Α-amylase Gene Research Articles

Genome-wide characterization and expression analysis of α-amylase and β-amylase genes underlying drought tolerance in cassava

BackgroundStarch hydrolysates are energy sources for plant growth and development, regulate osmotic pressure and transmit signals in response to both biological and abiotic stresses. The α-amylase (AMY) and the β-amylase (BAM) are important enzymes that catalyze the hydrolysis of plant starch. Cassava (Manihot esculenta Crantz) is treated as one of the most drought-tolerant crops. However, the mechanisms of how AMY and BAM respond to drought in cassava are still unknown.ResultsSix MeAMY genes and ten MeBAM genes were identified and characterized in the cassava genome. Both MeAMY and MeBAM gene families contain four genes with alternative splicing. Tandem and fragment replications play important roles in the amplification of MeAMY and MeBAM genes. Both MeBAM5 and MeBAM10 have a BZR1/BES1 domain at the N-terminus, which may have transcription factor functions. The promoter regions of MeAMY and MeBAM genes contain a large number of cis-acting elements related to abiotic stress. MeAMY1, MeAMY2, MeAMY5, and MeBAM3 are proven as critical genes in response to drought stress according to their expression patterns under drought. The starch content, soluble sugar content, and amylase activity were significantly altered in cassava under different levels of drought stress.ConclusionsThese results provide fundamental knowledge for not only further exploring the starch metabolism functions of cassava under drought stress but also offering new perspectives for understanding the mechanism of how cassava survives and develops under drought.

A rice seed-specific glycine-rich protein OsDOR1 interacts with GID1 to repress GA signaling and regulates seed dormancy

Seed dormancy is an important agronomic trait under the control of complex genetic and environmental interactions, which have not been yet comprehensively understood. From the field screening of rice mutant library generated by a Ds transposable element, we identified a pre-harvest sprouting (PHS) mutant dor1. This mutant has a single insertion of Ds element at the second exon of OsDOR1 (LOC_Os03g20770), which encodes a novel seed-specific glycine-rich protein. This gene successfully complemented the PHS phenotype of dor1 mutant and its ectopic expression enhanced seed dormancy. Here, we demonstrated that OsDOR1 protein binds to the GA receptor protein, OsGID1 in rice protoplasts, and interrupts with the formation OsGID1-OsSLR1 complex in yeast cells. Co-expression of OsDOR1 with OsGID1 in rice protoplasts attenuated the GA-dependent degradation of OsSLR1, the key repressor of GA signaling. We showed the endogenous OsSLR1 protein level in the dor1 mutant seeds is significantly lower than that of wild type. The dor1 mutant featured a hypersensitive GA-response of α-amylase gene expression during seed germination. Based on these findings, we suggest that OsDOR1 is a novel negative player of GA signaling operated in the maintenance of seed dormancy. Our findings provide a novel source of PHS resistance.

Promoter-proximal introns impact recombinant amylase expression in Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) of starch requires recombinant Saccharomyces cerevisiae strains that produce raw starch-degrading enzymes and ferment the resultant sugars to ethanol in a single step. In this study, the native S. cerevisiae COX4 and RPS25A promoter-proximal introns were evaluated for enhanced expression of amylase genes (ateA, temA or temG_Opt) under the control of an S. cerevisiae promoter (ENO1P, TEF1P, TDH3P, or HXT7P). The results showed that different promoters and promoter-intron combinations differentially affected recombinant amylase production: ENO1P-COX4i and TDH3P-RPS25Ai were the best promoters for AteA, followed closely by HXT7P. The latter was also the best promoter for TemA and TemG production, followed closely by TDH3P-RPS25Ai for both these enzymes. Introducing promoter-proximal introns increased amylase activity up to 62% in Y294[ENO-COX-AteA] and Y294[TDH3-RPS-TemA], a significant improvement relative to the intron-less promoters. Strains co-expressing both an α-amylase and glucoamylase genes yielded up to 56g/L ethanol from 20%w/v raw starch, with a higher carbon conversion observed with strains co-expressing TDH3P-RPS25Ai-temG_Opt than HXT7P-temG_Opt. The study showed that promoter-proximal introns can enhance amylase activity in S. cerevisiae and suggest that these alternative cassettes may also be considered for expression in more efficient ethanol-producing industrial yeast strains for raw starch CBP.

Análise por cromatografia gasosa-espectrometria de variações no óleo essencial de folhas de seis espécies de eucalipto e alelopatia de componentes principais

ABSTRACT: The Eucalyptus plant releases allelopathic chemicals into the environment mostly through the essential oils volatilized from the leaves. This study discussed the composition of the leaf oils of few seven-year-old varieties like Eucalyptus pellita (E. pel), Eucalyptus camaldulensis (E. cama), Eucalyptus grandis (E. gra), Eucalyptus dunni (E. dun), Eucalyptus saligna (E. sal), and E. grandis × E. urophylla (E. gra×E. uro) and three-year-old E. grandis × E. urophylla (E. gra × E. uro (three)). It determined the allelopathic mechanism and the types of chemical components playing the leading role. Essential oil was obtained by hydrodistillation and analyzed by the Gas Chromatography-Mass Spectrometry (GC-MS) method. In order to determine the effect of allelopathy, seed germination experiments were carried out at different concentrations (10-100 mL/L) of the E. Gra × E. uro leaf oil (EO) and the major components. The wheat seeds germinated by adding 1,8-cineole were used to determine the activity of α-amylase. Moreover, the mRNA expression of α-amylase in seeds was studied. The major chemical class in the essential oil was oxygenated monoterpene; 1,8-cineole (20.2-67.5%) displayed the highest content. Other substances that were high in content and ubiquitous included α-pinene (0.3-21.8%), α-terpineol (0.44-19.24%), and borneol (0.81-3.05%). The four chemical constituents and EO influenced the germination and growth of the three plants. Among them, 1,8-cineole exhibited the strongest inhibitory effect. The α-amylase activity of the 1,8-cineole-treated wheat seeds had decreased significantly. Molecular evidence suggested that 1,8-cineole decreased the α-amylase gene (AMY) expression.

Endotoxin-free gram-negative bacterium as a system for production and secretion of recombinant proteins.

Gram-negative bacteria are common and efficient protein expression systems, yet their outer membrane endotoxins can elicit undesirable toxic effects, limiting their applicability for parenteral therapeutic applications, e.g., production of vaccine components. In the bacterial genus Sphingomonas from the Alphaproteobacteria class, lipopolysaccharide (LPS) endotoxins are replaced with non-toxic glycosphingolipids (GSL), rendering it an attractive alternative for therapeutic protein production. To explore the use of sphingomonas as a safe expression system for production of proteins for therapeutic applications, in this study, Sphingobium japonicum (SJ) injected live into embryonated hen eggs proved safe and nontoxic. Multimeric viral polypeptides derived from Newcastle disease virus (NDV) designed for expression in SJ, yielded soluble proteins which were specifically recognized by antibodies raised against the whole virus. In addition, native signal peptide (SP) motifs coupled to secreted proteins in SJ identified using whole-genome computerized analysis, induced secretion of αAmylase (αAmy) and mCherry gene products. Relative to the same genes expressed without an SP, SP 104 increased the secretion of αAmy (3.7-fold) and mCherry (16.3-fold) proteins and yielded accumulation of up to 80µg/L of the later in the culture medium. Taken together, the presented findings demonstrate the potential of this unique LPS-free gram-negative bacterial family to serve as an important tool for protein expression for both research and biotechnological purposes, including for the development of novel vaccinesand as a live bacteria delivery system for protein vaccines. KEY POINTS: • Novel molecular tools for protein expression in non-model bacteria. • Bacteria with GSL instead of LPS as a potential vector for protein delivery.

Extrinsic role of gibberellin mitigating salinity effect in different rice genotypes.

The overall effects of gibberellic acid (GA3) with NaCl on different rice genotypes are inadequately understood. The present study determines the effect of different GA3 (50 and 100 µM) concentrations on the morphophysiological, molecular and biochemical effects of 120 mM NaCl salt stress in rice seedlings. Salt stress reduced germination percentages and seedling growth and decreased bioactive GA content. It also downregulated the relative expression of α-amylase-related genes - OsAmy1A, OsAmy1C, and OsAmy3C in the salt-sensitive IR28 cultivar. Salt stress differentially regulated the expression of GA biosynthetic genes. Salt stress increased antioxidant activity in all rice genotypes tested, except in IR28. GA3 mitigates the effect of salt stress, rescuing seed germination and growth attributes. GA3 significantly increased bioactive GA content in Nagdong and pokkali (50 µM) and Cheongcheong and IR28 (100 µM) cultivars. The α-amylase genes were also significantly upregulated by GA3. Similarly, GA3 upregulated OsGA2ox1 and OsGA2ox9 expression in the Cheongcheong and salt-sensitive IR28 cultivars. The present study demonstrated that salt stress inactivates bioactive GA - inhibiting germination and seedlings growth - and decreases bioactive GA content and GSH activity in IR28 and Pokkali cultivars. Further, GA3 significantly reversed the effects of 120 mM NaCl salt stress in different rice genotypes. The current study suggested that the known coastal area salinity concentration can be significantly recovered with the application of exogenous GA3. Thus, it could be possible to grow eco-friendly rice close to the coastal zone in order to reduce the damage caused by salinity.

Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis

Terminators serve as the regulatory role in gene transcription termination; however, few researches about terminator optimization have been conducted, which leads to the lack of available and universal terminator for gene expression regulation in Bacillus. To solve this problem and expand synthetic biology toolbox of Bacillus licheniformis, the terminator T1 of endogenous α-amylase gene (amyL) was characterized in this research, with a termination efficiency of 87.81%. Then, we explored and optimized the termination strength of terminator T1 from four aspects: the distance between stop codon and terminator, GC content at the bottom of stem structure, loop size, and U-tract length, and the best terminator T24 was attained by combination optimization strategy, which termination efficiency was increased to 97.97%, better than the commonly used terminator T7 (T7P) from Escherichia coli. Finally, terminator T24 was applied to protein expression, which, respectively, led to 33.00%, 25.93%, and 11.78% increases of green fluorescence intensity, red fluorescence intensity, and keratinase activity, indicating its universality in protein expression. Taken together, this research not only expands a plug-and-play synthetic biology toolbox in B. licheniformis but also provides a reference for the artificial design of versatile intrinsic terminator.Graphical

Herbicidal activity and biochemical characteristics of the botanical drupacine against Amaranthus retroflexus L.

Botanical herbicide has been a hot topic in the research and development of novel pesticides. The herbicidal activity and biochemical characteristics of the botanical compound drupacine were studied by evaluating its effects on seed germination, seedling growth, morphological and physiological characteristics of Amaranthus retroflexus. Drupacine inhibited seed germination and seedling growth, and had a median inhibition concentration (IC50) value of 38.99 mg L−1 against A. retroflexus root. The α-amylase activity and soluble sugar content in treated plants were significantly lower than that of the control. The expression of α-amylase gene was dosage-dependently inhibited compared to the untreated control. This suggested that inhibition of α-amylase activity was a mode of action on seed germination. The root hairs were significantly decreased and part of the root cap fell off after treatment with drupacine. The ultrastructure observation showed that cell damage of root tips increased with the treatment time. Drupacine also increased the relative conductivity and malondialdehyde (MDA) content. Peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities were significantly enhanced in the treatment compared to the control. These findings indicated that the physiological and biochemical reaction changes leading to morphological and membrane injuries were the main effects of drupacine on the inhibition of seedling growth. Drupacine can be developed as a botanical herbicide.

Molecular mechanism underlying cadmium tolerance differentiation in Lentinula edodes as revealed by mRNA and milRNA analyses

Lentinula edodes is an important candidate for heavy metal mycoremediation. This study explored the molecular mechanism of cadmium (Cd) tolerance differentiation of L. edodes through comparative mRNA and milRNA analyses of two strains, YS45 (Cd sensitive) and YS119 (Cd tolerant). Results showed the emergence of more possible protein aggregates in YS45 than in YS119 under Cd stress. The number of differentially expressed genes (DEGs) and milRNAs (DEMs) was identified as 2036 and 370 as well as five and three in YS45 and YS119, respectively. Comparison of DEGs and DEMs between the two strains revealed that genes and milRNAs involved in cell wall remodeling, transport, heavy metal chelation, lipid and carbohydrate metabolism, transcription regulation, redox homeostasis, proteolysis, signaling, DNA repair, cell cycle, may play important roles in Cd stress response, and their different expression patterns, especially those associated with proteolysis and carbohydrate metabolism, might lead to Cd tolerance differentiation between the two strains. The function of the α-amylase gene LeAmy in enhancing Cd tolerance in L. edodes was further verified by overexpression. This study has shed light on the molecular mechanism of Cd tolerance differentiation in L. edodes, and laid a foundation for breeding elite strains for heavy metal mycoremediation.

Revealing the difference of α-amylase and CYP6AE76 gene between polyphagous Conogethes punctiferalis and oligophagous C. pinicolalis by multiple-omics and molecular biological technique

BackgroundConogethes pinicolalis has been thought as a Pinaceae-feeding variant of the yellow peach moth, Conogethes punctiferalis. The divergence of C. pinicolalis from the fruit-feeding moth C. punctiferalis has been reported in terms of morphology, ecology, and genetics, however there is a lack of detailed molecular data. Therefore, in this study, we investigated the divergence of C. pinicolalis from C. punctiferalis from the aspects of transcriptomics, proteomics, metabolomics and bioinformatics.ResultsThe expression of 74,611 mRNA in transcriptome, 142 proteins in proteome and 218 metabolites in metabolome presented significantly differences between the two species, while the KEGG results showed the data were mainly closely related to metabolism and redox. Moreover, based on integrating system-omics data, we found that the α-amylase and CYP6AE76 genes were mutated between the two species. Mutations in the α-amylase and CYP6AE76 genes may influence the efficiency of enzyme preference for a certain substrate, resulting in differences in metabolic or detoxifying ability in both species. The qPCR and enzyme activity test also confirmed the relevant gene expression.ConclusionsThese findings of two related species and integrated networks provide beneficial information for further exploring the divergence in specific genes, metabolism, and redox mechanism. Most importantly, it will give novel insight on species adaptation to various diets, such as from monophagous to polyphagous.

Sex Modulates Cardiovascular Effects of Icodextrin-Based Peritoneal Dialysis Solutions.

Background/Aims: Some previous observations have noted that after six months of peritoneal dialysis (PD) treatment with icodextrin solutions, blood pressure (BP) and NT-proBNP tend to return to baseline values. This may be due to accumulation of icodextrin products that exert a colloid osmotic effect, which drives water into the bloodstream, causing the rise in blood pressure. Since icodextrin is metabolized by α-Amylase and its gene copies are lower in females than in males, we hypothesized icodextrin metabolites reach higher concentrations in females and that cardiovascular effects of icodextrin are influenced by sex. Methods: Secondary analysis of a RCT comparing factors influencing fluid balance control in diabetic PD patients with high or high average peritoneal transport receiving icodextrin (n = 30) or glucose (n = 29) PD solutions. Serum icodextrin metabolites, osmolality, body composition and Inferior Vena Cava (IVC) diameter were measured at baseline, and at 6 and 12 months of follow-up. Results: After six months of treatment, icodextrin metabolites showed higher levels in females than in males, particularly G5-7 and >G7, serum osmolality was lower in females. In spite of reduction in total and extracellular body water, ultrafiltration (UF) was lower and IVC diameter and BP increased in females, suggesting increment of blood volume. Conclusion: Females undergoing PD present with higher levels of icodextrin metabolites in serum that may exert an increased colloid-osmotic pressure followed by less UF volumes and increment in blood volume and blood pressure. Whether this could be due to the lesser number of α-Amylase gene copies described in diabetic females deserves further investigation.

Deletion of α-amylase genes via CRISPR/Cas9 decreases the side effects of hydrolysis towards nonreducing maltoheptaose preparation

Maltodextrin is a significant ingredient extensively used in food industries, but its application is limited due to uneven polymerization and high reducibility. Hence, preparation of nonreducing maltodextrin with narrow distribution of DP is essential. A dual-enzyme cascade enzymatic reaction for preparing nonreducing maltoheptaose (N-G7) is feasible via recombinant cyclomaltodextrinase (EC 3.2.1.54, CDase) and maltooligosyltrehalose synthase (EC 5.4.99.15, MTSase) heterologously overexpressed in Escherichia coli BL21(DE3). However, during this process, N-G7 further was hydrolyzed into small molecule maltooligosaccharides by the amylases homologously expressed in the host. In this study, two α-amylase genes (ycjM and malS) were deleted to avoid host hydrolysis in N-G7 via CRISPR/Cas9 system. Gene deletion had a significant decrease on the yield of maltooligosaccharides of 5 and 6 glucose units. The defective strains ΔycjM and ΔycjM-ΔmalS increased the yield of N-G7 by 21.94% and 25.47%, correspondingly. Marginal impact was made in cell growth and protein secretion of the host. YcjM and MalS were overexpressed in ΔycjM-ΔmalS and the hydrolytic activity of YcjM was twice that of MalS. They constituted a complex multiple hydrolysis system for N-G7 with the Mal series enzymes involved in a maltose system in the host.

Gibberellins in developing wheat grains and their relationship to late maturity α-amylase (LMA)

Main conclusionα-Amylase synthesis by wheat aleurone during grain development (late maturity α-amylase) appears to be independent of gibberellin unlike α-amylase synthesis by aleurone during germination or following treatment with exogenous GA.Late-maturity α-amylase (LMA) in wheat (Triticum aestivum L.) involves the synthesis of α-amylase by the aleurone tissue during grain development. Previous research identified a putative ent-copalyl diphosphate synthase gene, coding for an enzyme that controls the first step in gibberellin biosynthesis, that underlies the major genetic locus involved in variation in LMA phenotype. The reported results for gene transcript analysis, preliminary gibberellin analysis and the effects of DELLA mutants on LMA phenotype appeared to be consistent with involvement of gibberellin but did not provide definitive proof of a causal link. Conversely, several observations do not appear to be consistent with this hypothesis. In this current study, LMA phenotype, gibberellin profiles and ABA content were recorded for experiments involving susceptible and resistant genotypes, gibberellin biosynthesis inhibitors, genetic lines containing different LMA quantitative trait loci and treatment of distal halves of developing grains with exogenous gibberellin. The results suggested that gibberellin may not be a prerequisite for LMA expression and further that the mechanism involved in triggering α-amylase synthesis did not correspond to the model proposed for germination and gibberellin challenged aleurone of ripe grain. The results provide new insight into LMA and highlight the need to investigate alternate pathways for the induction of α-amylase gene transcription, the function of novel 1-β-OH gibberellins and other functions of DELLA proteins in developing grains.

Effects of Ag/SiO2 Nanoparticles on Gene Expression of Digestive a-Amylase in Colorado Potato Beetle

Insecticidal properties of nanoparticles (NPs) have been considered widely in different studies. In this study, synthesis of Ag/SiO2 NPs is reported and the effect of the nanoparticles on the digestive a-amylase gene expression in the Colorado potato beetle (CPB), Lepidoptera decemlineata say is investigated. For this purpose, the inhibition of fourth instar larval digestive a-amylase with NPs was analyzed by different concentrations, and the highest inhibitory effect was recorded (72%) at 1000 ppm of Ag/SiO2 NPs. Mortality for the reared larvae from the first instar on potato leaves coated with 1000 ppm Ag/SiO2 NPs was 65%, while it is 8% for the control trial. To investigate the process, we conjugated the nanoparticles with extracted RNA, and the photoluminescent (PL) spectra of the obtained materials were measured and investigated. The results indicate that the FRET phenomenon occurs when the RNA reacts with nanoparticles. These results are according to the PL spectra of RNAs extracted from the larval fed with nanoparticles. The structure is simulated and calculated by the DFT method. The obtained results confirm chemical interaction between nanoparticles and nitrogenous bases on the RNA which can decrease gene expression (0.47 fold) compared to control and causes inhibition of larvae’s growth.

Fermentation optimization and amylase activity of endophytic Bacillus velezensis D1 isolated from corn seeds

To acquire quality amylase adopted in practical applications, endophytic bacteria were identified as Bacillus velezensis strain D1 which was isolated from corn seeds. The fermentation conditions and amylase properties of the strain were investigated. The strain D1 was identified via morphological, physiological and 16S rDNA phylogenetic analysis. The fermentation conditions of secreting amylase were optimized by single-factor and orthogonal experiments. The α-amylase gene was expressed in E. coli and purified by means of immobilized metal ion affinity chromatography (IMAC), upon which the enzyme activity of purified recombinant α-amylase was determined. The results outlined that (1) The strain D1 was identified as Bacillus velezensis. (2) The optimized fermentation conditions for maximum amylase yields included 44°C for 48h at pH 7.5. (3) The enzyme had an optimal reaction temperature of 60°C with the highest activity at 50°C and tolerance to 4-h incubation at 70°C. (4) The enzyme was strong acid resistant and tolerated at pH 5.0-6.0 while the optimal pH was 8.0. (5) Besides, the amylase activity was elevated by the presence of Ca2+ and Cu2+ . (6) The activity of purified recombinant amylase was 20.59U/ml under optimal conditions, nearly seven times that of crude amylase preparations. The amylase produced by Bacillus velezensis D1 is strongly tolerant towards acid and high temperatures. Amylases with thermophilic and acid-resistant characteristics are useful for a wide range of applications in food, brewing, textile, starch, paper and deterrent industries. The enzyme from Bacillus velezensis D1 can be effectively used in different areas of industries.

CLONING, PURIFICATION AND STUDY OF THE BIOCHEMICAL PROPERTIES OF Α-AMYLASE FROM BACILLUS LICHENIFORMIS T5 STRAIN

Amylases are rather important enzymes in modern biotechnology. Alpha-amylases hydrolyze starch to form glucose and maltose and are involved in the conversion of starch-containing raw materials. In comparison with α-amylases of eukaryotic organisms, bacterial α-amylases have a number of advantages, namely, the preservation of enzymatic activity at high temperatures, which is important in enzymatic starch hydrolysis technologies. In this work, the α-amylase gene from the Kazakh strain of Bacillus licheniformis T5 was cloned into the pET-28c(+) vector. Recombinant α-amylase was obtained by plasmid expression in Escherichia coli strain ArcticExpressRP(DE3). Studies have shown that the pH and temperature optimum for the recombinant alpha-amylase was 6.5 and 80°C, respectively. As has been established that α-amylase is a calcium-independent enzyme. The study of the temperature stability of α-amylase showed that the enzyme retains more than 70% of its activity when α-amylase is incubated at 80°C for 30 minutes. The obtained results show a high prospect of using α-amylase from Bacillus licheniformis T5 as a thermostable enzyme in the technologies of enzymatic hydrolysis of starch-containing raw materials.

Bacillus velezensis Identification and Recombinant Expression, Purification, and Characterization of Its Alpha-Amylase

Amylases account for about 30% of the global market of industrial enzymes, and the current amylases cannot fully meet industrial needs. This study aimed to identify a high α-amylase producing bacterium WangLB, to clone its α-amylase coding gene, and to characterize the α-amylase. Results showed that WangLB belonged to Bacillus velezensis whose α-amylase gene was 1980 bp coding 659 amino acids designated as BvAmylase. BvAmylase was a hydrophilic stable protein with a signal peptide and a theoretical pI of 5.49. The relative molecular weight of BvAmylase was 72.35 kDa, and was verified by SDS-PAGE. Its modeled structure displayed that it was a monomer composed of three domains. Its optimum temperature and pH were 70 °C and pH 6.0, respectively. It also showed high activity in a wide range of temperatures (40–75 °C) and a relatively narrow pH (5.0–7.0). It was a Ca2+-independent enzyme, whose α-amylase activity was increased by Co2+, Tween 20, and Triton X-100, and severely decreased by SDS. The Km and the Vmax of BvAmylase were 3.43 ± 0.53 and 434.19 ± 28.57 U/mg. In conclusion, the α-amylase producing bacterium WangLB was identified, and one of its α-amylases was characterized, which will be a candidate enzyme for industrial applications.

Toward a More Comprehensive View of α-Amylase across Decapods Crustaceans.

Simple SummaryDecapod crustaceans live in practically all marine, freshwater, and semi-terrestrial habitats on Earth, and exhibit a remarkable variation in their feeding behavior, from filter feeding, grazing, and scavenging to hunting. However, most knowledge about digestive biochemistry in crustaceans has come from studies on a few economically relevant species due to the importance of optimized formulated feeds for aquaculture success. Moreover, most data on α-amylases in decapods derived from studies in herbivore and omnivore species. There are few reviews addressing different aspects of the digestive physiology of decapods, including data on digestive enzymes, but no comprehensive review is available on α-amylases in this group and, in general, information on carnivorous species is often neglected. This review summarizes the information obtained on decapods’ α-amylases and uses recent data from a carnivorous lobster as a connecting thread to compare features of α-amylases from species with different feeding habits, drawing a more comprehensive view of the role of α-amylases across decapods crustaceans.Decapod crustaceans are a very diverse group and have evolved to suit a wide variety of diets. Alpha-amylases enzymes, responsible for starch and glycogen digestion, have been more thoroughly studied in herbivore and omnivore than in carnivorous species. We used information on the α-amylase of a carnivorous lobster as a connecting thread to provide a more comprehensive view of α-amylases across decapods crustaceans. Omnivorous crustaceans such as shrimps, crabs, and crayfish present relatively high amylase activity with respect to carnivorous crustaceans. Yet, contradictory results have been obtained and relatively high activity in some carnivores has been suggested to be a remnant trait from ancestor species. Here, we provided information sustaining that high enzyme sequence and overall architecture conservation do not allow high changes in activity, and that differences among species may be more related to number of genes and isoforms, as well as transcriptional and secretion regulation. However, recent evolutionary analyses revealed that positive selection might have also occurred among distant lineages with feeding habits as a selection force. Some biochemical features of decapod α-amylases can be related with habitat or gut conditions, while less clear patterns are observed for other enzyme properties. Likewise, while molt cycle variations in α-amylase activity are rather similar among species, clear relationships between activity and diet shifts through development cannot be always observed. Regarding the adaptation of α-amylase to diet, juveniles seem to exhibit more flexibility than larvae, and it has been described variation in α-amylase activity or number of isoforms due to the source of carbohydrate and its level in diets, especially in omnivore species. In the carnivorous lobster, however, no influence of the type of carbohydrate could be observed. Moreover, lobsters were not able to fine-regulate α-amylase gene expression in spite of large changes in carbohydrate content of diet, while retaining some capacity to adapt α-amylase activity to very low carbohydrate content in the diets. In this review, we raised arguments for the need of more studies on the α-amylases of less studied decapods groups, including carnivorous species which rely more on dietary protein and lipids, to broaden our view of α-amylase in decapods crustaceans.

Novel cold-adapted raw-starch digesting α-amylases from Eisenia fetida: Gene cloning, expression, and characterization

We identified the raw-starch-digesting α-amylase genes a earthworm Eisenia fetid α amylase I and II (Ef-Amy I and Ef-Amy II). Each gene consists of 1,530 base pairs (bp) that encode proteins of 510 amino acids, as indicated by the corresponding mRNA sequences. Ef-Amy I and II showed an 89% amino acid identity. The amino acid sequences of Ef-Amy I and II were similar to those of the α-amylases from porcine pancreas, human pancreas, Tenebrio molitor, Oryctolagus cuniculus, and Xenopus (Silurana) tropicalis. Each gene encoding mature Ef-Amy I and II was expressed in the GS115 strain of Pichia pastoris. The molecular masses of the recombinant Ef-Amy I and II were 57 kDa each, and catalytically important residues of α-amylases of the GH family 13 were conserved in both proteins. These amylases exhibited raw-starch-digesting activity at 4 °C. The substrate specificities of rEf-Amy I and II were dissimilar. rEf-Amy I and II were shown to be active even in 40% ethanol, 4 M NaCl, and 4 M KCl.

Description and functional analysis of the transcriptome from malting barley

The present study aimed to establish an early model of the malting barley transcriptome, which describes the expression of genes and their ontologies, identify the period during malting with the largest dynamic shift in gene expression for future investigation, and to determine the expression patterns of all starch degrading enzyme genes relevant to the malting and brewing industry. Large dynamic increases in gene expression occurred early in malting with differential expressed genes enriched for cell wall and starch hydrolases amongst many malting related categories. Twenty-five of forty starch degrading enzyme genes were differentially expressed in the malting barley transcriptome including eleven α-amylase genes, six β-amylase genes, three α-glucosidase genes, and all five starch debranching enzyme genes. Four new or novel α-amylase genes, one β-amylase gene (Bmy3), three α-glucosidase genes, and two isoamylase genes had appreciable expression that requires further exploration into their potential relevance to the malting and brewing industry.