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Biochemical characteristics of α-amylase in the digestive system and head glands of Cryptolaemus montrouzieri, a key predator of citrus mealybug, Planococcus citri (Pseudococcidae), were studied. The major isoform of α-amylase with the same molecular weight was detected in both gut and head glands loaded on polyacrylamide-starch gel electrophoresis. Moreover, a minor band with much lower intensity was observed in zymogram analysis of gut. Results showed that the specific activity of α-amylase from head glands (0.89 ± 0.02 μmol/min/mg protein) was significantly more than that of digestive system (0.76 ± 0.01 μmol/min/mg protein) in common condition (temperature equal to 25 ± 1 ^oC). The optimal pH and temperature for α-amylases activity were determined to be nearly 4 and 50°C in digestive system and 6 and 60 °C in head glands, respectively. EDTA (Ethylenediamine tetra acetic acid), Mg²⁺, Na⁺, Co²⁺, Fe²⁺ and Ca²⁺ inhibited the enzyme activity but Ba²⁺, Zn²⁺, Hg⁺ and K⁺ enhanced enzyme activity in digestive system. EDTA and all tested metal ions except Ba²⁺ inhibited the enzyme activity of head glands. Detectable levels of α-amylase activity in the insect reflect adaptation of the coccinellid for using starch granules or sugars (honeydew; sugary excreta of homopterans; and nectar) as a source of food in addition to predatory habits.

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The rose sawfly, Arge rosae Linnaeus, is one of the most destructive pests of rose bushes in the north of Iran. Nowadays, many attempts have been made to reduce pesticide application by looking for new methods of pest control. A non chemical method for controlling insect pests including A. rosae can be achieved by using genetically engineered plants expressing carbohydrase inhibitors. Therefore, in present study we characterized biochemical properties of digestive carbohydrases in the gut of A. rosae for achieving a new method for control of this pest. The specific activity of α-amylase in the digestive system of last larval instars of A. rosae was obtained as 9.46 ± 0.06 μmol min^-1 mg^-1 protein. Also, the optimal pH and temperature for α-amylase were found to be at pH 8 and 50 °C. As calculated from Lineweaver-Burk plots, the K_m and V_maxvalues for α-amylase were 0.82 mg/ml and 7.32 µmol min^-1 mg^-1 protein, respectively, when starch was used as substrate. The effects of ions on amylolytic activity showed that Mg²⁺ and Na⁺ significantly increased amylase activity, whereas SDS and EDTA decreased the enzyme activity. The highest activities of α-/β-glucosidase and β-galactosidase were obtained at pH 5.0. By the native PAGE, three, one, one and two bands were clearly detected for α-amylase, α-/β-glucosidase and β-galactosidase, respectively. No bands were found for α-galactosidase that confirmed the absence or low activity of this carbohydrate in the digestive system of A. rosae. These results could provide the knowledge needed to produce transgenic plants for control of this pest.  

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The carob moth is one of the most devastating pests of pomegranate and some other products. Various pest control measures have been undertaken in order to control this pest but none of them has been successful so far. In the current study the effects of cereal seed proteinaceous extracts including triticale and three wheat cultivars (MV17, Aflak, and Zare) have been studied on α-amylase and protease activity of salivary glands of this insect.Initial screening showed 38, 44, 28 and 76% inhibitory effect for triticlae, MV-17, Aflak, and Zare cereal seed extracts respectively on α-amylase activity. Further studies were performed with Zare wheat cultivar using various concentrations including 13, 6.5, 3.25, 1.625 and 0.8125 µg protein on the enzyme activity and results showed that they inhibited the enzyme activity by 76, 75, 68, 60, and 42%, respectively. Gel assays confirmed the spectrophotometric data i.e the effect of the seed extract on the enzyme was dose dependant. The same trend was observed when seed extracts were tested against proteinase activity. These data suggest that plants produce different proteins with different specificity toward herbivores digestive enzymes some of which could be used for insect control in IPM program.

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Willow leaf beetle, Plagiodera versicolora is an important pest of willow trees that feeds on the leaves both as larvae and as adults. -Amylases (EC 3.2.1.1) are the major insect digestive enzymes that catalyze the endohydrolysis of long -1,4-glucan chains such as starch and glycogen. In the current study, -amylase activity was studied in the midgut of larvae and adults of P. versicolora. Amylase activity in the midgut of larvae and adults was 0.6807 and 0.1162 µmol/min/mg protein, respectively. The optimal pH for -amylase activity of larvae and adults was 4 and 8, respectively and the optimal temperature for both was 35 °C. The enzyme activity of larvae was inhibited by the addition of Na²⁺, K⁺ and Zn²⁺. K⁺ (at 5 mmol) had the most positive effect on α-amylase activity in adults. EGTA had significant influence on decreasing the enzyme activity in larvae. EDTA had the most effect on increasing the activity of the enzyme in adults. Plant amylase inhibitors play important role against insect pests. Therefore, the characterization of digestive enzymes and the examination of inhibitors on the enzyme activity could be useful in tackling insect pests.    

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In the  Bacillusamyloliquefaciens α-amylase (BAA), the loop (residues from 177-185; region І) is the constructive part of the cage responsible for attachment to calcium. It has two more amino acid residues than the α-amylase from Bacillus licheniformis (BLA). Arg176 in this region makes an ionic interaction with Glu126 from region ІІ (residues 118-131) but this interaction is lost in BLA due to substitution of R176Q and E126V. It is the common feature of α-amylases that calcium ion is required for their thermal stability. The present work quantitatively estimates the effect of ionic interaction on the overall stability of the enzyme. To clarify the functional and structural significance of corresponding salt bridge, first an automated homology model of the mutant enzyme (∆E126) was built by the Swiss-Model Protein Modeling Server.  Bacillus amyloliquefaciens α-amylase (3BH4.pdb) was used as the template and examined by GETAREA and WHAT IF programs, then Glu126 was deleted (∆E126) by site-directed mutagenesis and the thermostability was examined for the wild-type and mutant enzymes. Modeling results showed that deletion of salt bridge affected on the hydrophobic and hydrophilic residues orientation of two discussed regions (Ι, ΙΙ). The mutant enzyme also exhibited lower thermostability relative to the wild-type enzyme. Thus, it may be suggested that salt bridge could affect on accessible surface area of the discussed regions, decrease water diffusion,  prevent diffusion of cations and improve the thermostability of the whole protein.

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Asparaginase and amylase are widely used enzymes in various industries, which can be produced by endophytic fungi. In this study, the ability of producing these two enzymes in endophytic fungi isolated from six species of Thymus has been reported for the first time in the world. Among 89 isolates of the test, 34 isolates produced asparaginase among which M24 (Fusarium subglutinans) displayed the greatest enzyme activity. Thirty three isolates showed the ability to produce amylase while the greatest enzyme activity belonged to M53 (Curvularia akaii). This study can be regarded as a preliminary work and endophytic fungi of high activity are proposed as possible resources for control of cancer in humans and for industrial applications.

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 Maltogenic Amylases (MAase) are a subfamily of Į-amylase family that can hydrolyze multiple substrates including starch, pullulan and cyclodextrins however, they prefer cyclodextrins to others, and unlike other Į-amylases, they are intracellular. This enzyme has the potential for use in many industrial processes such as food, fermentation and pharmacy. The effect of different concentrations of Ca2+ and K+ ions on irreversible thermoinactivation of the enzyme at 65 ÛC showed that Ca2+ and K+ decreased and increased its thermal stability. The CD spectra of the enzyme in the presence and absence of metal ions were measured to detect changes in the secondary structure contents. The spectra showed a decrease in the Į-helix content in the presence of 1 and 10 mM of Ca2+, but in the presence of 5 mM, a drastic increase in Į-helix content of the enzyme was witnessed. In the presence of 1 and 5 mM of Na+ the Į-helix content decreased, while it was increased in the presence of 10 mM. The results from intrinsic fluorescence of the protein (excitation at 280 nm) indicated that Ca2+ ion at 1 and 5 mM caused an increase in tertiary structure of the enzyme; however, at 10 mM, a decrease was observed in its tertiary structure. K+ ion at all concentrations increased the tertiary structure of the enzyme. These spectroscopic results are in a good agreement with the thermostability data. It was shown that destabilizing effect of calcium was enthalpic (decrease in ǻH#) whereas the stabilizing effect of potassium was entropic (decrease in ǻS#).

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The effect of simultaneous application of probiotic and digestive enzyme on the intestinal digestive activity of the whiteleg shrimp, Litopenaeus vannamei, was evaluated. For this purpose, the juvenile shrimps were fed for 30 days with a dietary probio-enzyme (containing a combination of six exogenous enzymes and four probiotic bacterial strains) at four concentrations of 0, 0.25, 0.5, and 1 g kg^-1 feed. Shrimps (5.04±0.39 g ind^-1) were randomly distributed in 12 aquaria (4 treatments × 3 replications); each aquarium contained 15 individual shrimps. Results indicated significantly (p≤0.05) higher growth performance, amylase and lipase activity at 0.5 g kg^-1 treatment as compared to other treatments. Protease activity was, however, significantly (p≤ 0.05) higher at 1.0 g kg^-1treatment as compared to other groups. Results also indicated that increase in the concentration of probiotic and enzymes supplementation was not associated with increase in all the digestive enzyme activity. In other words, probiotics and enzymes onlywithin specific range can have positive effect on growth performance and digestive enzyme activity of L. vannamei, above or below.

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The elm leaf beetle, Xanthogalerucella luteola (Muller) (Col.: Chrysomelidae) is a serious pest of elm trees and it has been distributed all over the world. The current study was undertaken to investigate the inhibitory effects of protein extracts of three weed seeds including datura Datura stramonium L., amaranth Amaranthus retroflexus L. and wild oat Avena fatua L. against X. luteola α-amylase using spectrophotometric assay as well as in gel assays. The effects of five concentrations of each seed proteinaceous extracts were tested on α-amylase activity of the larval gut. The results showed a dose dependent manner in inhibition of the insect enzyme. At the highest concentration of protein extracts (12 μg protein) of all three seed extracts including amaranth, wild oat and datura, the inhibition was 71, 79 and 31%, respectively. Whilst, at low concentration (0.75 μg protein), the inhibition observed was 15, 36 and 5%, respectively. Thus, the greatest inhibition percentage was obtained when proteinaceous extract of wild oat seed was used. These results were confirmed when in gel assays were performed. All three seed proteinaceous extracts had an optimum pH inhibition of 6.0. Thus, it is concluded that wild oat seed proteins are potentially good for detailed investigation in order to get a clear picture of its active compound/s and its structure-function relationship.    

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The tomato leaf miner, Tuta absoluta (Meyrick) is an imported pest and serious threat to tomato production in farms and greenhouses of Iran. Use of genetically engineered plants expressingcarbohydrase inhibitors is one of the non-chemical methods for controlling insect pests, and knowledge about enzymatic properties of carbohydrases will help us to achieve this goal. Therefore, in present study we characterized biochemical properties of digestive carbohydrases in the midgut of last larval instar of T. absoluta fed on different tomato cultivars (Kingston, Riogrande, Super Luna, Super Chief, Super strain B and Calj). While the highest amylolytic activity was on Super strain B, the lowest was on Super Chief. The optimal pH and temperature for α-amylase were found to be at pH 9.0 and 45 °C, respectively. As calculated from Lineweaver-Burk plots, the highest Km and Vmax values for α-amylase obtained in Super Chief and Super Luna cultivars were 0.565 ± 0.11mM and 2.287 ± 0.4mM/min, respectively. The effects of different compounds on amylolytic activity indicated that CaCl2, MgCl2, NaCl and KCl increased amylase activity, whereas EDTA, ZnCl2 and BaCl2 decreased the enzyme activity in Super Luna cultivar. The highest activity of α-/ß-glucosidases was observed at pH 6.0 and 7.0, respectively, whereas the optimal pH for α/ß-galactosidases was at 5.0. The highest specific activity of α-/ß-glucosidases was determined in Riogrande-fed larvae, whereas the highest α/ß-galactosidases activity was in the larvae fed on Riogrande and Calj cultivars, respectively. By the native- PAGE, two bands were clearly detected for α-amylase. Since the larvae reared on Kingston showed lowest carbohydrase activities, this cultivar could possibly be suggested as the least suitable host for feeding of T. absoluta.

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Human activin A is a homodimer of βA subunit which is synthesized in the form of prepro-activin with 426 amino acids; mature activin A with 116 amino acids is processed from this larger precursor protein. This protein which was extracted for the first time from follicular fluid is a strong stimulator of FSH biosynthesis. The functions have been found to be exerted by activin, including roles in cell proliferation, differentiation, apoptosis and survival of neurons. As this protein plays a considerable role in the treatment of neurodegenerative disease such as Alzheimer,s disease and wound repair, in this study for the first time was expressed in three different strains of E.coli. Activin A has disulfide bonds in its native and functional structure, so the cytoplasmic reducing environment of E.coli is not appropriate for its expression. Therefore, the oxidative space of periplasm for production of correctly folded activin A was considered. In this study, h-activin A cDNA and modified Iranian Bacillus Licheniformis α-amylase signal peptide obtained from NCBI data bank after codon optimization was cloned in pET21b(+) vector and transformed to BL21(DE3)pLysS, BL21(DE3)Rosetta gami and BL21(DE3) strains of E.coli. Expression occurred via induction of promoter with IPTG. Consequently, extracted proteins from these three strains were compared with each other using SDS-PAGE, Dot blot and western blot techniques. The data shows activin A expression especially in BL21(DE3) and BL21(DE3)Rosetta gami strains of E.coli.

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Efficacy of quercetin on α-amylase, lipase and protease activities via crustacean cardioactive peptide (CCAP) content of the midgut of the diamondback moth, Plutella xylostella (L.) was investigated. Fresh cabbage leaf discs were dipped in quercetin solution at different concentrations (100, 500 and 1000ppm) for 10 seconds. Third instar larvae of P. xylostella were fed on leaf dipped in quercetin solutions for 5 days. α-Amylase, lipase and protease activities were evaluated for 5 days. Quercetin significantly decreased lipase, protease and α-amylase activities in the midgut. The results of competitive ELISA showed that different concentration of quercetin had no effect on short neuropeptide F, tachykinin-4 and allatostatin content of the midgut, but it was shown that quercetin (500 and 1000ppm) decreased CCAP content of the midgut. Moreover, incubation of dissected midgut with CCAP increased α-amylase, lipase and protease activities. The injection of CCAP into the hemocoel clearly increased α-amylase, lipase and protease activities. Here, for the first time, it was confirmed that feeding on leaf dipped in quercetin, decreases CCAP content in the midgut of P. xylostella, that itself led to decrease of α-amylase, protease and lipase activities.
 
 

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  Enzymes of wheat flour have an important role on the quality of various backing products, especially bread. The knowledge of their activites can assist us to produce the high quality products. On the other hand, the most wheat flours in the countery have low α-amylase activity. Therefore, optimization of flour α-amylase activity is important for production of high quality bread. In the present work, chemical properties of three wheat flours varieties (Mahdavi, Kavir and M7318 that are produced in Isfahan province) were investigated. To show the changes of α-amylase activities, the activities of enzyme were measured during the germ growth times up to 72 hours. α-amylase activity of flours was also optimized according to falling number 250. The effect of this optimization on the bread texture in four different times after baking were then evaluated. The results showed that all three varieties had lower levels of α-amylase activity than the optimum level. The results indicated an increase in the activity of α-amylase during the growth of germ up to 72 hours. The results of the determination of the best blending percentage of germinated wheat flour with baking flour indicated that optimization of α-amylase activity is essential in terms of both wheat varieties of flour and germination times of wheat germ. Bread texture analysis indicated that bread produced with optimized α-amylase activity flours using appropriate percentage of germinated wheat flour have softer texture and lower staling. Wheat variety had also an important effect on staling so that bread produced with Kavir variety flour had softer texture than other varieties. Breads produced with optimized flour using 72-hours germinated wheat flour had softer texture than the breads made by optimized flours with other germination times.

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Mediterranean flour moth, Ephestia kuehniella Zeller, is important for mass rearing of parasitoid insects such as Bracon wasps and it's feeding on cereals leads to economic losses in flour mills. In current research, the effects of four different diets: wheat, barley, oat, and maize flours, on protein content of larval and pupal whole-body, gut and fat bodies of the last instars and the digestive α-amylase activity were evaluated. In addition, their protein patterns were compared using polyacrylamide gel electrophoresis (SDS-PAGE). The protein contents of fifth instars whole-body, fat bodies, gut, and also pupa varied in different diets. On the whole, the lowest protein levels were found in all experiments in oat and the highest in barley and wheat. Other biological parameters such as insect weight and digestive α-amylase activity were also significantly affected by the decrease in protein content. The results obtained from the estimation of protein content by a colorimetric method and SDS-PAGE were consistent with each other; the low or high protein contents were also clearly visible in the gels. According to the results of enzyme activity and protein contents of insects reared on different diets, barley and wheat are reported to be appropriate diets for this pest, while oat and maize are not suitable diets.
 

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The Sunn pest, Eurygaster integriceps (Puton) (Hem.: Scutelleridae), is an economic pest of wheat that reduces the quantitative and qualitative properties of wheat products. We investigated the effect of E. integriceps feeding on six different wheat cultivars (Ghermez (Red), Noodle, Antanius, Sabalan, Azar 2 (with two types of cultivation), and Sardari) on the protein content in the adult’s gut and fat body and their digestive enzymatic activity. All qualified values of the insect feeding on wheat cultivars differed significantly. The least amount of adult weight and protein content of gut and fat body were observed in the insects fed on Ghermez (Red) cultivar, and the highest amount belonged to the Sardari cultivar. The same results for protein content were obtained from SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Also, the lowest and highest gut α-amylase, pectinase, and protease activities were in Ghermez and Sardari cultivars, respectively. Therefore, it was concluded that the type of wheat cultivar affects the food preference of this insect and, thereby, physiological parameters of the insect gastrointestinal tract. Planting a wheat cultivar like “Ghermez,” which may be resistant, can be a suitable and cost-effective method to decrease the chemicals applied against this pest.

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Glucoamylase, is an important economic enzyme due to its ability to hydrolyze starch and β-D-glucose polymers. Understanding of factors affecting the thermal stability of the glucoamylase enzyme is critical in the production of isoenzymes with high heat or cold stability.  In this study, the effect of temperature on the structure and properties of each of the isoenzymes of the mesophilic, thermophilic and psychrophilic glucoamylase were studied. For this purpose, molecular dynamics simulation was used to assess these factors and structural differences. 240 nanosecond of MD simulation was done for three isoenzymes of glucoamylase in four temperatures at 300, 350, 400 and 450 K. The variations of each of these parameters were compared for three isoenzymes, and it was found that among the computable factors in molecular dynamics simulation, electrostatic energy of protein with water, van der Waals energy between proteins and water, free energy solubility (∆G_solvation), instability parameter, nonpolar solvent accessible surface, and total solvent accessible surface can be used to predict thermal stability of a protein during increase of temperature.
 

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The aim of this study was to isolate and identify a-amylase-producing bacteria present in mangrove ecosystems on Qeshm Island, Hormozgan, Iran. Samples of mangrove leaves and roots were screened for a-amylase activity using Lugol’s solution. Crude extracts were prepared of positive samples, and their a-amylase activity was determined by the Bernfeld method. The two strains with the highest activity were identified by molecular analysis of their 16S rRNA genes. a-Amylase production and activity were optimized by varying temperature and pH. 46 bacterial strains were isolated from mangrove tree leaf and root samples. Of these, 28 strains were capable of producing a-amylase. 16S rRNA gene sequence analysis of two strains with the highest enzyme production identified them as Bacillus sp. strain HR10 and Bacillus sp. strain HR11. The optimum temperature for enzyme production was 35 and 30 °C for strains HR10 and HR11, respectively, and the optimum pH was pH 8 for both strains. The highest enzyme activity was observed at 70 °C and 60 °C for the HR10 and HR11 strains, respectively, and the optimum pH was pH 8 for both strains. In conclusion, we have shown that bacteria isolated from mangrove leaf and root samples are potential source of a-amylases, tolerating a wide range of temperature and pH. Such a-amylases may be of interest for use in environmentally friendly industries.

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The experiment was conducted to determine α-amylase activity and the effect of seven plant species extracts including Punica granatum L. (Punicaceae), Rheum officinale B. (Polygonaceae), Rhus coriaria L. (Anacardiaceae), Artemisia sieberi B. ( Compositae), Peganum harmala L. (Nitrariaceae) , Datura stramonium L. (Solanaceae) and Thymus vulgaris L. (Lamiaceae) on α-amylase activity of four stored insect pests including Callosobruchus maculatus F. (Coleoptera: Bruchidae), Rhyzopertha dominica F. (Coleoptera: Bostrichidae), Sitophilus granarius L. (Coleoptera: Curculionidae), and Trogoderma granarium E. (Coleoptera : Dermestidae). Also, gut pH and optimum temperature for α-amylase activity of these insects were determined. It was found that α -amylases midgut pH of all four insect species was acidic and optimum temperature was between 30 and 40 °C. Beyond these temperatures, the α-amylases activities sharply decreased. Plant extracts caused inhibitory activity on insect α-amylases varying from nearly 4% to 95% inhibition. D. stramonium and R. officinali extracts had the highest amylase inhibitory activity among the tested extracts, while methanolic extracts of P. harmala, and T. vulgaris (except for S. granaries α-amylase) showed the lowest inhibitory activity. Gel assays revealed that more than one isoform of α -amylase detected in midgut crude extracts of the four insect pests examined could be inhibited by the plant extracts.

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