Microbial α-amylases: a biotechnological perspective

Basic biochemical mechanisms behind the health benefits of polyphenols.

Pomegranate (Punica granatum L.) is an ancient fruit that is widely consumed as fresh fruit and juice. The use of pomegranate fruit dates from ancient times and reports of its therapeutic qualities have echoed throughout the ages. Both in vitro and in vivo studies have demonstrated how this fruit acts as antioxidant, antidiabetic, and hypolipidemic and shows antibacterial, antiinflammatory, antiviral, and anticarcinogenic activities. The fruit also improves cardiovascular and oral health. These beneficial physiological effects may also have preventive applications in a variety of pathologies. The health benefits of pomegranate have been attributed to its wide range of phytochemicals, which are predominantly polyphenols, including primarily hydrolyzable ellagitannins, anthocyanins, and other polyphenols. The aim of this review was to present an overview of the functional, medical, and physiological properties of this fruit.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1541-4337.2010.00131.x

Pomegranate and its Many Functional Components as Related to Human Health: A Review

https://repositorio.unb.br/bitstream/10482/6389/1/ARTIGO_ApplicationMicrobialAmylase.pdf

Application of microbial α-amylase in industry - A review

Enzymes are the large biomolecules that are required for the numerous chemical interconversions that sustain life. They accelerate all the metabolic processes in the body and carry out a specific task. Enzymes are highly efficient, which can increase reaction rates by 100 million to 10 billion times faster than any normal chemical reaction. Due to development in recombinant technology and protein engineering, enzymes have evolved as an important molecule that has been widely used in different industrial and therapeutical purposes. Microbial enzymes are currently acquiring much attention with rapid development of enzyme technology. Microbial enzymes are preferred due to their economic feasibility, high yields, consistency, ease of product modification and optimization, regular supply due to absence of seasonal fluctuations, rapid growth of microbes on inexpensive media, stability, and greater catalytic activity. Microbial enzymes play a major role in the diagnosis, treatment, biochemical investigation, and monitoring of various dreaded diseases. Amylase and lipase are two very important enzymes that have been vastly studied and have great importance in different industries and therapeutic industry. In this review, an approach has been made to highlight the importance of different enzymes with special emphasis on amylase and lipase in the different industrial and medical fields.

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A Broader View: Microbial Enzymes and Their Relevance in Industries, Medicine, and Beyond

α-Amylases (α-1,4-glucan-4-glucanohydrolases; EC 3.2.1.1) are classical calcium-containing enzymes, which constitute a family of endo -amylases catalysing the cleavage of α- d -(1-4) glycosidic bonds in starch and related carbohydrates with retention of the α-anomeric configuration in the products. They can be found in microorganisms, plants and higher organisms where they play a dominant role in carbohydrate metabolism. This study characterizes the substrate binding sites of Bacillus licheniformis α-amylase (BLA), human salivary α-amylase (HSA) and its Y151M mutant. It describes the first subsite maps, namely, number of subsites, position of cleavage sites and apparent subsite energies. The product pattern and cleavage frequencies were determined by HPLC, utilising a homologous series of chromophore-substituted maltooligosaccharides of degree of polymerisation (DP) 3–10 as model substrates. 2-Chloro-4-nitrophenyl (CNP) and 4,6- O -benzylidene-modified 4-nitrophenyl (Bnl-NP) β-maltooligosaccharides (DP 4–8) were synthesised from cyclodextrins using a chemical procedure. For the preparation of CNP-maltooligosides of longer chain length a new chemoenzymatic procedure was developed using rabbit skeletal muscle glycogen phosphorylase b. Our results confirmed the presence of eight binding sites in BLA, five glycone sites (−5,−4,−3,−2,−1), three aglycone sites (+1,+2,+3) and the catalytic site is located between subsites (−1 and +1). In addition, the subsite map revealed a barrier site at the reducing end of active site which repulses the glucose residue. The binding region of HSA is composed of four glycone and three aglycone-binding sites, while that of Tyr151Met mutant is composed of four glycone and two aglycone-binding sites. The subsite maps show that Y151M has strikingly decreased binding energy at subsite (+2), where the mutation has occurred (−2.6 kJ/mol), compared to the binding energy at subsite (+2) of HSA (−12.0 kJ/mol).

α-Amylases of medical and industrial importance

Inhibitory effects of tannin on human salivary α-amylase

Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley α-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (β/α)8-barrel and the noncatalytic C-terminal domain, respectively. Site-directed mutagenesis of Trp278 and Trp279, stacking onto adjacent ligand glucosyl residues at SBS1, and of Tyr380 and His395, making numerous ligand contacts at SBS2, suggested that SBS1 and SBS2 act synergistically in degradation of starch granules. While SBS1 makes the major contribution to binding and hydrolysis of starch granules, SBS2 exhibits a higher affinity for the starch mimic β-cyclodextrin. Compared to that of wild-type AMY1, the Kd of starch granule binding by the SBS1 W278A, W279A, and W278A/W279A mutants thus increased 15−35 times; furthermore, the kcat/Km of W278A/W279A was 2%, whereas both affinity and activity for Y380A at SBS2 were 10% of the wild-type values. Dual site double and triple SBS1/SBS2...

Two secondary carbohydrate binding sites on the surface of barley α-amylase 1 have distinct functions and display synergy in hydrolysis of starch granules

The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two residues Trp58 and Trp59, which belong to beta2-alpha2 loop of the catalytic (beta/alpha)(8) barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k(cat), an increase in K(m) and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2, -3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amylase.

Human salivary α-amylase Trp58 situated at subsite -2 is critical for enzyme activity

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2802%2902649-2

Lili Kandra

Papers

α-Amylases of medical and industrial importance

Inhibitory effects of tannin on human salivary α-amylase

Two secondary carbohydrate binding sites on the surface of barley α-amylase 1 have distinct functions and display synergy in hydrolysis of starch granules

Human salivary α-amylase Trp58 situated at subsite -2 is critical for enzyme activity

Action pattern and subsite mapping of Bacillus licheniformis α-amylase (BLA) with modified maltooligosaccharide substrates