Induction of xylulose-5-phosphate phosphoketolase in a variety of yeasts grown ond-xylose: the key to efficient xylose metabolism

Activity of a pentulose (xylulose 5-phosphate) phosphoketolase was detected in 20 out of 25 yeasts examined. No significant activity was detected in any yeast grown with glucose, and the enzyme was induced by up to 70-fold when the yeasts were grown on xylose as sole carbon source. Biomass yields from xylose were greater than, or approximately equal to, those from glucose in 15 of the 19 yeasts which possessed phosphoketolase activity. The molar yield of C₂ units from xylose, by metabolism via the pentose phosphate pathway, can be calculated to be insufficient to account for the high yields of biomass and ethanol obtained from xylose. We have shown that the presence of a phosphoketolase system can account for such yields by producing 2 mol C₂ from 1 mol C₅. This pathway must therefore be regarded as a major route of pentose dissimilation in such yeasts.     

The specific activity of aldolase in the livers of old and young rats.

The catalytic activity of liver fructose-biphosphate aldolase (EC 4.1.2.13) of young and old rats in units per manomole sequence has been calculated. No evidence of the accumulation with age of altered enzyme molecules of low catalytic activity was obtained. This is contrary to results obtained using mice and rabbits and indicates that the accumulation of altered enzymes may not always be associated with aging. The possibility that altered proteins are formed, but do not accumulate, cannot be ruled out.     

Immobilization of phospholipase A2 from Central Asian cobra venom on polyamide sorbents

The effect of the immobilization technique and the ligand nature on catalytic properties of phospholipase A2 from the cobra venom was studied. Preparations of phospholipase A2 adsorbed on and covalently bound to polyamide sorbents were obtained. The enzyme was coupled to polyamide beads modified with glutaraldehyde. In this case only 9% of the enzyme activity was retained. The enzyme adsorbed on polyamide modified with phosphatidylethanolamine retained up to 20% of the initial activity. The binding selectivity of phospholipase A2 was maximum in case of the sorbent with a binary ligand, e. g. phosphatidylethanolamine+cytotoxin, the sorbent capacity for the bound enzyme increased 2-3 times (460-600 units/g sorbent. The specific activity of the adsorbed phospholipase A2 was 17-40 units/g sorbent in contrast to 8.6 units/g sorbent for the covalently bound enzyme. Immobilization of the enzyme on polyamide sorbents resulted in changes of the pH-optimum, sensitivity to Ca2+ ions and the character of the enzyme-substrate interactions. Heart stability of the adsorbed phospholipase A2 was lower than that of the covalently bound enzyme. However, the adsorbed enzyme can be used, for example, in affinity chromatography due to its higher specific activity, selectivity and reversibility of the sorption.     

Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli.

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.     

Production of SCP and cellulase by Aspergillus terreus from bagasse substrate

The fermentation of 1.0% untreated bagasse under optimum cultural and nutritional conditions with Aspergillus terreus GN1 indicated that the maximum rate of protein and cellulase production could be obtained during three days of submerged fermentation. Even though 16.4% protein recovery, 0.55 units CMCase/mL, and 0.027 FPase units/mL were obtained on the seventh day, the rates of increase in protein recovery and cellulase production were slower than those obtained up to these days, which were 14.3% protein recovery, 0.45 units CMCase/mL, and 0.019 units FPase/mL. There was an initial lag in the utilization of cellulose up to two days due to the utilization of the water-soluble carbohydrate present in untreated bagasse. Cellulose utilization and water-soluble carbohydrate content during fermentation were correlated with protein recovery and enzyme production. The protein and cellulase production during three days fermentation with 1.0% untreated and treated bagasse were compared and the protein content of the total biomass was calculated and treated bagasse were compared and the protein content of the biomass was calculated into constituent protein contributed by the fungal mycelium and the under graded bagasse. The total biomass recovered with untreated and treated bagasse was 1020 and 820 mg/g bagasse substrate, respectively, and contained 14.3 and 20.6% crude protein, respectively. The contribution of fungal biomass and under graded bagasse was 309 and 711, and 373 and 447 mg/g untreated and treated bagasse substrates, respectively. In an 8-L-flask trial during three days of fermentation, the recovery of SCP and cellulase were 66 g and 32,400 units (Sigma) for treated bagasse and 82 g and 8200 units (Sigma) for untreated bagasse, respectively.     

Purification of rat liver phenylalanine hydroxylase by affinity chromatography

Rat liver phenylalanine hydroxylase has been purified to homogeneity on a totally synthesized affinity matrix. The affinity matrix consisted of a succinylated diaminodipropylamine arm linked to Sepharose-4B, to which the cofactor, 6,7-dimethyl-5,6,7,8-tetrahydropterin, was covalently linked. The pure enzyme was eluted with buffered 50% ethylene glycol, 1 m KCl in one step after the 50% ammonium sulfate fraction of the rat liver homogenate was applied to the affinity column. Specific activities ranging from 1.4 to 3.0 units/mg of protein were obtained. The enzyme has been shown to be homogeneous by: (i) discontinuous gel electrophoresis, and (ii) sodium dodecyl sulfate gel electrophoresis. The subunit molecular weight was determined by the same technique and was calculated to be between 51,000 and 55,000.     

Aggregation Properties of the Acetylcholinesterase from the Central Nervous System of Manduca sexta

Abstract: The CNS of the tobacco hornworm, Manduca sexta , provides a rich source of true acetylcholinesterase (AChE, acetylcholine hydrolase, EC 3.1.1.7.). Optimal extraction of the enzyme was obtained with a nonionic detergent at high ionic strength (1% Triton X-100, 0.5 M NaCl). Velocity sedimentation of the Triton + salt-extracted enzyme demonstrated a single peak whose sedimentation coefficient was dependent upon the enzyme concentration layered on top of the gradient. When more than 20 units were applied to the gradient, a sedimentation coefficient of 8.6 S (205,000) was obtained, and extrapolation to zero units yielded a 5.7 S (110,500) species. Sedimentation in the absence of detergents (1.0 M NaCl or 10 mM phosphate buffer, pH 7.4) yielded pelleted enzyme and species with mean values of 18.6 S (650,000) and 17.5 S (600,000), respectively. The detergent-extracted enzyme also demonstrated a concentration-dependent size in gel filtration experiments. When less than 300 units were applied to the column, a single species was recovered, with a molecular radius of 40.15 ± 2.08 Å, (108,000) or 43.4 ± 2.38 Å, (117,000) calculated by different methods. If the sample contained 300 to 1300 units, two species were observed, with molecular radii of 40.15 ± 2.08 Å or 43.4 ± 2.38 Å and 78.4 ± 3.94 Å, (319,000) or 80.25 ± 3.01 Å (326,000). Velocity sedimentation and gel filtration of AChE have demonstrated that the enzyme has a minimum molecular weight of approximately 110,000 and also exists as higher-molecular-weight aggregates of this value.     

Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films

Summary The cytochemical determination of the activity of glucose-6-phosphate dehydrogenase (G6PDH) with tetranitro blue tetrazolium (TNBT) was studied with model films of polyacrylamide gel incorporating purified enzyme. This model system enabled a quantitative study to be made of different parameters involved with the cytochemical assay as it is applied to sections or smears. The enzyme activity of G6PDH incorporated in the model films was also assayed biochemically. Optimal conditions for retaining the maximum amount of enzymic activity are described. The behaviour of G6PDH towards enzyme inhibitors was found to be similar in model films and in solution. With TNBT, absorbance measurements at a single wavelength (535 nm) were used to estimate the enzyme activity quantitatively. When carried out under standardized conditions, both the cytochemical and biochemical assay showed a linear relation with the time of incubation and obeyed the Beer-Lambert law. The correlation between biochemical and cytochemical data was very high, which enabled cytochemical data to be converted into absolute units of enzyme activity. The data obtained in this way closely resembled the data of enzyme activity calculated from the absorbance of formazan produced inside polyacrylamide model films and afterwards extracted into a suitable solvent.     

Studies on a wild strain of Schizophyllum commune: Cellulase and xylanase production and formation of the extracellular polysaccharide Schizophyllan

A wild strain of schizophyllum commune (Fr:Fr:) isolated in Bangladesh produced cellulase and xyianase in high yields as well as the exobiopolymer schizophyllan. It was found experimentally that concentrations of 4% Avicel, 3.5% peptone, and 0.5% Ca(NO(3))(2).4H(2)O were optimal for growth and product formation. Bacto-peptone was found to be the most suitable substrate of a number of casein, mycological, and meat peptone preparations for enzyme production. Young plate-culture inocula (4 days) were found to be better than comparatively aged fungal cultures (14 days). With the optimized medium, 5 units filter paper (FP) cellulase, 1244 units xylanase, 108 units beta-glucosidase, and 65 units of carboxymethyl (CM) cellulase per mL culture filtrate were obtained in shake flasks. In a laboratory fermentor the respective enzyme activities were 4.5 units FP-cellulase, 1200 units xylanase, 100 units beta-glucosidase, and 60 units CM-cellulase per mL culture filtrate. A biopolymer, reported to be active against can cerous cells, was an additional product in addition to the enzymes.     

Acetopyruvate hydrolase production by Pseudomonas putida O1--optimization of batch and fed-batch fermentations

The main objective of this work was the optimization of the production of the beta-ketolase, acetopyruvate hydrolase, from Pseudomonas putida O1. Orcinol was used as an inducer for enzyme production. The growth medium was optimized in two steps. In the first step, screening for optimal glucose concentration was performed. In the second step, a central composite design was used to optimize carbon and nitrogen sources in the medium. After this optimization procedure, a medium was obtained which produced seven times more biomass than the initial medium. Acetopyruvate hydrolase enzyme production was optimized by determining the optimal time of feed and amount of orcinol, using statistical methods. In a subsequent step, the maximal orcinol-degradation rate was determined. The results obtained were used to find an optimal feeding profile for enzyme production. By using the optimized fed-batch process, acetopyruvate hydrolase activity was enhanced from 10 units l(-1)to 400 units l(-1), in comparison with previously reported fermentation experiments. Productivity could even be increased by a factor of 75, to a value of 20 units l(-1 )h(-1).     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

Mode of action of a family 75 chitosanase from Streptomyces avermitilis

Chitooligosaccharides (CHOS) are oligomers composed of glucosamine and N-acetylglucosamine with several interesting bioactivities that can be produced from enzymatic cleavage of chitosans. By controlling the degree of acetylation of the substrate chitosan, the enzyme, and the extent of enzyme degradation, CHOS preparations with limited variation in length and sequence can be produced. We here report on the degradation of chitosans with a novel family 75 chitosanase, SaCsn75A from Streptomyces avermitilis . By characterizing the CHOS preparations, we have obtained insight into the mode of action and subsite specificities of the enzyme. The degradation of a fully deacetylated and a 31% acetylated chitosan revealed that the enzyme degrade these substrates according to a nonprocessive, endo mode of action. With the 31% acetylated chitosan as substrate, the kinetics of the degradation showed an initial rapid phase, followed by a second slower phase. In the initial faster phase, an acetylated unit (A) is productively bound in subsite -1, whereas deacetylated units (D) are bound in the -2 subsite and the +1 subsite. In the slower second phase, D-units bind productively in the -1 subsite, probably with both acetylated and deacetylated units in the -2 subsite, but still with an absolute preference for deacetylated units in the +1 subsite. CHOS produced in the initial phase are composed of deacetylated units with an acetylated reducing end. In the slower second phase, higher amounts of low DP fully deacetylated oligomers (dimer and trimer) are produced, while the higher DP oligomers are dominated by compounds with acetylated reducing ends containing increasing amounts of internal acetylated units. The degradation of chitosans with varying degrees of acetylation to maximum extents of degradation showed that increasingly longer oligomers are produced with increasing degree of acetylation, and that the longer oligomers contain sequences of consecutive acetylated units interspaced by single deacetylated units. The catalytic properties of SaCsn75A differ from the properties of a previously characterized family 46 chitosanase from S. coelicolor (ScCsn46A).     

A comparative investigation of various cellulase assay procedures

The cellulolytic activity of crude enzyme preparations from different cellulolytic fungi (namely Trichoderma viride, Trichoderma Koningii, Fusarium solani, Sporotrichum pulverulentum, Sporotrichum thermophile) was assayed comparatively with several common analytical procedures described in the literature. The investigation was carried out with the objective of evaluating, with raw culture filtrates, the different cellulase tests in relation to their specificity for endo- and exo-cellulase action as well as to allow comparisons to be made between results from different research groups using different methods. (1)Cellulase activity was tested viscometrically as well as chemically (determination of reducing end groups) with different carboxymethylcelluloses as substrates. Essentially constant ratios between both kinds of activities were obtained, indicating that they are directly related. Nevertheless, international units of activity, calculated from viscometric measurements (glycosidic bonds broken per unit time) were considerably lower than international units deduced from the increase in reducing power (glucose equivalents liberated per unit time), this discrepancy most likely accounted for by the predominant influence of the exo-cellulase component in cellulase tests based on the determination of reducing eng groups. (2) By estimating cellulase activity with insoluble cellulosic substrates no direct relationship could be established with the above-described activities except in the case where the cellulose was amorphous. The ratio profile between activities thus obtained and endo-cellulase activities determined viscometrically shows that some enzyme preparations (such as those from both Trichoderma sp.) are clearly more active than others against crystalline cellulose reflecting quantitative differences in enzyme composition. Nevertheless, for a biological understanding of cellulolysis. analytical procedures using crystalline celluloses are not adequate for specifically monitoring exo-cellulase activity in crude enzyme solutions for essentially two reasons: (a) they are not sufficiently sensitive to detect small changes in enzyme activity during the early phase of growth, and (b) exo-cellulase activity in crude enzyme solutions also depends on the endo-cellulase activity present.     

Purification and characterization of neuraminidase from Clostridium perfringens

Neuraminidase (EC 3.2.1.18) has been purified from the culture medium of Clostridium perfringens ATCC 10543, through steps of gel filtration on Sephadex G-75 column, DEAE-cellulose DE 23 anion exchange chromatography, and isochromatofocusing. A homogeneous enzyme was obtained with a 7552-fold increase in specific activity to 295 units/mg protein. The yield was about 25%. The enzyme consists of a single polypeptide with a molecular weight of 69,000 as determined by SDS-polyacrylamide gel electrophoresis. Kinetic studies showed that Km is 1.5 mM for sialyllactose and Vmax is 0.41 mumole/min/ml at the enzyme concentration of 0.14 microgram/ml. The enzyme is stable at pH 5.2-8.0 with an optimal pH of 6.0. A concentrated solution of the purified enzyme was stable over one year at 4 degrees C. The purified enzyme hydrolyzed human alpha 1-acid glycoprotein completely; thus, it can be used in the clinical assay of N-acetylneuraminic acid in the serum.     

蚯蚓体内超氧化物歧化酶分析

蚯蚓体内SOD含量甚高,35℃饲养的蚯蚓其SOD比活最高,因此,纯化前将蚯蚓在35℃养殖4周以上.采用硫酸铵分级沉淀和柱层析的方法,从蚯蚓体内分离得到纯的铜锌超氧化物歧化酶.每100g组织得到SOD制品总活力为17,190 U,比活7995 U/mg,回收率为35%.该酶呈淡蓝绿色,最大紫外吸收波长为270nm.该酶分子量为33,000,亚基分子量为16,500.该酶亚基含156个氨基酸残基,不含酪氨酸.N-末端为丙氨酸,等电聚焦为三条谱带,等电点分别为5.30 、5.59和6.22.     

The application of triazine dye affinity chromatography to the large-scale purification of glycerokinase from Bacillus stearothermophilus

Gram quantities of homogeneous glycerokinase have been prepared from the thermophilic bacterium, Bacillus stearothermophilus, using three major steps: precipitation of debris at pH 5.1, ion-exchange chromatography on DEAE-Sephadex, and affinity chromatography on Procion Blue MX-3G-Sepharose. This method is a considerable improvement over conventional techniques; the purified enzyme was obtained with a 40% recovery and a specific activity of 120 units (mumol/min)/mg protein. A modified culture medium enabled yields of 3.4 X 10(6) units of enzyme to be obtained from 400-liter production cultures.     

Rice chitinases: sugar recognition specificities of the individual subsites

Sugar recognition specificities of class III (OsChib1a) and class I (OsChia1cDeltaChBD) chitinases from rice, Oryza sativa L., were investigated by analyzing (1)H- and (13)C-nuclear magnetic resonance spectra of the enzymatic products from partially N-acetylated chitosans. The reducing end residue of the enzymatic products obtained by the class III enzyme was found to be exclusively acetylated, whereas both acetylated and deacetylated units were found at the nearest neighbor to the reducing end residue. Both acetylated and deacetylated units were also found at the nonreducing end residue and its nearest neighbor of the class III enzyme products. Thus, only subsite (-1) among the contiguous subsites (-2) to (+2) of the class III enzyme was found to be specific to an acetylated residue. For the class I enzyme, the reducing end residue was preferentially acetylated, although the specificity was not absolute. The nearest neighbor to the acetylated reducing end residue was specifically acetylated. Moreover, the nonreducing end residue produced by the class I enzyme was exclusively acetylated, although there was a low but significant preference for deacetylated units at the nearest neighbor to the nonreducing end. These results suggest that the three contiguous subsites (-2), (-1), and (+1) of the class I enzyme are specific to three consecutive GlcNAc residues of the substrate. In rice plants, the target of the class I enzyme might be a consecutive GlcNAc sequence probably in the cell wall of fungal pathogen, whereas the class III enzyme might act toward an endogenous complex carbohydrate containing GlcNAc residue.     

An adenosine 3':5' monophosphate dependent protein kinase from sea urchin spermatozoa.

A cyclic AMP dependent protein kinase (EC 2.7.1.37) from sea urchin sperm as purified to near homogeneity and characterized. A 68-fold purification of the enzyme was obtained. This preparation had a specific activity of 389 000 units/mg protein with protamine as the substrate. On the basis of the purification required, it may be calculated that the protein kinase constitutes as much as 1.5% of the soluble protein in sperm. There appeared to be a single form of the enzyme in sea urchin sperm, based on the behavior of the enzyme during DEAE-cellulose and Sephadex G-200 column chromatography. Magnesium ion was required for enzyme activity. The rate of phosphorylation of protamine was stimulated 2.5-fold by an optimal concentration of 0.9 M NaCl. The Km for ATP (minus cyclic AMP) was 0.119 +/- 0.013 (S.D.) and 0.055 mM +/- 0.009 (S.D.) in the presence of cyclic AMP. The specificity of the enzyme toward protein acceptors, in decreasing order of phosphorylation, was found to be histone f1 protamine, histone f2b, histone f3 and histone f2a; casein and phosvitin were not phosphorylated. The holoenzyme was found to have an apparent molecular weight of 230 000 by Sephadex G-200 chromatography. In the presence of 5 - 10(-6) M cyclic AMP, the holoenzyme was dissociated on Sephadex G-200 to a regulatory subunit of molecular weight 165 000 and a catalytic subunit of Mr 73 000. The dissociation could also be demonstrated by disc gel electrophoresis in the presence and absence of cyclic AMP.     

Presence of two endo-beta-N-acetylglucosaminidases in human kidney

We have isolated for the first time two kinds of endo-beta-N-acetylglucosaminidases (E-beta-GNases) simultaneously from human kidney. E-beta-GNase 1 was purified by water extraction, ammonium sulfate fractionation, and chromatography on Sephadex-G-200, DEAE-Sephadex, concanavalin A-Sepharose and Hypatite C columns. After the DEAE-Sephadex step, 107 units of E-beta-GNase 1 with a specific activity of 0.53 units/mg was obtained and after hydroxyapatite column, the enzyme recovery was 26 units with a specific activity of 10.4 units/mg. This enzyme hydrolyzed the high mannose-type asparaginylglycopeptide efficiently and had little activity toward the complex-type glycopeptide. This enzyme had an pH optimum at about 4.5 and was not inhibited by acetate ion. The Asn residue in a glycopeptide appeared not to be an important recognition site for E-beta-GNase 1 to express its activity because the acetylation or the dansylation of Asn residues as well as the elimination of Asn residue from the glycopeptide did not change the susceptibility of the oligosaccharide to E-beta-GNase 1. E-beta-GNase 2 was purified by water extraction, ammonium sulfate fractionation, and chromatography on Sephadex G-200, DEAE-Sephadex, concanavalin A-Sepharose, and Mono S columns. This enzyme was purified about 110-fold with 6.6% recovery. E-beta-GNase 2 was found to be a novel type of E-beta-GNase that hydrolyzed both the high mannose-type and the complex-type oligosaccharide with chitobiosyl group at the reducing end and without the Asn. E-beta-GNase 2 activity was found to be dependent on a L-aspartamido-beta-D-N-acetylglucosamine amidohydrolase (Asn-GNase) for the hydrolysis of asparaginylglycopeptide. Asn-GNase cleaved off the Asn residue from the glycopeptide, and the resulting oligosaccharide was hydrolyzed by E-beta-GNase 2. Because the acetylation or the dansylation of Asn residue in a glycopeptide rendered the glycopeptide resistant to Asn-GNase, the use of the modified asparaginylglycopeptide could not reveal the existence of E-beta-GNase 2 activity. The pH optimum of E-beta-GNase was found to be about 3.5. Like beta-hexosaminidases, this enzyme was inhibited by acetate ion, suggesting the recognition of GlcNAc moiety by this enzyme.