereas the insertion of a longer peptide between the original α-amylase C-terminus and the 6-His tag (DPNSSSVDKLAAALEHHHHHH) caused a substantial decrease (more than 40%) in comparison with the specific activity of the original protein. The recombinant α-amylases were preliminary tested for their ability to hydrolyze 1% and 10% corn starch suspensions. Two types of starch were used: Meritena 100 (amylose starch) and Meritena 300 (amylopectin starch). Concerning the α-amylase concentration sufficient to reach the industrial requirements, i.e. dextrose equivalent 6–12% in 3 h, one unit of enzyme activity at defined conditions per mg starch can be considered as optimal concentration with 10% starch suspension. It was found that even a simple and partial purification (boiling the enzyme supernatant for 5 min) is enough for improving the course of corn starch hydrolysis in order to accomplish the technological parameters in a reasonable time.

CpSufE Activates the Cysteine Desulfurase CpNifS for Chloroplastic Fe-S Cluster Formation

CpNifS, a cysteine desulfurase required to supply sulfur for ironsulfur cluster biogenesis in Arabidopsis thaliana chloroplasts, belongs to a class of NifS-like enzymes with low endogenous cysteine desulfurase activity. Its bacterial homologue SufS is stimulated by SufE. Here we characterize the Arabidopsis chloroplast protein CpSufE, which has an N-terminal SufE-like domain and a C-terminal BolA-like domain unique to higher plants. CpSufE is targeted to the chloroplast stroma, indicated by green fluorescent protein localization and immunoblot experiments. Like CpNifS, CpSufE is expressed in all major tissues, with higher expression in green parts. Its expression is light-dependent and regulated at the mRNA level. The addition of purified recombinant CpSufE increased the Vmax for the cysteine desulfurase activity of CpNifS over 40-fold and decreased the KM toward cysteine from 0.1 to 0.043 mm. In contrast, CpSufE addition decreased the affinity of CpNifS for selenocysteine, as indicated by an increase in the KM from 2.9 to 4.17 mm, and decreased the Vmax for selenocysteine lyase activity by 30%. CpSufE forms dynamic complexes with CpNifS, indicated by gel filtration, native PAGE, and affinity chromatography experiments. A mutant of CpSufE in which the single cysteine was changed to serine was not active in stimulating CpNifS, although it did compete with WT CpSufE. The iron-sulfur cluster reconstitution activity of the CpNifS-CpSufE complex toward apoferredoxin was 20-fold higher than that of CpNifS alone. We conclude that CpNifS and CpSufE together form a cysteine desulfurase required for iron-sulfur cluster formation in chloroplasts.

Inactivation of Saccharomyces cerevisiae and Polyphenoloxidase in Mango Nectar Treated with UV Light

Fresh mango nectar was processed by UV light at five flow rates (0.073 to 0.451 liter/min) and five UV light doses (75 to 450 kJ/m2) to evaluate total microbial load, Saccharomyces cerevisiae survival, and polyphenoloxidase activity. UV systems containing an inner mercury lamp (254 nm) each with intensity of 25 mW/cm2 were used as germicidal sources. In addition, mango nectar was treated for 15 min at 0.073 and 0.451 liter/min, stored at 3°C, and evaluated periodically for total microbial count, yeast count, color, and polyphenoloxidase activity. The first-order kinetics modeling found that DUV-values in mango nectar ranged from 27.9 to 10.9 min (R2 > 0.950) and 26.0 to 11.8 min (R2 > 0.962) for total microbial count and yeast count, respectively. The maximum log reduction (CFU per milliliter) was 2.71 and 2.94 for total microbial count and yeast count, respectively, after 30 min of UV treatment at 0.451 liter/min. DUV-values ranging from 156 to 204 min were observed for polyphenoloxidase activity. The remaining polyphenoloxidase activity after 30 min of UV treatment at 0.451 liter/min was 19 ± 4%. Initial microbial load and yeast in stored mango nectar were reduced in the range 2.86 to 3.41 and 1.82 to 1.97 log (CFU/ml) cycles, respectively. No substantial microbial growth was observed prior to 20 days of storage. Averages of 1,055 ± 32, 803 ± 32, and 710 ± 37 enzyme activity units were observed in mango nectar UV processed at 0, 0.073, and 0.451 liter/min, respectively, during the entire storage period. However, mango nectar treated at 0.073 and 0.451 liter/min maintained a yellow and yellow-orange color, respectively, after 26 days of storage.

Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor

During dye decoloration by Trametes versicolor ATCC 20869 in modified Kirk's medium, manganese peroxidase (MnP) and laccase were produced, but not lignin peroxidase, cellobiose dehydrogenase or manganese-independent peroxidase. Purified MnP decolorized azo dyes [amaranth, reactive black 5 (RB5) and Cibacron brilliant yellow] in Mn(2+)-dependent reactions but did not decolorize an anthraquinone dye [Remazol brilliant blue R (RBBR)]. However, the purified laccase decolorized RBBR five to ten times faster than the azo dyes and the addition of a redox mediator, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), did not alter decoloration rates. Amaranth and RB5 were decolorized the most rapidly by MnP since they have a hydroxyl group in an ortho position and a sulfonate group in the meta position relative to the azo bond. During a typical batch decoloration with the fungal culture, the ratio of laccase:MnP was 10:1 to 20:1 (based on enzyme activity) and increased to greater than 30:1 after decoloration was complete. Since MnP decolorized amaranth about 30 times more rapidly than laccase per unit of enzyme activity, MnP should have contributed more to decoloration than laccase in batch cultures.

Identification and Molecular Cloning of a Novel Glycoside Hydrolase Family of Core 1 Type O-Glycan-specific Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum

We found endo-alpha-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590-1381 amino acid residues) exhibited 31-53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Galbeta1,3GalNAc disaccharide from Galbeta1,3GalNAcalpha1pNP and asialofetuin, but did not release GalNAc, Galbeta1,3(GlcNAcbeta1,6)GalNAc, GlcNAcbeta1,3GalNAc, and Galbeta1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by (1)H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-alpha-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).

MJ0917 in Archaeon Methanococcus jannaschii Is a Novel NADP Phosphatase/NAD Kinase

NAD kinase phosphorylates NAD(+) to form NADP(+). Conversely, NADP phosphatase, which has not yet been identified, dephosphorylates NADP(+) to produce NAD(+). Among the NAD kinase homologs, the primary structure of MJ0917 of hyperthermophilic archaeal Methanococcus jannaschii is unique. MJ0917 possesses an NAD kinase homologous region in its C-terminal half and an inositol-1-phosphatase homologous region in its N-terminal half. In this study, MJ0917 was biochemically shown to possess both NAD kinase and phosphatase activities toward NADP(+), NADPH, and fructose 1,6-bisphosphate, but not toward inositol 1-phosphate. With regard to the phosphatase activity, kinetic values indicated that NADP(+) is the preferred substrate and that MJ0917 would function as a novel NADP phosphatase/NAD kinase showing conflicting dual activities, viz. synthesis and degradation of an essential NADP(+). Furthermore, in vitro analysis of MJ0917 showed that, although MJ0917 could supply NADP(+), it prevented excess accumulation of NADP(+); thus, it has the ability to maintain a high NAD(+)/NADP(+) ratio, whereas 5'-AMP would decrease this ratio. The evolutionary process during which MJ0917 arose is also discussed.

Energetics of Substrate Binding and Catalysis by Class 1 (Glycosylhydrolase Family 47) α-Mannosidases Involved in N-Glycan Processing and Endoplasmic Reticulum Quality Control

Nascent glycoproteins are subject to quality control in the lumen of the endoplasmic reticulum (ER) where they can either be effectively folded with the aid of a collection of ER chaperones or they can be targeted for disposal in a process known as ER-associated degradation. Initiation of the ER disposal process involves selective trimming of N-glycans by ER alpha-mannosidase I and subsequent recognition by the ER degradation-enhancing alpha-mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The kinetics and energetics of substrate binding and catalysis by members of this family were investigated here by the analysis of wild type and mutant forms of human ER alpha-mannosidase I. The contributions of several amino acid residues and an enzyme-associated Ca(2+) ion to substrate binding and catalysis were demonstrated by a combination of surface plasmon resonance and enzyme kinetic analyses. One mutant, E330Q, shown previously to alter general acid function within the catalytic site, resulted in an enzyme that possessed increased glycan binding affinity but compromised glycan hydrolysis. This mutant protein was used in a series of glycan binding studies with a library of mannose-containing ligands to examine the energetics of Man(9)GlcNAc(2) substrate interactions. These studies provide a framework for understanding the nature of the unusual substrate interactions within the family 47 mannosidases involved in glycan maturation and ER-associated glycoprotein degradation.

High Hydrostatic Pressure Processing of Mango Puree Containing Antibrowning Agents

Natural mango puree and purees containing cysteine ( Cys, 200 ppm) or ascorbic acid ( AA, 500ppm) were treated at high hydrostatic pressure, HHP (379 to 586 MPa) for selected times (0, 0.033, 5, 10, 15 and 20min) to assess changes in polyphenoloxidase (PPO) activity. Mango purees were stored at 31°C. Colour parameters ( L*, a* and b*) were analysed to evaluate colour stability. PPO activity was inhibited from 93.8 to 89.7 or 85.5 enzyme activity units (EAU) after adding AAor Cys, respectively. Such inhibition was improved after applying HHP, by an average of 69.5± 10.6, 4.2 ± 2.2 and 47.0 ± 35.2 EAU for natural purees, purees containing ascorbic acid or cysteine, respectively. No significant differences (p≥ 0.05) were observed in PPO activity at 448, 517 and 586 MPa after 10 or 20 min of treatment for natural mango puree and puree containing cysteine. The total colour difference (Δ E*) increased as storage time increased. However, this colour difference was barely noticeable ( p≥ 0.05) after 27 days of storage for natural puree (9.1 ± 0.9), puree containing ascorbic acid (8.8 ± 1.2), and cysteine (16.4 ± 3.5).

Production of tannase by Aspergillus niger HA37 growing on tannic acid and Olive Mill Waste Waters

Production of tannase (tannin acyl hydrolase, EC 3.1.1.20) by Aspergillus nigerHA37 on a synthetic culture medium containing tannic acid at different concentrations has been studied. Maximal enzymatic activity increased according to the initial concentration of tannic acid; respectively 0.6, 0.9 and 1.5 enzyme activity units (EU) ml−1 medium in the presence of 0.2%, 0.5% and 1% of tannic acid. Tannase production by A. niger HA37 on fourfold diluted olive mill waste waters (OMWW) as substrate, was between 0.37 and 0.65 EU ml−1. Enzyme production on the diluted OMWW remained globally stable during more than 30 h. Growth of A. niger HA37 on OMWW was correlated with about 70% degradation of phenolic compounds present in the waste. This strain has therefore the capacity to degrade complex wastewaters which cause environmental damage to aquatic streams.

Discovery of Pectin-degrading Enzymes and Directed Evolution of a Novel Pectate Lyase for Processing Cotton Fabric

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.

Complete Reversal of Coenzyme Specificity of Xylitol Dehydrogenase and Increase of Thermostability by the Introduction of Structural Zinc

Pichia stipitis NAD(+)-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD(+) to NADP(+) and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp(207), Ile(208), Phe(209), and Asn(211) in the discrimination between NAD(+) and NADP(+). Single mutants (D207A, I208R, F209S, and N211R) improved 5 approximately 48-fold in catalytic efficiency (k(cat)/K(m)) with NADP(+) compared with the wild type but retained substantial activity with NAD(+). The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k(cat)/K(m) with NADP(+), but they still preferred NAD(+) to NADP(+). The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k(cat)/K(m) with NADP(+) than the wild-type enzyme, reaching values comparable with k(cat)/K(m) with NAD(+) of the wild-type enzyme. Because most NADP(+)-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP(+).

Catalytic Mechanism of Chlamydia trachomatis Flavin-dependent Thymidylate Synthase

Here we report on a Chlamydia trachomatis gene that complements the growth defect of a thymidylate synthase-deficient strain of Escherichia coli. The complementing gene encodes a 60.9-kDa protein that shows low level primary sequence homology to a new class of thymidylate-synthesizing enzymes, termed flavin-dependent thymidylate synthases (FDTS). Purified recombinant chlamydial FDTS (CTThyX) contains bound flavin. Results with site-directed mutants indicate that highly conserved arginine residues are required for flavin binding. Kinetic characterization indicates that CTThyX is active as a tetramer with NADPH, methylenetetrahydrofolate, and dUMP required as substrates, serving as source of reducing equivalents, methyl donor, and methyl acceptor, respectively. dTMP and H(4)folate are products of the reaction. Production of H(4)folate rather than H(2)folate, as in the classical thymidylate synthase reaction, eliminates the need for dihydrofolate reductase, explaining the trimethoprim-resistant phenotype displayed by thyA(-) E. coli-expressing CTThyX. In contrast to the extensively characterized thyA-encoded thymidylate synthases, which form a ternary complex with substrates dUMP and CH(2)H(4)folate and follow an ordered sequential mechanism, CTThyX follows a ping-pong kinetic mechanism involving a methyl enzyme intermediate. Mass spectrometry was used to localize the methyl group to a highly conserved arginine, and site-directed mutagenesis showed this arginine to be critical for thymidylate synthesizing activity. These differentiating characteristics clearly distinguish FDTS from ThyA, making this class of enzymes attractive targets for rational drug design.

Performance, nutrient digestibility and intestinal disaccharidase activity of weaner/grower pigs given diets containing extruded Chinese stored brown rice with exogenous enzyme supplements

AbstractOne performance and one ileal apparent digestibility (IAD) trial were conducted to investigate the performance, nutrient digestibility and intestinal disaccharidase activity of weaner/grower pigs given diets containing Chinese stored brown rice (CSBR) extruded and with exogenous enzyme supplements. In experiment 1, 96 crossbred (Duroc X Landrace X Large White) piglets weaned at 24 days of age were randomly divided into four groups according to a 2 X 2 factorial design. The animals were given four diets: (1) CSBR without enzyme supplementation, (2) CSBR supplemented with 625 mg a-amylase (2509 enzyme activity units) and 200 mg glucoamylase per kg (20018 enzyme activity units) per kg diet, (3) a normal temperature extruded CSBR (NTECSBR) without enzyme supplementation, and (4) NTECSBR supplemented with 625 mg a-amylase (2508 enzyme activity units) and 200 mg glucoamylase (20010 enzyme activity units) per kg diet. Growth, food consumption and specific activity of disaccharides in sections of the digestive tract were measured. In experiment 2, six male grower pigs with a mean initial body weight 21 kg, fitted with a simple ‘T’ cannula at the terminal ileum were used in a 6X6 Latin-square design. In addition to CSBR and NTECSBR diets with and without enzymes, a low temperature extruded diet LTECSBR with and without enzymes (as for other diets) was introduced. IAD of nutrients was measured. Extrusion resulted in a poorer (P < 0.05) food/gain for weaned pigs. Extrusion and enzyme supplementation had no influence (P > 0.05) on average daily food intake and average daily gain. Extrusion tended to improve (P = 0.075) IAD of starch but IAD of energy, dry matter, crude protein and amino acids were not influenced (P > 0.05). Enzyme supplementation had no effect on IAD of amino acids (P > 0.05), except for increasing IAD of lysine, threonine and isoleucine (P < 0.05). Extrusion and enzyme supplementation resulted in a higher (P < 0.05) maltase activity in the duodenum at day 14 of the trial. However, extrusion decreased (P < 0.05) the activity of maltase, isomaltase, sucrase and lactase in the jejunum at day 28. Enzyme supplementation increased (P < 0.05) the activities of maltase, isomaltase, trehalase and lactase in the jejunum at day 28. There was a positive extrusion X enzyme interaction (P < 0.05) for the activity of maltase and trehalase in the duodenum at day 14; a negative interaction was observed for the activity of lactase, maltase, isomaltase and trehalase in the jejunum at day 28. The results suggest that neither extrusion nor enzyme supplementation are necessary for CSBR to be used in pig diets.

A dose-response study with the feed enzyme beta-mannanase in broilers provided with corn-soybean meal based diets in the absence of antibiotic growth promoters

An experiment was designed to assess the effects of graded levels of beta-mannanase on performance and body weight uniformity of male broilers provided with diets based on corn and soybean meal and devoid of antibiotic growth promoters or coccidiostats. Four dietary treatments contained 0, 50, 80, and 110 MU of Hemicell/ ton (where 1 MU = 10(6) enzyme activity units, 100 MU/ton is manufacturer's recommendation). Each treatment contained 15 pens with 40 birds/pen. Individual bird weights were determined on d 0, 21, and 42. From 21 to 42 d of age, feed intake for the 80 MU/ton treatment was significantly greater than the 50 MU/ton treatment. beta-Mannanase inclusion at 80 or 110 MU/ton induced improvements (P < 0.05) in weight gain (3.9 to 4.8%) and feed efficiency (3.5 to 3.8%) over the control, whereas inclusion of 50 MU/ton resulted in no significant benefit. There were no significant differences between 80 or 110 MU/ton. The experiment demonstrated that dietary inclusion of beta-mannanase at approximately 50 MU/ton is not sufficient for maximum response. Inclusion at 80 MU/ ton improved broiler gains and feed conversion and increasing to 110 MU/ton resulted in no significant additional response.

Digestive capabilities reflect the major food sources in three species of talitrid amphipods

Digestive enzyme activities of three talitrid amphipods were examined to investigate the relationship between their digestive capabilities and diet. Laminarinase, cellobiase, carboxymethyl-cellulase, xylanase, α- and β-glucosidase and lipase were detected in all three species suggesting talitrid amphipods can readily digest dietary carbohydrate and lipid, including complex polysaccharides. Relatively high specific enzyme activity (Units (mg −1 digestive tract protein) −1) of laminarinase and lipase was detected in Talorchestia marmorata, a supralittoral kelp feeder which is coherent with the digestion of lipid-esters and β-glucans (laminarin) which are the main lipid and storage polysaccharides of brown seaweeds. Talorchestia sp., a low shore intertidal feeder, had high enzymatic activity of α- and β-glucosidase, cellobiase and xylanase, which is consistent with the digestion of diatoms. Keratroides vulgaris, a forest litter feeder had a relatively low specific activity of all enzymes. It is possible that leaf litter is partially digested prior to ingestion by bacteria and fungi present in the rotting vegetation, with bacterial and fungal enzymes contributing to this species' ability to hydrolyse its diet. This study provides the first quantitative data on digestive capacity in these three talitrid amphipods and confirms the relationship between dietary preference and digestive enzyme complement.

Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: purification and characterization

The chitin deacetylase gene from Colletotrichum lindemuthianum UPS9 was isolated and cloned in Pichia pastoris as a tagged protein with six added terminal histidine residues. The expressed enzyme was recovered from the culture supernatant and further characterized. A single-step purification based on specific binding of the histidine residues was achieved. The purified enzyme has a molecular mass of 25 kDa and is not glycosylated as determined by mass spectrometry. The activity of the recombinant chitin deacetylase on chitinous substrates was investigated. With chitotetraose as substrate, the optimum temperature and pH for enzyme activity are 60 °C and 8.0, respectively. The specific activity of the pure protein is 72 U/mg. One unit of enzyme activity is defined as the amount of enzyme that produces 1 μmol of acetate per minute under the assay conditions employed. The enzyme activity is enhanced in the presence of Co 2+ ions. A possible use of the recombinant chitin deacetylase for large-scale biocatalytic conversion of chitin to chitosan is discussed.

Treatment of simulated wastewater containing toxic amides by immobilized Rhodococcus rhodochrous NHB-2 using a highly compact 5-stage plug flow reactor

Biodegradation of toxic amides by immobilized Rhodococcus rhodochrous NHB-2 has been studied to generate data for future development of reactors for the treatment of simulated wastewater containing various toxic amides. The whole resting cells were immobilized in different matrices like agar, polyacrylamide and alginate. Agar gel beads were selected for the treatment of simulated wastewater containing 100mM each acetamide, propionamide, and 10mM of acrylamide and packed in a highly compact five-stage plug flow reactor. The immobilized bacterium worked well in a broad pH range from 5 to 10, with an optimum at 8.7. The apparent K m-value for the turnover of acetamide for the resting cells was determined to be around 40mM at pH 8.5 and 55°C, whereas the K m-value of the purified amidase was predicted to be about 20 mM. This organism exhibited greater turnover of aliphatic amides as compared to aromatic amides. Although these cells showed maximal amide-degrading activity at 55°C, simulated wastewater treatment was carried out at 45°C, because of the greater stability of the amidase activity at that temperature. Of note, indices for overall temperature stability, based on the temperature dependence of apparent first order kinetic temperature denaturation constants, were determined to be −7.9±1.1×10−4, and −13.7±1.3×10−4, −14.5±0.7×10−4, and −13.7±0.8×10−4°Cmin, for free cells and cells immobilized in alginate, agar and polyacrylamide respectively. After 250min the reactor showed maximum degradation of acetamide, propionamide and acrylamide of about 97, 100 and 90%, respectively by using 883 enzyme activity units per reactor stage. The results of this investigation showed that R. rhodochrous NHB-2 expressing thermostable amidase could be used for the efficient treatment of wastewater containing toxic amides. Therefore, we suggest that this microbe has a very high potential for the detoxification of toxic amides from industrial effluents and other wastewaters.

PfPKB, a novel protein kinase B-like enzyme from Plasmodium falciparum: I. Identification, characterization, and possible role in parasite development.

Extracellular signals control various important functions of a eukaryotic cell, which is often achieved by regulating a battery of protein kinases and phosphatases. Protein Kinase B (PKB) is an important member of the phosphatidylinositol 3-kinase-dependent signaling pathways in several eukaryotes, but the role of PKB in protozoan parasites is not known. We have identified a protein kinase B homologue in Plasmodium falciparum (PfPKB) that is expressed mainly in the schizonts and merozoites. Even though PfPKB shares high sequence homology with PKB catalytic domain, it lacks a pleckstrin homology domain typically found at the N terminus of the mammalian enzyme. Biochemical studies performed to understand the mechanism of PfPKB catalytic activation suggested (i) its activation is dependent on autophosphorylation of a serine residue (Ser-271) in its activation loop region and (ii) PfPKB has an unusual N-terminal region that was found to negatively regulate its catalytic activity. We also identified an inhibitor of PfPKB activity that also inhibits P. falciparum growth, suggesting that this enzyme may be important for the development of the parasite.