Toward a More Comprehensive View of α-Amylase across Decapods Crustaceans: New Insights from Carnivorous

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 studies 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 respect to carnivorous. 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. Also, 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 broad our view of α-amylase in decapods crustaceans.

Biochemical Characterization of the Amylase Activity from the New Haloarchaeal Strain Haloarcula sp. HS Isolated in the Odiel Marshlands

Alpha-amylases are a large family of α,1-4-endo-glycosyl hydrolases distributed in all kingdoms of life. The need for poly-extremotolerant amylases encouraged their search in extreme environments, where archaea become ideal candidates to provide new enzymes that are able to work in the harsh conditions demanded in many industrial applications. In this study, a collection of haloarchaea isolated from Odiel saltern ponds in the southwest of Spain was screened for their amylase activity. The strain that exhibited the highest activity was selected and identified as Haloarcula sp. HS. We demonstrated the existence in both, cellular and extracellular extracts of the new strain, of functional α-amylase activities, which showed to be moderately thermotolerant (optimum around 60 °C), extremely halotolerant (optimum over 25% NaCl), and calcium-dependent. The tryptic digestion followed by HPLC-MS/MS analysis of the partially purified cellular and extracellular extracts allowed to identify the sequence of three alpha-amylases, which despite sharing a low sequence identity, exhibited high three-dimensional structure homology, conserving the typical domains and most of the key consensus residues of α-amylases. Moreover, we proved the potential of the extracellular α-amylase from Haloarcula sp. HS to treat bakery wastes under high salinity conditions.

Recent Patents on the Industrial Application of Alpha-amylases

Background: Alpha-amylases are enzymes capable of degrading polysaccharides, such as starch and glycogen. Found in various organisms, such as fungi and bacteria, these enzymes have great biotechnological potential due to their insertion in several industrial sectors ranging from food to biofuels. Objective: The aim of this study was to analyze patents deposited in intellectual property databases on alpha-amylases in the fields of food, beverages, detergents, animal feeds, biofuels, pharmaceuticals and textiles, with the search period being 5 years. Methods: This study targeted the use of alpha-amylases in various industrial sectors, so searches were carried out on the intellectual property database Espacenet website (European Patent Office - EPO) which contains more than 90 million patents deposited in its database. Results: During the search for patents filed in the last 5 years, 186 were found related to the use of alpha-amylases. These were disturbed as follows: 84 (biofuel), 41 (drinks), 16 (pharmaceuticals), 15 (detergents), 11 (food), 10 (animal feed), 9 (textiles). From the total number of patents found, we selected 6 from each area, except pharmaceutical products, to discuss and provide information on the application of this enzyme. Conclusion: This study demonstrated that the sectors of beverages and animal feed have preferences for thermostable alpha-amylases while sectors such as food, biofuels and textiles only regarded the importance of enzymatic efficiency. The detergent sector presented the greatest use of alpha-amylases which had distinct biochemical characteristics as solvent resistance and thermostability. The pharmaceutical sector was the one that presented less patents related to the application of alpha amylases. In addition, this work showed that China is the country with the highest patent registration for the use of alpha-amylases in the analyzed period.

Microbial Alpha-Amylase Production: Progress, Challenges and Perspectives

Alpha-amylase reputes for starch modification by breaking of 1-4 glycosidic bands and is widely applied in different industrial sectors. Microorganisms express unique alpha-amylases with thermostable and halotolerant characteristics dependent on the microorganism’s intrinsic features. Likewise, genetic engineering methods are applied to produce enzymes with higher stability in contrast to wild types. As there are widespread application of α-amylase in industry, optimization methods like RSM are used to improve the production of the enzyme ex vivo. This study aimed to review the latest researches on the production improvement and stability of α-amylase.

Horizontal Transfer and Gene Loss Shaped the Evolution of Alpha-Amylases in Bilaterians

The subfamily GH13_1 of alpha-amylases is typical of Fungi, but it is also found in some unicellular eukaryotes (e.g., Amoebozoa, choanoflagellates) and non-bilaterian Metazoa. Since a previous study in 2007, GH13_1 amylases were considered ancestral to the Unikonts, including animals, except Bilateria, such that it was thought to have been lost in the ancestor of this clade. The only alpha-amylases known to be present in Bilateria so far belong to the GH13_15 and 24 subfamilies (commonly called bilaterian alpha-amylases) and were likely acquired by horizontal transfer from a proteobacterium. The taxonomic scope of Eukaryota genomes in databases has been greatly increased ever since 2007. We have surveyed GH13_1 sequences in recent data from ca. 1600 bilaterian species, 60 non-bilaterian animals and also in unicellular eukaryotes. As expected, we found a number of those sequences in non-bilaterians: Anthozoa (Cnidaria) and in sponges, confirming the previous observations, but none in jellyfishes and in Ctenophora. Our main and unexpected finding is that such fungal (also called Dictyo-type) amylases were also consistently retrieved in several bilaterian phyla: hemichordates (deuterostomes), brachiopods and related phyla, some molluscs and some annelids (protostomes). We discuss evolutionary hypotheses possibly explaining the scattered distribution of GH13_1 across bilaterians, namely, the retention of the ancestral gene in those phyla only and/or horizontal transfers from non-bilaterian donors.

Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase

Background PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. Results In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. Conclusions In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA–amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.