The SGNH-hydrolase of Streptomyces coelicolor has (aryl)esterase and a true lipase activity

The Streptomyces coelicolor A3(2) gene SCI11.14c was overexpressed and purified as a His-tagged protein from heterologous host, Streptomyces lividans. The purification procedure resulted in 34.1-fold increase in specific activity with an overall yield of 21.4%. Biochemical and physical properties of the purified enzyme were investigated and it was shown that it possesses (aryl)esterase and a true lipase activity. The enzyme was able to hydrolyze p-nitrophenyl-, α- and β-naphthyl esters and poly(oxyethylene) sorbitan monoesters (Tween 20–80). It showed pronounced activity towards p-nitrophenyl and α- and β-naphthyl esters of C₁₂–C₁₆. Higher activity was observed with α-naphthyl esters. The enzyme hydrolyzed triolein (specific activity: 91.9 U/mg) and a wide range of oils with a preference for those having higher content of linoleic or oleic acid (C18:2; C18:1, cis). The active-site serine specific inhibitor 3,4-dichloroisocoumarin (DCI) strongly inhibited the enzyme, while tetrahydrofurane and 1,4-dioxane significantly increased (2- and 4- fold, respectively) hydrolytic activity of lipase towards p-nitrophenyl caprylate. The enzyme exhibited relatively high temperature optimum (55 °C) and thermal stability. CD analysis revealed predominance of α-helical structure (54% α-helix, 21% β-sheet) and a T_m value at 66 °C.     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

Crystal Structure of an Essential Enzyme in Seed Starch Degradation: Barley Limit Dextrinase in Complex with Cyclodextrins

Barley limit dextrinase [Hordeum vulgare limit dextrinase (HvLD)] catalyzes the hydrolysis of α-1,6 glucosidic linkages in limit dextrins. This activity plays a role in starch degradation during germination and presumably in starch biosynthesis during grain filling. The crystal structures of HvLD in complex with the competitive inhibitors α-cyclodextrin (CD) and β-CD are solved and refined to 2.5 Å and 2.1 Å, respectively, and are the first structures of a limit dextrinase. HvLD belongs to glycoside hydrolase 13 family and is composed of four domains: an immunoglobulin-like N-terminal eight-stranded β-sandwich domain, a six-stranded β-sandwich domain belonging to the carbohydrate binding module 48 family, a catalytic (β/α)₈-like barrel domain that lacks α-helix 5, and a C-terminal eight-stranded β-sandwich domain of unknown function. The CDs are bound at the active site occupying carbohydrate binding subsites + 1 and + 2. A glycerol and three water molecules mimic a glucose residue at subsite − 1, thereby identifying residues involved in catalysis. The bulky Met440, a unique residue at its position among α-1,6 acting enzymes, obstructs subsite − 4. The steric hindrance observed is proposed to affect substrate specificity and to cause a low activity of HvLD towards amylopectin. An extended loop (Asp513-Asn520) between β5 and β6 of the catalytic domain also seems to influence substrate specificity and to give HvLD a higher affinity for α-CD than pullulanases. The crystal structures additionally provide new insight into cation sites and the concerted action of the battery of hydrolytic enzymes in starch degradation.     

Characterization and application of a detergent-stable alkaline α-amylase from Bacillus subtilis strain AS-S01a

A strain AS-S01a, capable of producing high-titer alkaline α-amylase, was isolated from a soil sample of Assam, India and was taxonomically identified as Bacillus subtilis strain AS-S01a. Optimized α-amylase yield by response surface method (RSM) was obtained as 799.0 U with a specific activity of 201.0 U/mg in a process control bioreactor. A 21.0 kDa alkaline α-amylase purified from this strain showed optimum activity at 55 °C and pH 9.0, and it produced high molecular weight oligosaccharides including small amount of glucose from starch as the end product. The K_m and V_max values for this enzyme towards starch were determined as 1.9 mg/ml and 198.21 μmol/min/mg, respectively. The purified α-amylase retained its activity in presence of oxidant, surfactants, EDTA and various commercial laundry detergents, thus advocating its suitability for various industrial applications.     

Multi-objective optimization of bioethanol production during cold enzyme starch hydrolysis in very high gravity cassava mash

Cold enzymatic hydrolysis conditions for bioethanol production were optimized using multi-objective optimization. Response surface methodology was used to optimize the effects of α-amylase, glucoamylase, liquefaction temperature and liquefaction time on S. cerevisiae biomass, ethanol concentration and starch utilization ratio. The optimum hydrolysis conditions were: 224 IU/g_starch α-amylase, 694 IU/g_starch glucoamylase, 77 °C and 104 min for biomass; 264 IU/g_starch α-amylase, 392 IU/g_starch glucoamylase, 60 °C and 85 min for ethanol concentration; 214 IU/g_starch α-amylase, 398 IU/g_starch glucoamylase, 79 °C and 117 min for starch utilization ratio. The hydrolysis conditions were subsequently evaluated by multi-objectives optimization utilizing the weighted coefficient methods. The Pareto solutions for biomass (3.655-4.380 × 10⁸ cells/ml), ethanol concentration (15.96-18.25 wt.%) and starch utilization ratio (92.50-94.64%) were obtained. The optimized conditions were shown to be feasible and reliable through verification tests. This kind of multi-objective optimization is of potential importance in industrial bioethanol production.     

Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L.

In this paper, polysaccharides were extracted from the seeds of Plantago asiatica L. with hot water and separated into three fractions PLP-1 (18.9%), PLP-2 (52.6%) and PLP-3 (28.5%) by Sephacryl™ S-400 HR column chomatography. The main fraction PLP-2's structure was elucidated using oxalic acid hydrolysis, partial acid hydrolysis, methylation, GC, GC-MS, 1D and 2D NMR. PLP-2 was composed of Rha, Ara, Xyl, Man, Glc and Gal, in a molar ratio of 0.05:1.00:1.90:0.05:0.06:0.10. Its uronic acid was GlcA. PLP-2 was highly branched heteroxylan which consisted of a β-1,4-linked Xylp backbone with side chains attached to O-2 or O-3. The side chains consisted of β-T-linked Xylp, α-T-linked Araf, α-T-linked GlcAp, β-Xylp-(1 → 3)-α-Araf and α-Araf-(1 → 3)-β-Xylp, etc. Based on these results, the structure of PLP-2 was proposed.     

Effects of poly(ethylene glycol) and salt on the binding of α-amylase from the fermentation broth of Bacillus amyloliquefaciens by Cu2+-β-CD affinity adsorbent

α-Amylase from Bacillus amyloliquefaciens was purified by the immobilized metal ion affinity adsorbent, β-CD_cl-IDA-Cu²⁺. The adsorbent was prepared by reacting the cross-linked β-cyclodextrin (β-CD) with the ligand, iminodiacetic acid (IDA). The copper ion was further linked to the adsorbent. Poly(ethylene glycol) (PEG) was added to the fermentation broth to improve the adsorption efficiency of the adsorbent toward α-amylase. The effort was to provide hydrophobic interactions with the impurities which might interfere with the adsorption of α-amylase. It also provided a polymer shielding effect to prevent non-specific interactions. With the addition of PEG, the adsorption efficiency could be increased to 98%. Imidazole containing a phosphate buffer and NaCl was used to elute the bound α-amylase. By consecutive adsorption/desorption steps, up to 81% of the α-amylase activity could be recovered. Regarding the reutilization of the affinity adsorbents, α-amylase could be adsorbed and desorbed six times consecutively without a significant loss of α-amylase activity.     

Galactans from Cryptonemia species. Part II: Studies on the system of galactans of Cryptonemia seminervis (Halymeniales) and on the structure of major fractions

Cryptonemia seminervis biosynthesizes a family of d,l-hybrid galactans based on the classical 3-linked β-d-galactopyranosyl→4-linked α-d- and α-l-galactopyranosyl alternating sequence (A-units→B-units) with major amounts of α-d- and α-l-galactose and 3,6-anhydro-d- and l-galactose and lesser percentages of 3,6-anhydro-2-O-methyl-l-galactose, 2-O-methyl-, 4-O-methyl- and 6-O-methylgalactoses. The dispersion of structures in this family is based on five structural factors, namely: (a) the amount and position of substituent groups as sulfate (major), pyruvic acid ketals, methoxyl and glycosyl side-chain (4-O-methyl galactopyranosyl and/or xylosyl); (b) the ratio galactose/3,6-anhydrogalactose in the B-units; (c) the ratio d,l-galactoses and d,l-3,6-anhydrogalactoses also in the B-units, (d) the formation of diads and (e) the sequence of the diads in the linear backbone. Considering these variables it is not unexpected to find in the fractions studied at least 18 structural units producing highly complex structures. Structural studies carried out in two major fractions (S2S-3 and S2S-4) showed that these galactans were formed mainly by β-d-galactopyranosyl 2-sulfate (20 and 11.9 mol %), β-d-galactopyranosyl 2-sulfate 4,6-O-(1′-carboxyethylidene) (8.9 and 6.0 mol %) and β-d-galactopyranosyl 2,6-sulfate (5.4 and 18.6 mol %), together with 3,6-anhydro-α-l-galactopyranosyl (11.4 and 7.3 mol %) and 3,6-anhydro-α-l-galactopyranosyl 2-sulfate (4.9 and 15.4 mol %) and minor quantities of 12-15 other structural units.Preparative alkaline treatment carried out on fraction (S2S-3) produced a quantitative formation of 3,6-anhydro α-l-galactopyranosyl units from precursor units (α-l-galactose 6-sulfate and α-l-galactose 2,6-sulfate). Kinetic studies on this 3,6-anhydro cyclization show a rate constant of 5.2 × 10⁴ s⁻¹ indicating diads of the type G→L6S/2,6S. Data from chemical, spectroscopic and kinetic studies suggest that, in S2S-3, the agaran block in the d,l-hybrid galactan is composed of the following diads: G(6R)→L6S/2,6S and G2S(P)(2,6S)→LA(2S)(2R)(2M) and the carrageenan block of G2S(P)→D(2S)(2,3S)(3S)(3,6S) in a molar ratio of agaran to carrageenan structures of ∼2:1.     

Yeast alpha glucosidase inhibitory and antioxidant activities of six medicinal plants collected in Phalaborwa,South Africa

Recent decades have experienced a sharp increase in the incidence and prevalence of diabetes mellitus. One antidiabetic therapeutic approach is to reduce gastrointestinal glucose production and absorption through the inhibition of carbohydrate-digesting enzymes such as α-amylase and α-glucosidase and α-amylase. The aim of the current study was to screen six medicinal plant species, with alleged antidiabetic properties for α-glucosidase inhibitory activities. Powdered plant materials were extracted with acetone, and tested for ability to inhibit baker's yeast α-glucosidase and α-amylase activities. The largest mass (440 mg from 10 g) of the extract was obtained from Cassia abbreviata, while both Senna italica and Mormordica balsamina yielded the lowest mass of the extracts. Extracts of stem bark of C. abbreviata inhibited baker's yeast α-glucosidase activity with an IC₅₀ of 0.6 mg/ml. This plant species had activity at low concentrations, with 1.0 mg/ml and above resulting in inhibition of over 70%. The other five plant extracts investigated had IC₅₀ values of between 1.8 and 3.0 mg/ml. Senna italica only managed to inhibit the activity of enzyme-glucosidase at high concentrations with an IC₅₀ value of 1.8 mg/ml, while Tinospora fragosa extracts resulted in about 55% inhibition of the activity of the enzyme at a concentration of 3.5 mg/ml, with an estimated IC₅₀ value of 2.8 mg/ml. The bark extract of C. abbreviata was the most active inhibitor of the enzyme, based on the IC₅₀ values (0.6 mg/ml). The bark extract of C. abbreviata contains non-competitive inhibitor(s) of α-glucosidase, reducing V_max value of this enzyme from 5 mM·s^–1 to 1.67 mM·s^–1, while K_m remained unchanged at 1.43 mM for para-nitrophenyl glucopyranoside. Antioxidant activity of the extracts was also investigated. The C. abbreviata extract was more active as an antioxidant than the positive control, trolox. The extracts did not inhibit alphaamylase activity more than about 20% at the highest concentration tested.     

Modified α-amylase activity among insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais

Fitness cost is usually associated with insecticide resistance and may be mitigated by increased energy accumulation and mobilization. Preliminary evidence in the maize weevil (Coleoptera: Curculionidae) suggested possible involvement of amylases in such phenomenon. Therefore, α-amylases were purified from an insecticide-susceptible and two insecticide-resistant strains (one with fitness cost [resistant cost strain], and the other without it [resistant no-cost strain]). The main α-amylase of each strain was purified by glycogen precipitation and ion-exchange chromatography (≥70-fold purification, ≤19% yield). Single α-amylase bands with the same molecular mass (53.7 kDa) were revealed for each insect strain. Higher activity was obtained at 35-40 °C and at pH 5.0-7.0 for all of the strains. The α-amylase from the resistant no-cost strain exhibited higher activity towards starch and lower inhibition by acarbose and wheat amylase inhibitors. Opposite results were observed for the α-amylase from the resistant cost strain. Although the α-amylase from the resistant cost strain exhibited higher affinity to starch (i.e., lower K_m), its V_max-value was the lowest among the strains, particularly the resistant no-cost strain. Such results provide support for the hypothesis that enhanced α-amylase activity may be playing a major role in mitigating fitness costs associated with insecticide resistance.     

Preparation and physical properties of chitin fatty acids esters

Trifluoroacetic anhydride is an effective promoter for the preparation of chitin single- and mixed-acid esters. Complete dissolution is achieved within 30 min when powdered chitin is heated at 70 °C in a mixed solution of carboxylic acid(s) and trifluoroacetic anhydride. Chitin esters prepared are chitin acetate, chitin butyrate, chitin hexanoate and chitin octanoate, chitin co-acetate/butyrate, chitin co-acetate/hexanoate, chitin co-acetate/octanoate, chitin co-acetate/palmitate, each from a solution of the respective reactants. The products have degrees of O-acyl substitution in a range of DS 1-2 depending on the nature of acyl group, as analyzed by gas-liquid and high-pressure liquid chromatography. Acetic acid as a mutual component for the mixed-acid esters increases the total degree of substitution, and the acetyl substitution is close to the relative distribution in the reaction mixture for chitin co-acetate/butyrate. It is favored over hexanoate, octanoate, and palmitate. The parent molecules, as calculated by the composition of the chitin esters and their molecular weights by light-scattering spectroscopy, are 30 kDa for the smallest and 150-151 kDa for the largest. Films of these chitin derivatives when cast from solution are strong and flexible with limited extensibility. By dynamic mechanical analysis of the ester film, it was found that both the glass transition temperature (T_g) and the tensile modulus (E′ at 25 °C) are highest for chitin acetate (218 °C and 5.8 GPa), and lowest for chitin octanoate (182 °C and 1.5 GPa). For the other esters, these values lie between the above-cited values, where the T_g and the E′ decrease with an increase in the chain length of the acyl constituent.     

O-Methylated mannogalactan from the microalga Coccomyxa mucigena, symbiotic partner of the lichenized fungus Peltigera aphthosa

A structural study of the carbohydrates from Coccomyxa mucigena, the symbiotic algal partner of the lichenized fungus Peltigera aphthosa, was carried out. It produced an O-methylated mannogalactan, with a (1 → 6)-linked β-galactopyranose main-chain partially substituted at O-3 by β-Galp, 3-OMe-α-Manp or α-Manp units. There were no similarities with polysaccharides previously found in the lichen thallus of P. aphthosa. Moreover, the influence of lichenization in polysaccharide production by symbiotic microalgae and the nature of the photobiont in carbohydrate production in lichen symbiosis are also discussed.     

Maltose-forming α-amylase from the hyperthermophilic archaeon Pyrococcus sp. ST04

The deduced amino acid sequence from a gene of the hyperthermophilic archaeon Pyrococcus sp. ST04 (Py04_0872) contained a conserved glycoside hydrolase family 57 (GH57) motif, but showed <13 % sequence identity with other known Pyrococcus GH57 enzymes, such as 4-α-glucanotransferase (EC 2.4.1.25), amylopullulanase (EC 3.2.1.41), and branching enzyme (EC 2.4.1.18). This gene was cloned and expressed in Escherichia coli, and the recombinant product (P yrococcus sp. ST04 maltose-forming α-amylase, PSMA) was a novel 70-kDa maltose-forming α-amylase. PSMA only recognized maltose (G2) units with α-1,4 and α-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzed pullulan very poorly. G2 was the primary end product of hydrolysis. Branched cyclodextrin (CD) was only hydrolyzed along its branched maltooligosaccharides. 6-O-glucosyl-β-cyclodextrin (G1-β-CD) and β-cyclodextrin (β-CD) were resistant to PSMA suggesting that PSMA is an exo-type glucan hydrolase with α-1,4- and α-1,6-glucan hydrolytic activities. The half-saturation value (K _m) for the α-1,4 linkage of maltotriose (G3) was 8.4 mM while that of the α-1,6 linkage of 6-O-maltosyl-β-cyclodextrin (G2-β-CD) was 0.3 mM. The k _cat values were 381.0 min^?1 for G3 and 1,545.0 min^?1 for G2-β-CD. The enzyme was inhibited competitively by the reaction product G2, and the K _i constant was 0.7 mM. PSMA bridges the gap between amylases that hydrolyze larger maltodextrins and α-glucosidase that feeds G2 into glycolysis by hydrolyzing smaller glucans into G2 units.     

Effect of adrenergic agents on α-amylase release and adenosine 3′,5′-monophosphate accumulation in rat parotid tissue slices

The role of cyclic AMP in stimulus-secretion coupling was investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20–30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both α-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effective. A parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fraction. The results suggest that these drugs are acting on the parotid acinar cell through a β₁-adrenergic mechanism.At the lowest concentrations tested, each of the adrenergic agonists stimulated significant α-amylase release with no detectable stimulation of cyclic AMP accumulation. Even in the presence of theophylline, phenylephrine at several concentrations increased α-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intracellular concentration of cyclic AMP may not be necessary for stimulation of α-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of α-amylase release by isoproterenol.Stimulation of α-amylase release by phenylephrine was only partially blocked by either α- or β-adrenerg blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolamine. Phenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and α-amylase release. However, phenoxybenzamine also potentiated the stimulation of α-amylase release by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using α-adrenergic blocking agents as tools for investigation of α- and β-adrenergic antagonism.     

Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch

The gene for a membrane-bound, halophilic, and thermostable α-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove—the N-C groove—some 30 Å away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 Å resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with α-cyclodextrin and maltoheptaose at 2.2 Å resolution, a maltotetraose molecule is bound in subsites − 1 to + 3, spanning the cleavage point at − 1/+ 1, with the − 1 glucosyl residue present as a ²S_o skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across − 1/+ 1 and ring distortion. We observe three methionine side chains that serve as “binding platforms” for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch.     

Biochemical properties of latices from the Euphorbiaceae

Latex sera from 18 Euphorbia species were surveyed for protein, carbohydrate and total solid contents. Protease, esterase, alkaline and acid phosphatase, N-acetyl-β-glucosaminidase, α-mannosidase, α- and β-galactosidase, α- and β-glucosidase, α-amylase, lysozyme, leucine amino peptidase and cellulase activities were also measured. The effects of nine protease inhibitors were assayed as were haemagglutinating (lectin) contents. Two-dimensional (isoelectric focusing and SDS) PAGE maps of the sera were made. The results obtained show that the latices have widely varying biochemical properties.     

Molecular cloning and characterization of a novel family VIII alkaline esterase from a compost metagenomic library

A metagenomic library was constructed from completely fermented compost using a fosmid vector. From a total of 23,400 clones, 19 esterase-positive clones were selected on LB plates containing 1% glyceryl tributyrate as the substrate. The esterase gene of an esterase-positive clone, est2K, was on an ORF of 1299 bp and encoded a protein of 432 amino acids. Est2K had a SMTK motif and was a family VIII esterase. Unlike most family VIII esterases, Est2K had a signal peptide of 27 amino acids. The molecular mass and pI of the mature Est2K was calculated to be 44,668 Da and 4.48, respectively. The amino acid sequence of Est2K showed 72% identity with that of EstC, an esterase of an uncultured bacterium from leachate. The purified Est2K was optimally active at pH 10.0 and 50 °C. Est2K was stable in the presence of 30% methanol and exhibited a 2.4-fold higher activity in the presence of 5% methanol than in the presence of 1% isopropanol. Est2K preferred short to medium length p-nitrophenyl esters, especially p-nitrophenyl butyrate, as the substrate. Est2K did not hydrolyze β-lactam antibiotics ampicillin and nitrocefin, even though Est2K showed the highest similarity to EstC.     

Partial biochemical characterization of α- and β-glucosidases of lesser mulberry pyralid, Glyphodes pyloalis Walker (Lep.: Pyralidae)

The Lesser Mulberry Pyralid, Glyphodes pyloalis, is an important pest of mulberry. This pest feeds on mulberry leaves, and causes some problems for the silk industries in the north of Iran. The study of digestive enzymes is highly imperative to identify and apply new pest management technologies. Glucosidases have an important role in the final stages of carbohydrate digestion. Some enzymatic properties of α- and β-glucosidases from midgut and salivary glands of G. pyloalis larvae were determined. The activities of α- and β-glucosidase in the midgut and salivary glands of 5th instar larvae were obtained as 0.195, 1.07, 0.194 and 0.072 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively. Activity of α- and β-glucosidase from whole body of larval stages was also determined. Data showed that the highest activity of α- and β-glucosidase was observed in the 5th larval stage, 0.168 and 0.645 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively and the lowest activity in the 2nd larval stage, 0.042 and 0.164 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively. Results showed that the optimal pH for α- and β-glucosidase activity in midgut and salivary glands were 7.5, 5.5, 8-9 and 8-9 respectively. Also, the optimal temperature for α- and β-glucosidase activity in the midgut was obtained as 45 °C. The addition of CaCl₂ (40 mM) decreased midgut β-glucosidase activity whereas α-glucosidase activity was significantly increased at this concentration. The α-glucosidase activity, in contrast to β-glucosidase, was enhanced with increasing in concentration of EDTA. Urea (4 mM) and SDS (8 mM) significantly decreased digestive β-glucosidase activity. Characterization studies of insect glucosidases are not only of interest for comparative investigations, but also understanding of their function is essential when developing methods of insect control such as the use of enzyme inhibitors and transgenic plants to control insect pest.     

Enhancing purification of α-amylase by superparamagnetic complex with alginate/chitosan/β-cyclodextrin/TPP

The main aim of this study was to synthesize the superparamagnetic nanoparticles coated by alginate/chitosan/β-cyclodextrin to purify α-amylase. Isolated bacteria were identified by morphological, biochemical and taxonomic molecular studies. FTIR- spectrometer, VSM, X-ray instruments and Malvern Zetasizer were used to characterize nanoparticles characteristics. The morphological structures and the elemental composition of the nanoparticles were studied by using FESEM and EDS, respectively. The molecular weight of enzyme was determined using SDS-PAGE, and the enzyme activity detected by zymographic analysis. FTIR studies showed the presence of Fe–O–Fe in the Fe₃O₄ and verified the interaction between chitosan, β-cyclodextrin and alginate. The saturation magnetization for superparamagnetic and coated superparamagnetic nanoparticles was indicated 39 and 1.9?emu?g^?1, respectively. The maximum intensity of the XRD peak indicated the presence of the Fe₃O₄. FESEM and EDS analysis showed that the nanoparticles were regular and spherical in shape and corresponded to the Fe and O elements. Enzyme purification by synthesized nanoparticles was achieved 13.84?U?mg^?1; purification fold of 3.50. The molecular weight of α-amylase was about 22?kDa. The highest activity of α-amylase was observed at 70?°C, pH 9.3 and Ca²⁺-independent. As a conclusion, the coated superparamagnetic nanoparticles showed more applications in enzyme purification comparing to the conventional methods.     

Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica

EhCP-B9, a cysteine protease (CP) involved in Entamoeba histolytica virulence, is a potential target for disease diagnosis and drug design. After purification from inclusion bodies produced in Escherichia coli, the recombinant EhCP-B9 precursor (ppEhCP-B9) can be refolded using detergents as artificial chaperones. However, the conformational changes that occur during ppEhCP-B9 refolding remain unknown. Here, we comprehensively describe conformational changes of ppEhCP-B9 that are induced by various chemical detergents acting as chaperones, including non-ionic, zwitterionic, cationic and anionic surfactants. We monitored the effect of detergent concentration and incubation time on the secondary and tertiary structures of ppEhCP-B9 using fluorescence and circular dichroism (CD) spectroscopy. In the presence of non-ionic and zwitterionic detergents, ppEhCP-B9 adopted a β-enriched structure (ppEhCP-B9_β1) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a β-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an α + β structure, similar to that of papain but without proteolytic activity (ppEhCP-B9_α + β1). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a β-sheet rich structure (ppEhCP-B9_β2) that slowly transforms into a different type of α + β conformation that exhibited proteolytic activity (ppEhCP-B9_α + β2) suggesting that enzymatic activity is gained as slow transformation occurs.