Enzyme-coding genes as molecular clocks: The molecular evolution of animal alpha-amylases

Summary We constructed a cDNA library for the beetle,Tribolium castaneum. This library was screened using a cloned amylase gene fromDrosophila melanogaster as a molecular probe. Beetle amylase cDNA clones were isolated from this bank, and the nucleotide sequence was obtained for a cDNA clone with a coding capacity for 228 amino acids. Both the nucleotide sequence and predicted amino acid sequence were compared to our recent results forD. melanogaster alpha-amylases, along with published sequences for other alpha-amylases. The results show that animal alpha-amylases are highly conserved over their entire length. A borader comparison, which includes plant and microbial alpha-amylase sequences, indicates that parts of the gene are conserved between prokaryotes, plants, and animals. We discuss the potential importance of this and other enzyme-coding genes for the construction of molecular phylogenies and for the study of the general question of molecular clocks in evolution.     

A simple and reliable turbidimetric and kinetic assay for alpha-amylase that is readily applied to culture supernatants and cell extracts

Summary A simple, reliable and sensitive assay for alpha-amylase activity is reported, together with its theoretical derivation, that overcomes many of the problems encountered with other assays, especially when attempting to assay alpha-amylase activity in crude cell extracts or culture supernatants. The method relies on the reduction in turbidity that occurs upon digestion of a starch suspension with alpha-amylase. The initial rate of decrease in turbidity is shown to be proportional to a wide range of enzyme concentrations, permitting a rapid spectrophotometric and kinetic determination of alpha-amylase activity.     

Engineering of Hydrolysis Reaction Specificity in the Transglycosylase Cyclodextrin Glycosyltransferase

Abstract

Cyclodextrin glycosyltransferase (CGTase) is a member of the α-amylase family, a large group of enzymes that act on α-glycosidic bonds in starch and related compounds. Over twenty different reaction and product specificities have been found in this family. Although three-dimensional structure elucidation and the biochemical characterization of site-directed mutants have yielded a detailed insight into the mechanism of bond cleavage, the variation in reaction and product specificity is far from understood. This article gives an overview of recent developments in the undersanding and engineering of transglycosylation and hydrolysis specificity in CGTase, which is one of the best-studied α-amylase family enzymes.     

Sucrose and starch catabolism in the anther of <Emphasis Type="Italic">Lilium</Emphasis> during its development: a comparative study among the anther wall,locular fluid and microspore/pollen fractions

In order to better understand the various pathways of sucrose and starch catabolism in the anther of lily (Lilium hybrida var. “Enchantment”), invertase (EC 3.2.1.26) and amylase (EC 3.2.1.1, EC 3.2.1.2) activities were measured separately in different fractions (anther wall, locular fluid and microspore/pollen) and correlated with the sugar content during anther development. Our findings showed significant differences among the fractions analyzed, suggesting that the regulation of sucrose and starch catabolism could follow distinct pathways in each fraction. Glucose and fructose amounts progressively decreased from anther wall to fluid and from fluid to microspore/pollen. Thus, the developing pollen could act as a sink for the carbohydrates that reach the anther. In this sense, cell wall-bound invertases seem to play a major role in soluble sugar partitioning in the different fractions of the anther. Sucrose concentration was found to be substantially higher in the locular fluid than in the other fractions, indicating a probable site for storage. On the other hand, the anther wall tissues could have a buffering function, storing nutrient surplus in starch grains and thus regulating the availability of soluble sugars in the whole anther. All these results proved the advantages of the experimental model proposed here, as well as its usefulness to investigate sugar metabolism in Lilium anthers.     

Purification and properties of a hyperthermoactive α-amylase from the archaeobacterium Pyrococcus woesei

The cultivation of the hyperthermophilic archaeobacterium Pyrococcus woesei on starch under continuous gassing (80% H₂:20% CO₂) caused the formation of 250 U/l of an extremely thermoactive and thermostable -amylase. In a complex medium without elemental sulphur under 80% N₂ and 20% CO₂ atmosphere enzyme production could be elevated up to 1000 U/l. Pyrococcus woesei grew preferentially on poly-and oligosaccharides. The amylolytic enzyme formation was constitutive. Enzyme production was also observed in continuous culture at dilution rates from 0.1 to 0.4 h^-1. A 20-fold enrichment of -amylase was achieved after adsorption of the enzyme onto starch and its desorption by preparative gel electrophoresis. The -amylase consisted of a single subunit with a molecular mass of 70 000 and was catalytically active at a temperature range between 40°C and 130°C. Enzymatic activity was detected even after autoclaving at a pressure of 2 bars at 120°C for 5 h. The purified enzyme hydrolyzed exclusively -1,4-glycosidic linkages present in glucose polymers of various sizes. Unlike many -amylases from anaerobes the enzyme from P. woesei was unable to attack short chain oligosaccharides with a chain length between 2 and 6 glucose units.     

Physico-chemical Approach of the Amylolytic Action Pattern of a Thermostable Amylopullulanase

This paper describes the amylolytic action pattern of Thermococcus hydrothermalis recombinant amylopullulanase (Th-ApuΔ2) [E.C 3.2.1.41]. A comparison was made between amylose hydrolysis catalyzed by this enzyme and by two other amylolytic enzymes: α-amylase [E.C 3.2.1.1] (from Aspergillus oryzae) and glucoamylase [E.C. 3.2.1.3] (from Aspergillus niger), respectively taken as models for “endo” and “exo” catalytic patterns. Different independent physico-chemical methods were used to characterize the hydrolysis products obtained with the studied enzymes. Viscosity results were correlated to reducing sugars analysis to show a similarity between glucoamylase [E.C. 3.2.1.3] and Th-ApuΔ2 [E.C 3.2.1.41] behavior. On the other hand, whereas α-amylase [E.C 3.2.1.1] action rapidly decreased the viscosity of medium, glucoamylase and Th-ApuΔ2 hydrolysates have only shown a negligible reduction in viscosity. Glass transition temperatures of glucoamylase and Th-ApuΔ2 hydrolysates were found comparable (225–226°C) and significantly different from that of α-amylase (197°C). Thin-layer chromatographic analysis of hydrolysates mainly revealed the presence of glucose in the case of glucoamylase and Th-ApuΔ2 activities and in addition to glucose the Th-ApuΔ2 chromatograms have shown oligosaccharides with polymerization degree ranging from 2 to 7. These results incite us to conclude that Th-ApuΔ2 has a dual “endo” and “exo” catalytic action pattern. Analysis of the Fourier transform infrared (FTIR) results shows a comparable general aspect for all spectra. The presence of more numerous differentiated and intense peaks in the spectrum of Th-ApuΔ2 hydrolysate reveals the presence of short-chain oligosaccharides. These results confirm thin-layer chromatography results and support a dual action pattern.     

The stress-response-dampening effects of placebo

This experiment used both biological and self-report measures to examine how alcohol modifies stress responses, and to test whether the interaction between these two factors alters risk-taking in healthy young adults. Participants were divided into stress or no-stress conditions and then further divided into one of three beverage groups. The alcohol group consumed a binge-drinking level of alcohol; the placebo group consumed soda, but believed they were consuming alcohol; the sober group was aware that they were not consuming alcohol. Following beverage consumption, the stress group was subjected to the Trier Social Stress Test (TSST) while the no-stress group completed crossword puzzles; all participants subsequently completed a computerized risk-taking task. Exposure to the TSST significantly increased salivary levels of the hormone cortisol and the enzyme alpha-amylase, as well as subjective self-ratings of anxiety and tension. In the stress condition, both placebo and intoxicated groups reported less tension and anxiety, and exhibited a smaller increase in cortisol, following the TSST than did the sober group. Thus, the expectation of receiving alcohol altered subjective and physiological responses to the stressor. Neither alcohol nor stress increased risk taking, however the sober group demonstrated lower risk-taking on the computer task on the second session. These findings clearly demonstrate that the expectation of alcohol (placebo) alters subsequent physiological responses to stress.     

节肢动物α-淀粉酶系统进化树构建及分析

目的:构建节肢动物α-淀粉酶的系统进化树,探讨其进化关系,找出进化树中聚类在一起的α-淀粉酶的特异性序列。方法:在美国国立生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库中选取了56个节肢动物的α-淀粉酶氨基酸序列,利用CLUSTALX2.0进行序列比对、MEGA6.0建立进化树,通过BOXSHADE找到聚类的α-淀粉酶特异性序列。结果:56个α-淀粉酶聚类成A、B、C、D四大簇,A簇特异性序列为"VD NHD NQ",B簇特异性序列为"ID NHD NX",C簇特异性序列为"ID NHD NQ",D簇特异性序列为"XGN NHD X"。A、B、C、D四簇都含有保守的NHD(天冬酰胺-组氨酸-天冬氨酸)序列,但序列两端氨基酸种类不同。结论:56个节肢动物α-淀粉酶分为4簇,每簇都有其特异性序列,但都含有保守序列NHD。     

Design,and facile synthesis of 1,3 diaryl-3-(arylamino)propan-1-one derivatives as the potential alpha-amylase inhibitors and antioxidants

Diabetes is the most prevalent metabolic disorder causing a high rate of mortality and morbidity. Recently alpha-amylase is reported to be good drug design target for the treatment of diabetes mellitus. We have designed 116 molecules based on aza-Michael adduct of trans-chalcone as 1,3 diaryl-3-(arylamino)propan-1-ones which were studied by molecular docking and among them best six derivatives were synthesized easily via aza-Michael addition on trans-chalcone using KOH as a catalyst and evaluated for alpha-amylase inhibition along with antioxidant activity. It was observed that all compounds have alpha-amylase inhibitory activity but at different extents. The molecule 3e is the most potent alpha-amylase inhibitor of this series. 3a is the second most potent compound, whereas only one molecule 3d has shown antioxidant activity.