Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% ß-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Drying temperature affects rice seed vigor via gibberellin, abscisic acid, and antioxidant enzyme metabolism / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Seed vigor is a key factor affecting seed quality. The mechanical drying process exerts a significant influence on rice seed vigor. The initial moisture content (IMC) and drying temperature are considered the main factors affecting rice seed vigor through mechanical drying. This study aimed to determine the optimum drying temperature for rice seeds according to the IMC, and elucidate the mechanisms mediating the effects of drying temperature and IMC on seed vigor. Rice seeds with three different IMCs (20%, 25%, and 30%) were dried to the target moisture content (14%) at four different drying temperatures. The results showed that the drying temperature and IMC had significant effects on the drying performance and vigor of the rice seeds. The upper limits of drying temperature for rice seeds with 20%, 25%, and 30% IMCs were 45, 42, and 38 °C, respectively. The drying rate and seed temperature increased significantly with increasing drying temperature. The drying temperature, drying rate, and seed temperature showed extremely significant negative correlations with germination energy (GE), germination rate, germination index (GI), and vigor index (VI). A high IMC and drying temperature probably induced a massive accumulation of hydrogen peroxide (H2O2) and superoxide anions in the seeds, enhanced superoxide dismutase (SOD) and catalase (CAT) activity, and increased the abscisic acid (ABA) content. In the early stage of seed germination, the IMC and drying temperature regulated seed germination through the metabolism of H2O2, gibberellin acid (GA), ABA, and α-amylase. These results indicate that the metabolism of reactive oxygen species (ROS), antioxidant enzymes, GA, ABA, and α-amylase might be involved in the mediation of the effects of drying temperature on seed vigor. The results of this study provide a theoretical basis and technical guidance for the mechanical drying of rice seeds.

Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Highly efficient enzymatic preparation of isomalto-oligosaccharides from starch using an enzyme cocktail

Background: Current commercial production of isomalto-oligosaccharides (IMOs) commonly involves a lengthy multistage process with low yields. Results: To improve the process efficiency for production of IMOs, we developed a simple and efficient method by using enzyme cocktails composed of the recombinant Bacillus naganoensis pullulanase produced by Bacillus licheniformis, α-amylase from Bacillus amyloliquefaciens, barley bran ß-amylase, and α-transglucosidase from Aspergillus niger to perform simultaneous saccharification and transglycosylation to process the liquefied starch. After 13 h of reacting time, 49.09% IMOs (calculated from the total amount of isomaltose, isomaltotriose, and panose) were produced. Conclusions: Our method of using an enzyme cocktail for the efficient production of IMOs offers an attractive alternative to the process presently in use.

Salivary cortisol and alpha-amylase: subclinical indicators of stress as cardiometabolic risk

Currently, the potential for cardiovascular (CV) stress-induced risk is primarily based on the theoretical (obvious) side effects of stress on the CV system. Salivary cortisol and α-amylase, produced respectively by the hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic-adrenomedullary (SAM) system during stress response, are still not included in the routine evaluation of CV risk and require additional and definitive validation. Therefore, this article overviews studies published between 2010 and 2015, in which salivary cortisol and α-amylase were measured as stress biomarkers to examine their associations with CV/CMR (cardiometabolic risk) clinical and subclinical indicators. A comprehensive search of PubMed, Web of Science and Scopus electronic databases was performed, and 54 key articles related to the use of salivary cortisol and α-amylase as subclinical indicators of stress and CV/CMR factors, including studies that emphasized methodological biases that could influence the accuracy of study outcomes, were ultimately identified. Overall, the biological impact of stress measured by salivary cortisol and α-amylase was associated with CV/CMR factors. Results supported the use of salivary cortisol and α-amylase as potential diagnostic tools for detecting stress-induced cardiac diseases and especially to describe the mechanisms by which stress potentially contributes to the pathogenesis and outcomes of CV diseases.

BAY 41-2272 activates host defence against local and disseminated Candida albicans infections

In our previous study, we have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients. In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation. .

Application of decolourized and partially purified polygalacturonase and α-amylase in apple juice clarification

Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T660nm = 97.0%) of juice was attained after 2 h of incubation at 50 ºC in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.

Partial purification and some properties of -amylase from Bacillus subtilis KIBGE-HAS.

An extracellular -amylase from Bacillus subtilis KIBGE-HAS was partially purified by ultrafiltration and ammonium sulphate precipitation with 19.2-fold purification and specific activity of 4195 U/mg. The enzyme showed relatively high thermostability and retained 62% of its activity when kept at 70°C for 15 min. -Amylase was highly stable at -18°C and loss of activity was very low during stability study. Metal ions like Mn2+, Ca2+, Co2+, K+, Mg2+, and Fe3+ activated the enzyme, while Hg2+ Ba2+, Cu2+, Na+ and Al3+ strongly inhibited the activity. The α-amylase was highly stable in various surfactants and detergents. In the presence of surfactants such as SDS and Triton X-100 the enzyme activity was found 2.9 and 1.8-fold higher respectively than control. The non-ionic detergents (Tween 20 and Tween 80) exhibited slightly inhibitory effect on the enzyme activity.

Estrutura do grânulo de amido de banana e sua relação com as enzimas que atuam no metabolismo amido-sacarose / Structure of the banana starch granule and its relation with the enzymes that attack in the metabolism starch-sucrose

A banana é considerada um bom modelo para o estudo da transformação amido-sacarose, já que acumula um alto teor de amido durante o desenvolvimento que é rapidamente degradado durante o amadurecimento. Várias enzimas e provavelmente mais de uma via metabólica estão envolvidas neste processo. Com isso, o objetivo deste trabalho foi estudar as características estruturais dos grânulos, bem como, a atuação das enzimas envolvidas em sua degradação. Os grânulos de amidos foram isolados de bananas controle (não tratadas) e submetidas a diferentes tratamentos: etileno, 1-MCP, frutos mantidos a 13'graus'C e frutos tratados com etileno e mantidos a 13'graus'C. Os resultados obtidos mostraram alta atividade de enzimas 'alfa' e 'beta'-amilases ligadas ao grânulo tanto por ensaios in vitro como por géis de eletroforese contendo amilopectina como substrato...

Effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism in seedlings of wheat cultivars.

The effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism (alpha and beta amylases, sucrose phosphate synthase, sucrose synthase, acid and alkaline invertases) in wheat (Triticum aestivum L.) was investigated in the seedlings of drought-sensitive (PBW 343) and drought-tolerant (C 306) cultivars. The water deficit was induced by adding 6% mannitol (water potential -0.815 Mpa) in the growth medium. The water deficit reduced starch content in the shoots of tolerant seedlings as compared to the sensitive ones, but increased sucrose content in the shoots and roots of tolerant seedlings, indicating their protective role during stress conditions. It also decreased the alpha-amylase activity in the endosperm of seedlings of both the cultivars, but increased alpha and beta amylase activities in the shoots of tolerant ones. Sucrose phosphate synthase (SPS) activity showed a significant increase at 6 days of seedling growth (DSG) in the shoots of stressed seedlings of tolerant cultivar. However, SPS activity in the roots of stressed seedlings of sensitive cultivar was very low at 4 DSG and appeared significantly only at day 6. Sucrose synthase (SS) activity was lower in the shoots and roots of stressed seedlings of tolerant cultivar than sensitive ones at early stage of seedling growth. Higher acid invertase activity in the shoots of seedlings of tolerant cultivar appeared to be a unique characteristic of this cultivar for stress tolerance. Alkaline invertase activity, although affected under water deficit conditions, but was too low as compared to acid invertase activity to cause any significant affect on sucrose hydrolysis. In conclusion, higher sucrose content with high SPS and low acid invertase and SS activities in the roots under water deficit conditions could be responsible for drought tolerance of C 306.

Impact of cadmium and lead on Catharanthus roseus--a phytoremediation study.

The Madagascar Periwinkle, Catharanthus roseus (L.) G. Don (a valued medicinal plant) was exposed to different concentrations ofheavymetals like, CdCl, and PbCl, with a view to observe their bioaccumulation efficiency. Germination was inhibited by both the heavy metals in the seeds previously imbibed in GA, and KNO, for 24 hr. EC50 (the effective concentration which inhibits root length by 50%) was recorded as 180 microM for CdCl2, and 50 microM for PbCl2. Both alpha-amylase and protease activity were reduced substantially on treatment of seeds with increasing concentrations of CdCl2, and PbCl2. Malondialdehyde (MDA) a product of lipoxigenase (LOX) activity also increased due to the treatment of both CdCl, and PbCl2. When two-months-old plants grown in normal soil were transferred to soils containing increasing amounts of these two heavy metals, senescence of lower leaves and extensive chlorosis were noticed after four days of transfer However, plants gradually acclimatized and after 20 days the chlorophyll content was almost comparable to normal. Plants receiving CdCl2 treatment (250 microg g(-1) and less) became acclimatized after two weeks and started normal growth. But PbCl2 of 432 microg g(-1) and less could not affect the plant growth throughout, after a preliminary shock was erased. In case of CdCl2 treatment, a stunted growth with reduced leaf area, reduced biomass and sterility were recorded after six months, while plants show normal growth and flowering in case of PbCl2 treatment. Total alkaloid was also found to be decreased in the roots of CdCl2 treated plants. No change was observed in case of PbCl2. GA3 treatments to the CdCl2 treated plants show internode elongation and increase in leaf area with relatively elongated leaves and thinning of stem diameter AAS analyses of leaves of treated plants exhibited 5-10% accumulation of cadmium, but there was no accumulation of lead at all.

Isolation and identification of alpha-amylase producing Bacillus sp. from dhal industry waste.

A bacterial strain was isolated from dhal industry red gram waste and identified as Bacillus. A thermostable extracellular amylase was partially purified from the strain. Optimum temperature and pH for the enzyme were found to be 60 degrees C and 6.5, respectively. The maximum amylase production was achieved with maltose as carbon source. Among the nitrogen sources, peptone and yeast extract produced maximum amylase.

Chromium in tannery industry effluent and its effect on plant metabolism and growth.

Different dilution levels of tannery treated effluent and their corresponding concentration of chromium (Cr6+) were studied in a petridish culture experiment on seed germination and seedling growth in radish (Raphanus sativus L). The different concentrations of Cr6+ (2, 5 and 10 ppm) and treated tannery effluent (10, 25 and 50%) showed reduction in seedling growth and related enzymatic activities with increase in concentration of Cr6+ in treatments and effluent both. The low concentration of chromium (2 ppm) and effluent dilution (10%) showed significant growth reduction separately. At this concentration of chromium and effluent dilution chlorophyll content, amylase, catalase and protein contents remained unchanged while with increase in Cr6+ concentration (>2ppm) and effluent dilution (> 10%) in treatments showed growth inhibitory effects.

Bioregulation of carbohydrate metabolism in relation to source-sink operation during grain-filling phase of growth in wheat.

Mobilization of free sugars from vegetative tissues to grain and their transformation to starch in relation to activities of some relevant enzymes during growth and development were investigated in wheat (Triticum aestivum L.). Vegetative tissues, viz. flag-leaf, flag-leaf sheath, nodes and internodes contained high concentration of free sugars from 70 DAS to 18 DPA and that was in the order of accumulation--flag-leaf sheath> flag-leaf and internodes > nodes. In these tissues, major portion of 14C appeared in endogenous sucrose, irrespective of the nature of (U-14C]-sugars supplied. In photosynthetic structures above flag-leaf node, namely peduncle, rachis and bracts, the free sugar make-up was maximum at anthesis (90 DAS). Activity of soluble acid invertase (EC 3.2.1.26) was high in these tissues during early stages of grain growth but reverse was true for soluble neutral invertase (EC 3.2.1.27) activity. In apical and basal portions of grain, free sugars were more or less similarly distributed in concentration. Linear and rapid accumulation of starch in endosperm paralleled with a decline in accumulation of this polymer in pericarp-aleurone. In the latter tissue, the activities of starch hydrolyzing enzymes, i.e alpha- and beta-amylases (3.2.1.1 and 3.2.1.2) were high during initial stages of grain growth. During active grain-filling, alkaline inorganic pyrophosphatase (EC 3.6.1.1) seemed to play a vital role during starch accumulation in endosperm, whereas the involvement of 3-PGA phosphatase (EC 3.1.3.38) was almost confined to pericarp-aleurone. Impairement of ear head photosynthesis by shading depressed starch synthesis (approximately 50%) indicating, thereby, the significant role of current photosynthates during grain-filling. The results suggested that grain growth in wheat was influenced by an efficient operation of source as well as regulatory factors, including enzymes, constituting intrinsic potential of grain sink.

Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution.

For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, proteinase K was analyzed in detail using X-ray diffraction method. The overall structure of the enzyme was found to be similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrangement as some of the water molecules were either displaced or completely absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. Interlinked through water molecules, the sites occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in Ile 107 by rotating its side chain about C alpha-C beta bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change had earlier been observed in proteinase K when it was complexed to a substrate analogue, lactoferrin fragment.

Synergistic effects in enzymic reactions.

It has been shown that when two enzymes showing similar actions act in close proximity of each other they influence each other synergistically. The phenomenon of synergism is, however, not observed if the two enzymes are of dissimilar action type. The condition of closest proximity has been simulated by conducting the enzymic reactions inside the reversed micelles. In the present study we have experimented with alpha-amylase and invertase both hydrolysing enzymes and also with peroxidase and invertase which do not show similar actions.

Atividade dos inibidores de `alfaï-amilase do triticale parcialmente purificados sobre amilases de Acanthoscelides obtectus e Tenebrio molitor / Activity of `alphaï-amilase inhibitors from triticale partially purified on the Acanthoscelides obtectus and Tenebrio molitor amylases

Inibidores de `alfaï-amilase do triticale (Triticum x secale) foram purificados parcialmente através de fracionamento com sulfato de amônio, filtração em gel sobre Sephadex G-100 e cromatografia em DEAE-Celulose, e foram denominados como 23`B IND. 2ï, 54`B IND. 4ï e 23`C IND. 1ï, conforme variedade de triticale utilizada e ordem de eluição nas colunas cromatográficas. Os inibidores não apresentaram carboidratos na estrutura e os pesos moleculares aparentes foram estimados em 31,3, 30,2 e 27,6 kDaltons, respectivamente. Sob condições dissociantes observou-se uma banda com peso molecular estimado em 14 kDaltons, indicando duas subunidades polipeptídicas semelhantes. Os valores de pH isoelétrico foram 5,62 (23`B IND. 2ï) e 5,15 (54`B IND. 4ï)...

Enzymatic determination of soluble and insoluble dietary fiber in rice and wheat bran

The information about dietary fiber presents controversies in many research areas such as in nomenclature, related illnesses, recommended quantities and terminology, mainly because of lack of analytical data. Different needs and interests for the dietary fiber composition of foods and forages have led to a proliferation of methods for its analysis. This research, a further adaptation of the enzymatic method of Asp et al. (1983) for its appplication is proposed for rice and wheat bran, byproducts of agroindustries in the southern region of Rio Grande do Sul (Brazil). The inclusion of Amyloglucosidase in the proposed methodology contributed to the decrease in the content of residual starch at the end of the experiment, like Prosky et al (1992). To increase the efficiency of the enzyme system inthis type of samples, other changes were made with respect to incubation time and proteolytic enzyme concentration. In the final adaptation, a decrease of 51.33 per cent of the starch content was observed in rice bran (RB) and of 52.93 per cent in wheat bran (WB). This decrease was also verified in the model system (MS) (52.08 per cent), which demonstrates the adequacy of the proposed adaptation. With respect to the residual protein, it was verified that the measures adopted provoked a reduction of 42.15 per cent (RB), 52.19 per cent (WB) and 42.11 per cent (MS) as compared to the original method. Then the proposed conditions has been shown to be efficient in decreasing the level of interference (indigestible starch and protein) in the quantification of dietary fiber in rice and wheat bran.

Effect of pH and temperature on Bacillus subtilis ATCC 601 alfa-amylase production. Some properties of the crude enzyme

Neste trabalho foram estudadas a influência do pH inicial do meio de cultura e da temperatura de crescimento sobre a produçäo de alfa-amilase por Bacillus subtilis ATCC 601, bem como algumas propriedades da enzima bruta. Houve maior produçäo de enzima quando a fermentaçäo foi realizada a 37oC. A atividade enzimática específica global, Emax/Xmax' mostrou que a maior concentraçäo enzimática no caldo de cultura fermentado a 37oC näo foi devida a um maior crescimento celular e sim a uma maior síntese de enzima por unidade de massa celular. O estudo do efeito do pH inicial do meio de cultura sobre a produçäo da enzima mostrou que ela ocorreu preferencialmente a pH 7,0, também devido a um estímulo da sítese por unidade de massa celular. A temperatura ótima da enzima está ao redor de 50oC e o seu pH ótimo está entre 5,4 e 6,4. A alfa-amilase de Bacillus subtilis ATCC 601 mostrou-se pouco estável a 60oC foi completamente inativada quando mantida durante 10 minutos a 80oC. Ions Ca +2, nas concentraçöes de 10mM e 20mM, estabilizaram sensivelmente a enzima, enquanto que a adiçäo de EDTA 10mM a desestabilizou de tal modo que a enzima apresentou a 50oC um comportamento semelhante ao observado a 80oC. Näo houve formaçäo de glicose quando o amido foi hidrolisado por esta alfa-amilase. Os açúcares formados variaram de maltose à maltoheptaose