Major Hydrolysis Products Research Articles

Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn2+, Hg2+, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K m and V max of the enzyme for CMC were 3.4 mg ml−1 and 44.7 μmol min−1 (mg protein)−1, respectively.

Determination of phenyl-N-methylcarbamates and their hydrolysis products in water, using solid-phase extraction and reversed-phase liquid chromatography with UV and electrospray mass spectrometric detection

In this study, eight phenyl-N-methylcarbamates (PNMCs) were considered. Reversed-phase LC was set up for UV and mass spectrometry (MS) detection mode. Gradient elution was used, and the mobile phase was composed of acetonitrile and water. UV–vis was performed at 220 nm. The method was tested with different reversed-phase columns. Comparison between chromatographic parameters: retention time (tR), resolution (RS), and selectivity (α) was established. Hydrolysis kinetics of three of the PNMCs were reported. The major hydrolysis products were determined by LC–UV, and the effect of pH on hydrolysis was also studied. Also, chromatographic separation of a mixture of PNMCs and four of their hydrolysis products was carried out. The preconcentration of 12 studied solutes was realized by solid-phase extraction. C18 extraction cartridges of 1 g were used to extract solutes from a 100 mL volume of tap and surface water spiked at 10 µg/L. The recoveries were, respectively, between 68–86% and 62–83% with relative a standard deviation of less than 11%. Limits of detection (LODs) and limits of quantitation (LOQs) ranged, respectively, from 1–4 µg/L and from 4–10 µg/L. Since standard UV detection does not provide adequate selectivity for water samples, an electrospray (ES)-MS instrument equipped with a triple quadrupole mass filter was used. MS data acquisition was performed by a time-scheduled, selected-ion monitoring (SIM) program. Limits of quantitation gave values between 0.1–0.5 µg/L.Key words: phenyl-N-methylcarbamates, water analysis, solid-phase extraction, LC–UV–vis, LC–ES-MS.

466 Influence of the indole compounds on cellular level of glutathione and glutathione-s-transferase activity

Cruciferous vegetables such as cauliflower, cabbage, kale, kohlrabi, turnips or broccoli, contain μ g/g levels of glucosinolate glucobassicin (indolyl methyl glucosinolate). Indole-3-carbinol is the major hydrolytic product obtained from glucobrassicin. When the plant cells are damaged by cutting or chewing, a thioglucosidase-mediated autolytic process takes place generating indole-3-carbinol, glucose and thiocyanate ion. Recent reports have shown that indole compounds can modulate cellular response when cells are exposed to variety of environmental xenobiotics. In our previous work we have shown that indole-3-carbinol and cauliflower extract did not induce point mutations in Salmonella typhimurium TA98 and TA100. We have also shown that indole compounds have inhibited formation of point mutations when Salmonella typhimurium TA98 and TA100 were treated with two standard mutagens 4-nitroquinoline-1-oxide (direct acting mutagen) and 2-aminoanthracene (promutagen). In this work we have examined indole-3-carbinol and cauliflower extract for their ability to influence on phase II biotransformation enzyme activities in four cell lines: human laryngeal carcinoma cells (parental HEp2 and their cisplatin resistant subline CK2) and human cervix cancer (parental HeLa and their cisplatin resistant subline CK). We have measured and compared cellular concentration of glutathione and enzymatic activity of total glutathione-S-transferases after 72 hours of treatment of the cells with the highest nontoxic concentrations of indole-3-carbinol and cauliflower extract. Concentrations of glutathione in four cell lines were compared with the basal level of glutathione in untreated cells. It was shown that cauliflower extract and indole-3-carbinol have caused significant increase of glutathione in both, HEp2 and HeLa, parental cells. On the contrary, cisplatin resistant cells have increased basal level of glutathione, and cisplatin resistant cells have increased basal level of glutathione, and both drugs decreased the level of glutathione. The activity of glutathione-S-transferases were increased in parental HEp2 and HeLa cells after treatment with indole-3-carbinol and cauliflower extract. On the other hand, indole compounds caused significant decrease of glutathione-S-transferases activity in resistant cells.

Exo-mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP-23. A unique type of cellulase among Bacillales.

Sequence analysis of a Paenibacillus sp. BP-23 recombinant clone coding for a previously described endoglucanase revealed the presence of an additional truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding 3509-bp DNA fragment was isolated after gene walking and cloned in Escherichia coli Xl1-Blue for expression and purification. The encoded enzyme, a cellulase of 1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85, displayed a multidomain organization bearing a canonical family 48 catalytic domain, a bacterial type 3a cellulose-binding module, and a putative fibronectin-III domain. The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel. Maximum activity was achieved at 45 degrees C and pH 6.0 on acid-swollen cellulose, bacterial microcrystalline cellulose, Avicel and cellodextrins, whereas no activity was found on carboxy methyl cellulose, cellobiose, cellotriose, pNP-glycosides or 4-methylumbeliferyl alpha-d-glucoside. Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a processive cellobiohydrolase. Although no chromogenic activity was detected from pNP-glycosides, TLC analysis revealed the release of p-nitrophenyl-glycosides and cellodextrins from these substrates, suggesting that Cel48C acts from the reducing ends of the sugar chain. Presence of such a cellobiohydrolase in Paenibacillus sp. BP-23 would contribute to widen up its range of action on natural cellulosic substrates.

Uptake of N,N'-diacetylchitobiose [(GlcNAc)2] via the phosphotransferase system is essential for chitinase production by Serratia marcescens 2170.

The chiR gene of Serratia marcescens 2170, encoding a LysR-type transcriptional activator, was identified previously as an essential factor for expression of chitinases and a chitin-binding protein, CBP21. To identify other genes that are essential for chitinase production, transposon mutagenesis with mini-Tn5Km1 was carried out, and 25 mutants that were unable to produce chitinases and CBP21 were obtained. Analysis of the mutated gene of one of the mutants, N22, revealed the presence of a pts operon in this bacterium, and a mutation was found in ptsI in the operon. In addition to its inability to produce chitinase, N22 did not grow well on N-acetyl-D-glucosamine (GlcNAc), (GlcNAc)(2), and some other carbon sources, most of which were phosphotransferase system (PTS) sugars. Thus, the inability to produce chitinase was assumed to be caused by the defect in uptake of (GlcNAc)(2) via the PTS, considering that (GlcNAc)(2) is the minimal substrate for chitinase induction and the major product of chitin hydrolysis by chitinases of this bacterium. To confirm this assumption, the chb operon, encoding the (GlcNAc)(2)-specific enzyme II permease, was cloned by reference to its Escherichia coli counterpart, and the Serratia chb operon was shown to comprise chbB, chbC, bglA, chbR, and chbG. Disruption of chbC drastically reduced production of chitinases and CBP21 and impaired growth on colloidal chitin. These results indicate that uptake of (GlcNAc)(2) is mediated by the PTS and that the (GlcNAc)(2)-specific enzyme II permease constitutes its major pathway. Since (GlcNAc)(2) uptake is essential for induction of chitinases and CBP21 production, (GlcNAc)(2) appears to be the key molecule in recognition and utilization of chitin by S. marcescens.

Effects of alkali and simulated gastric and intestinal fluids on danazol stability

The degradation kinetics of methanolic solution of danazol (0.020% w/v) in aqueous buffers and sodium hydroxide was investigated using stability-indicating HPLC method. The drug degrades in alkaline medium through a base-catalysed proton abstraction rather than via an oxidative mechanism involving oxygen species. The degradation followed pseudo-first-order kinetics. The rates pH-profile exhibited specific base catalysis. The stability of the drug was found to be dependent on pH, buffer concentration, buffer species (acetate, borate, phosphate) and temperature. The ionic strength did not affect the stability of the drug. The energy of activation according to Arrhenius plot was estimated to be 22.62 kcal mol −1 at pH 12 and temperatures between 30 and 60 °C. The effect of simulated gastric and intestinal fluids on the drug stability was also investigated. Two major hydrolytic degradation products were separated and identified by IR, NMR and mass spectrometry and the degradative pathway suggested.

Reaction of Chlorpyrifos-methyl in Aqueous Hydrogen Sulfide/Bisulfide Solutions

The kinetics of the reactions of chlorpyrifos-methyl, an organophosphorus insecticide, with hydrogen sulfide (H(2)S) and bisulfide (HS(-)) were determined in well-defined aqueous solutions. The resulting pseudo-first-order rate constant for chlorpyrifos-methyl with bisulfide yielded a second-order rate constant of (2.1 +/- 0.3) x 10(-3) M(-1) s(-1). The second-order rate constant for chlorpyrifos-methyl with hydrogen sulfide is significantly slower than the second-order rate constant with bisulfide. The contribution of H(2)S to the observed degradation rate constant of chlorpyrifos-methyl at concentrations of up to 4 mM H(2)S is not significant. The second-order rate constant of chlorpyrifos-methyl with H(2)S was too low to be measured in this study. The results indicate that HS(-) present at environmentally relevant concentrations may represent an important sink for phosphorothionate triesters in a coastal marine environment, while H(2)S reacts too slowly to be environmentally relevant (pH 6-9). Trichloropyridinol, the major product of hydrolysis of chlorpyrifos-methyl, is only a minor product of the reaction of chlorpyrifos-methyl with bisulfide; however, trichloropyridinol was found to be stable under the experimental conditions.

Purification and characterization of an endo-β-(1→6)-galactanase from Trichoderma viride

An endo-β-(1→6)-galactanase from Onozuka R-10, a commercial cellulase preparation from Trichoderma viride, was purified 57-fold. Apparent M r values of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were 47,000 and 17,000, respectively. The enzyme was assayed with a galactan from Prototheca zopfii, which has a high proportion of β-(1→6)-linked galactosyl residues . It exhibited maximal activity toward the galactan at pH 4.3. The enzyme hydrolyzed specifically β-(1→6)-galactooligosaccharides with a degree of polymerization higher than 3 and their acidic derivatives with 4- O-methyl-glucosyluronic or glucosyluronic groups at the nonreducing terminals. The methyl β-glycoside of β-(1→6)-galactohexaose was degraded to reducing galactooligomers with a degree of polymerization 2–5 as the products at the initial stage of hydrolysis, and galactose and galactobiose at the final stage, indicating that the enzyme can be classified as an endo-galactanase. The extent of hydrolysis of the carbohydrate portion of a radish root arabinogalactan-protein (AGP) increased when α- l-arabinofuranosyl residues attached to β-(1→6)-linked galactosyl side chains of the AGP were removed in advance. The enzyme released galactose, β-(1→6)-galactobiose, and 4- O-methyl-β-glucuronosyl-(1→6)-galactose as major hydrolysis products when allowed to act exhaustively on the modified AGP.

Purification and characterization of a novel extracellular β-1,3-glucanase produced byBacillus clausii NM-1 isolated from ezo abaloneHaliotis discus hannai

A novel extracellular alkaline stable β-1,3-glucanase produced byBacillus clausii NM-1 isolated from the ezo abaloneHaliotis discus hannai was purified by ammonium sulfate precipitation, DEAE-Sepharose FF ion exchange chromatography and Sephacryl S-200HR gel filtration. The molecular weight of the purified enzyme was estimated to be 71 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was very stable at pH 5.3 to 11.5 but unstable at pH 4.0 to 4.5. The optimum temperature and thermostability of the enzyme increased in the presence of CaC1, The enzyme hydrolyzed R-1,3-glucan from marine organisms, but did not show activity against any other β-1,3-glucans. The major hydrolysis products of β-1,3-glucan fromLaminaria digitata andEisenia bicyclis were laminaritriose and laminaritetraose, respectively. The N-terminal amino acid sequence of the purified enzyme was similar to that of several β-1,3-glucanases in the glycoside hydrolase family 16.

Purification and Characterization of a Novel Extracellular .BETA.-1,3-Glucanase Produced by Bacillus clausii NM-1 Isolated from Ezo Abalone Haliotis discus hannai.

A novel extracellular alkaline stable β-1,3-glucanase produced byBacillus clausii NM-1 isolated from the ezo abaloneHaliotis discus hannai was purified by ammonium sulfate precipitation, DEAE-Sepharose FF ion exchange chromatography and Sephacryl S-200HR gel filtration. The molecular weight of the purified enzyme was estimated to be 71 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was very stable at pH 5.3 to 11.5 but unstable at pH 4.0 to 4.5. The optimum temperature and thermostability of the enzyme increased in the presence of CaC1, The enzyme hydrolyzed R-1,3-glucan from marine organisms, but did not show activity against any other β-1,3-glucans. The major hydrolysis products of β-1,3-glucan fromLaminaria digitata andEisenia bicyclis were laminaritriose and laminaritetraose, respectively. The N-terminal amino acid sequence of the purified enzyme was similar to that of several β-1,3-glucanases in the glycoside hydrolase family 16.

Purification and characterization of a new hyperthermostable, allosamidin-insensitive and denaturation-resistant chitinase from the hyperthermophilic archaeon Thermococcus chitonophagus.

A new chitinase (1,4-beta-D-N-acetyl-glucosaminidase, EC 3.2.1.14) was detected and purified to homogeneity in its native form from the chitinolytic enzyme system of the extremely thermophilic archaeon Thermococcus chitonophagus. This is the first nonrecombinant chitinase purified and characterized from archaea and also constitutes the first case of a membrane-associated chitinase isolated from archaea. The enzyme is a monomer with an apparent molecular weight of 70 kDa [therefore named chitinase 70 (Chi70)] and pI of 5.9; it is hydrophobic and appears to be associated with the outer side of the cell membrane. Chi70 is optimally active at 70 degrees C and pH 7.0 and exhibits remarkable thermostability, maintaining 50% activity even after 1 h at 120 degrees C, and therefore the enzyme is the most thermostable chitinase so far isolated. The enzyme was not inhibited by allosamidin, the natural inhibitor of chitinolytic activity, and was also resistant to denaturation by urea and SDS. On the other hand, guanidine hydrochloride significantly reduced enzymatic activity, indicating that, apart from the hydrophobic interactions, ion pairs located on the surface of the protein could be playing an important role in maintaining the protein's fold and enzyme activity. Chi70 showed broad substrate specificity for several chitinous substrates and derivatives. The lowest K(m) and highest K(cat) values were found for pNP(NAG)(2) as substrate and were determined to be 0.14 mM and 23 min(-1), respectively. The hydrolysis pattern was similar for oligomers and polymers, with N, N'-diacetylchitobiose [(NAG)(2)] being the final, major hydrolysis product. Chi70 was classified as an endochitinase due to its ability to release chitobiose from colloidal chitin. Additionally, the enzyme presented considerable cellulolytic activity. Analysis of the NH(2)-terminal amino acid sequence showed no detectable homology with other known sequences, suggesting that Chi70 is a new protein.

Thiodiglycol metabolism in Alcaligenes xylosoxydans subsp. denitrificans

The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp. denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C-S bonds in the intermediate products, thiodiglycolic and thioglycolic acids. The end products of these reactions are SO4(2-) ions and acetate, the latter being involved in the central metabolism of strain TD2. The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation. Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A. xylosoxydans subsp. denitrificans strain TD2 is proposed.

Monoacylglycerol Metabolism in Human Intestinal Caco-2 Cells: EVIDENCE FOR METABOLIC COMPARTMENTATION AND HYDROLYSIS

Free fatty acids (FFA) and sn-2-monoacylglycerol (MG), the two major hydrolysis products of dietary triacylglycerol (TG), are absorbed from the lumen into polarized enterocytes that line the small intestine. Intensive studies regarding FFA metabolism in the intestine have been published; however, little is known regarding the metabolism of MG. In these studies, we examined the metabolism of sn-2-monoolein (sn-2-18:1) by human intestinal Caco-2 cells. To mimic the physiological presentation of MG to the enterocyte, the metabolism of [(3)H]sn-2-monoolein was examined by adding taurocholate-mixed sn-2-18:1 and albumin-bound sn-2-18:1 at the apical (AP) and basolateral (BL) surfaces of the Caco-2 cell, respectively. The results demonstrate that more sn-2-18:1 was incorporated into TG from AP taurocholate-mixed sn-2-18:1, whereas more phospholipid was synthesized from BL albumin-bound sn-2-18:1. The TG:phospholipid ratio was approximately 5-fold higher for AP relative to BL MG incubation. Qualitatively similar results were observed for bovine serum albumin-bound MG added at the apical surface. It was also found that substantial sn-2-18:1 radioactivity was recovered in the FFA fraction, suggesting that sn-2-18:1 may be directly hydrolyzed within the Caco-2. We therefore used reverse transcription-PCR with primers designed from the murine MG lipase (MGL) gene, and detected the presence of MG lipase mRNA in Caco-2. The human MGL gene was cloned and found to be 83% identical to the murine MGL, and identical to a previously described lysophospholipase-like protein. Northern blot analysis showed the expression of MGL throughout Caco-2 differentiation. Thus, MG metabolism in Caco-2 cells may include not only well established anabolic processes, but also catabolic processes. Further, the observed polarity of MG metabolism suggests that, as for fatty acids, separate precursor and/or product pools of lipid may exist in the intestinal enterocyte.

Utilization of commercial non-chitinase enzymes from fungi for preparation of 2-acetamido-2-deoxy- d-glucose from β-chitin

Commercial non-chitinase enzymes from Aspergilus niger, Acremonium cellulolyticus and Trichoderma viride were investigated for potential utilization in the preparation of 2-acetamido-2-deoxy- d-glucose ( N-acetyl- d-glucosamine, GlcNAc) from chitin. Among the tested enzymes, cellulase A. cellulolyticus exhibited highest chitinolytic activity per weight toward amorphous chitin and β-chitin from squid pen. The optimum pH of the enzyme was 3 where it produced two major hydrolytic products, GlcNAc and N, N′-diacetylchitobiose ([GlcNAc] 2). The product ratio, GlcNAc:[GlcNAc] 2, increased while the total yield decreased as the pH was raised from 3. All of the [GlcNAc] 2 produced at pH 3 can be converted in situ to GlcNAc by mixing cellulase A. cellulolyticus with one of several other enzymes from A. niger resulting in a higher yield of GlcNAc. An appropriate mixing ratio of cellulase A. cellulolyticus to another enzyme was 9:1 (w/w) and an optimum substrate concentration was 20 mg/mL.

Determination of r-7,t-8,9,c-10-Tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene in Human Urine by Gas Chromatography/Negative Ion Chemical Ionization/Mass Spectrometry

r-7,t-8,9,c-10-Tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-BaP-tetraol) is the major hydrolysis product of r-7, t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the principal ultimate carcinogen of the environmental pollutant benzo[a]pyrene (BaP). As part of a program to establish activation/detoxification profiles of urinary metabolites of BaP in humans, we developed a method for quantifying trans-anti-BaP-tetraol. Urine was collected from three groups of individuals exposed to BaP: psoriasis patients treated with a coal tar-containing ointment, steel workers, and smokers. [(2)H(12)]-trans-anti-BaP-tetraol was added to the urine as an internal standard. The urine was treated with beta-glucuronidase and sulfatase, and then the BaP-tetraols were enriched by reverse-phase and phenylboronic acid solid-phase extraction. The resulting fraction was treated with sodium hydride and methylmethane sulfonate to convert BaP-tetraols to the corresponding tetramethyl ethers (BaP-TME). The mixture was purified by normal-phase HPLC and analyzed by gas chromatography/negative ion chemical ionization/mass spectrometry with selected ion monitoring. [(13)CH(3)](4)-trans-anti-BaP-TME was used as an external standard. Ions at m/z 376, 380, and 388 were monitored for quantitation of trans-anti-BaP-TME, [(13)CH(3)](4)-trans-anti-BaP-TME, and [(2)H(12)]-trans-anti-BaP-TME, respectively. The instrumental detection limit was approximately 1 fmol of trans-anti-BaP-TME. trans-anti-BaP-tetraol (as trans-anti-BaP-TME) was detected in 20 of 20 individuals receiving coal tar therapy (mean, 16 fmol/mL of urine), 13 of 13 exposed steel workers (mean, 4.1 fmol/mL of urine), and nine of 21 cigarette smokers (mean, 0.5 fmol/mL of urine). The means in these groups were significantly different (P < 0.0001). The urine of steel workers was also analyzed for cis-anti-BaP-tetraol and cys-syn-BaP-tetraol, but neither was found. The results of this study provide a quantitative method for determination of parts per trillion levels of trans-anti-BaP-tetraol in human urine. Ultimately, this method can be employed as part of a phenotyping approach for assessing BaP metabolites in human urine.

Simultaneous determination of fumonisin B(1) and hydrolyzed fumonisin B(1) in corn products by liquid chromatography/electrospray ionization mass spectrometry.

A method for the simultaneous determination of fumonisin B(1) (FB(1)) and its major hydrolysis product (HFB(1)), which is known to be formed during alkaline treatment of fumonisin-containing corn meal, was devised to analyze the levels of these mycotoxins in corn products available on the German market. Liquid chromatography/electrospray mass spectrometry in combination with selected ion monitoring (SIM) was used for unambiguous detection of FB(1) and HFB(1) after extraction of samples with acetonitrile/methanol/water (25:25:50) and solid-phase C18 cleanup. Quantitation was carried out using labeled fumonisin FB(1)-D(6) as an internal standard. The detection limits achieved with this method were 8 ng/g for HFB(1) (signal-noise ratio = 5:1) and 5 ng/g for FB(1) (s/n = 5:1) using the protonated molecule signals m/z 406 and 722 in the SIM mode. A screening of several corn-containing foodstuffs, among them extrusion products and alkali-processed corn food such as tortilla chips, showed HFB(1) and FB(1) contamination with levels of 8-80 and 5-450 ng/g, respectively.

Kinetics of carbofuran insecticide conversion under methanogenic conditions

This study reports on the kinetics of carbofiiran insecticide transformation under controlled aquatic methanogenic conditions. Attempts were also made to investigate the effects of culture acclimation on the kinetics of carbofiiran transformation. The results indicate that in an anaerobic system carbofuran was hydrolyzed to carbofiiran phenol and monomethyl amine by hydrolysis. Analysis of the kinetic parameters showed that active anaerobic cultures (both acclimated and unnacclimated) do not enhance the rate of hydrolysis of carbofuran. The hydrolysis product, monomethyl amine, was further mineralized by the methanogenic culture. The major product of carbofuran hydrolysis, carbofiiran phenol, was resistant to further degradation.

The effect of process conditions on the alpha-amylolytic hydrolysis of amylopectin potato starch: An experimental design approach.

The hydrolysis of amylopectin potato starch with Bacillus licheniformis alpha-amylase (Maxamyl) was studied under industrially relevant conditions (i.e. high dry-weight concentrations). The following ranges of process conditions were chosen and investigated by means of an experimental design: pH [5.6-7.6]; calcium addition [0-120 microg/g]; temperature [63-97 degrees C]; dry-weight concentration [3-37% [w/w]]; enzyme dosage [27.6-372.4 microL/kg] and stirring [0-200 rpm]. The rate of hydrolysis was followed as a function of the theoretical dextrose equivalent. The highest rate (at a dextrose equivalent of 10) was observed at high temperature (90 degrees C) and low pH (6). At a higher pH (7.2), the maximum temperature of hydrolysis shifted to a lower value. Also, high levels of calcium resulted in a decrease of the maximum temperature of hydrolysis. The pH, temperature, and the amount of enzyme added showed interactive effects on the observed rate of hydrolysis. No product or substrate inhibition was observed. Stirring did not effect the rate of hydrolysis. The oligosaccharide composition after hydrolysis (at a certain dextrose equivalent) did depend on the reaction temperature. The level of maltopentaose [15-24% [w/w]], a major product of starch hydrolysis by B. licheniformis alpha-amylase, was influenced mostly by temperature.

Catechols involved in sclerotization of cuticle and egg pods of the grasshopper, Melanoplus sanguinipes, and their interactions with cuticular proteins

N-Acetyldopamine (NADA) is the major catechol in the hemolymph of nymphal and adult grasshoppers, Melanoplus sanguinipes (F.), and mainly occurs as an acid-labile conjugate indicated to be a sulfate ester. Its concentration increases in last instar nymphs and peaks during adult cuticle sclerotization. Dopamine (DA), the precursor of NADA and melanic pigments, is about 10 times lower in concentration than NADA, but shows a similar pattern of accumulation. NADA also predominates in cuticle, but its concentration is lowest during the active period of sclerotization, reflecting its role as a precursor for quinonoid tanning agents. Two other catechols, 3,4-dihydroxybenzoic acid (DOBA) and 3,4-dihydroxyphenylethanol (DOPET), also occur in hemolymph and cuticle, and their profiles suggest a role in cuticle stabilization. Solid-state NMR analysis of sclerotized grasshopper cuticle (fifth instar exuviae) estimated the relative abundances of organic components to be 59% protein, 33% chitin, 6% catechols, and 2% lipid. About 99% of the catechols are covalently bound in the cuticle, and therefore are involved in sclerotization of the protein-chitin matrix. To determine the types of catechol covalent interactions in the exocuticle, samples of powdered exuviae were heated in Hcl under different hydrolytic conditions to release adducts and cross-linked products. 3,4-Dihydroxyphenylketoethanol (DOPKET) and 3,4-dihydroxyphenylketoethylamine (arterenone) are the major hydrolysis products in weak and strong acid, respectively, and primarily represent NADA oligomers that apparently serve as cross-links and filler material in sclerotized cuticle. Intermediate amounts of norepinephrine (NE) are released, which represent N-acetylnorepinephrine (NANE), a hydrolysis product of NADA bonded by the b-carbon to cuticular proteins and possibly chitin. Small quantities of histidyl-DA and histidyl-DOPET ring and side-chain C-N adducts are released by strong acid hydrolysis. Therefore, grasshopper cuticle appears to be sclerotized by both o-quinones and p-quinone methides of NADA and dehydro-NADA, which results in a variety of C-O and C-N covalent bonds linked primarily through the side-chain carbons of the catechol moiety to amino acid residues in cuticular proteins. The primary catechol extracted from both the female accessory glands/calyx and the proteinaceous frothy material of the egg pod is DOBA, which also commonly occurs in cockroach accessory glands and oothecae, presumably as a tanning agent precursor. 3,4-Dihydroxyphenylalanine (DOPA) was also detected in extracts of the accessory glands/calyx of grasshoppers, and may serve as a precursor for DOBA synthesis. Arch. Insect Biochem. Physiol. 40:119–128, 1999. © 1999 Wiley-Liss, Inc.

Catechols involved in sclerotization of cuticle and egg pods of the grasshopper, Melanoplus sanguinipes, and their interactions with cuticular proteins

N-Acetyldopamine (NADA) is the major catechol in the hemolymph of nymphal and adult grasshoppers, Melanoplus sanguinipes (F.), and mainly occurs as an acid-labile conjugate indicated to be a sulfate ester. Its concentration increases in last instar nymphs and peaks during adult cuticle sclerotization. Dopamine (DA), the precursor of NADA and melanic pigments, is about 10 times lower in concentration than NADA, but shows a similar pattern of accumulation. NADA also predominates in cuticle, but its concentration is lowest during the active period of sclerotization, reflecting its role as a precursor for quinonoid tanning agents. Two other catechols, 3,4dihydroxybenzoic acid (DOBA) and 3,4-dihydroxyphenylethanol (DOPET), also occur in hemolymph and cuticle, and their profiles suggest a role in cuticle stabilization. Solid-state NMR analysis of sclerotized grasshopper cuticle (fifth instar exuviae) estimated the relative abundances of organic components to be 59% protein, 33% chitin, 6% catechols, and 2% lipid. About 99% of the catechols are covalently bound in the cuticle, and therefore are involved in sclerotization of the protein-chitin matrix. To determine the types of catechol covalent interactions in the exocuticle, samples of powdered exuviae were heated in HCl under different hydrolytic conditions to release adducts and cross-linked products. 3,4-Dihydroxyphenylketoethanol (DOPKET) and 3,4-dihydroxyphenylketoethylamine (arterenone) are the major hydrolysis products in weak and strong acid, respectively, and primarily represent NADA oligomers that apparently serve as cross-links and filler material in sclerotized cuticle. Intermediate amounts of norepinephrine (NE) are released, which represent N-acetylnorepinephrine