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In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg−1. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg−1) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg−1). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5ʹ-dithio-bis-[2-nitrobenzoic acid (DNTB), β-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme’s activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (Km) and maximum reaction rate (Vmax), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL−1 and 1580.3 ± 183.7 μmol mg−1 protein min−1, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with β-glucosidase for 24 h.

Laminarinase, an enzyme with a specific affinity for laminarin—a complex polysaccharide found in the cell walls of brown algae and select marine organisms—was investigated in this study. We cloned and characterized a gene encoding a putative glycoside hydrolase family 16 (GH16) laminarinase derived from the Jermuk hot spring metagenome. The resulting product, named Jermuk-LamM, represents a novel 1,3-β- d -glucanase with 48.1% amino acid sequence similarity to previously characterized GH16 family members catalogued in the NCBI database. To date, this stands as the sole described endo-1,3-β- d -glucanase from the Fidelibacterota phylum, which was recently reclassified from Marinimicrobia . Jermuk-LamM, identified as an acidic laminarinase, exhibits optimal enzymatic activity at pH 5.0 and a temperature of 55 °C, maintaining its function for a duration of at least 7 h. Jermuk-LamM is an enzyme that efficiently hydrolyzes both soluble and insoluble (1,3)-β- d -glucans, as well as (1,3;1,4)-β- d -glucans, with a marked preference for laminarin. This enzymatic activity facilitates the valorization of macroalgal biomass by predominantly producing monosaccharides and disaccharides. These hydrolysis products can subsequently be converted into energy carriers such as alcohol, methane, and hydrogen. The enzyme’s specific activities, coupled with its resistance to various additives, render Jermuk-LamM a promising candidate for various industrial applications, encompassing the realms of biofuel and pharmaceutical production. Key points • Jermuk hot springs have significant potential as a source of novel enzymes . • Jermuk-LamM has less than 50% amino acid similarity to known enzymes . • It is the first enzyme characterized from the Fidelibacterota phylum .

Recently, there has been a revival of interest in the use of natural pigments in various industries. It is of great interest to obtain melanins from plant raw materials. An effective method has been developed for cleaning grape squeezes from impurities before the alkaline extraction of melanin. A schematic flow diagram of the isolation and purification of water-soluble melanin from grape squeezes is presented. The obtained pigment was identified by spectroscopic methods. It has been established by thermal treatment that the amorphous precipitate of melanin is stable at temperatures up to 120 °C, and the acid resistance of the obtained melanin solution is in the pH range of > 4.0. Gel filtration, electrophoresis and HPLC showed that the pigment preparation contained four fractions that differed by their molecular weights. The quantitative distribution and the molecular weights of the obtained melanin fractions were determined. Qualitative reactions and spectroscopic methods have shown that these fractions are typical melanins. The elemental composition of the obtained melanin and its antioxidant properties, the amino acid composition of the melanin protein, and its solubility were determined. The yield of melanin from the feedstock was 17.3%. Based on the physicochemical properties of the resulting melanin, it can be used as an effective natural food coloring and powerful antioxidant substance.

The production of recombinant enzymes, primarily used for obtaining pure and functional target molecules, holds significant importance in modern biotechnology. This study aimed to obtain and characterize recombinant, extracellularly expressed α-amylases (Amy3500 and Amy1974), derived from B. amyloliquefaciens MDC1974 and B. subtilis MDC3500, respectively, using the pBE-S shuttle vector. Both α-amylase genes were molecularly cloned into the E. coli/B. subtilis pBE-S shuttle vector, both with (Amy1974sig and Amy3500sig) and without their signal peptides (Amy1974 and Amy3500), along with a signal peptide originating from the plasmid, and tested in flask fermentations. For recombinant Amy3500, the amylase variants resulted in similar levels of volumetric activity (700–750 U/mL). In contrast, the expression of Amy1974 nearly doubled compared to Amy1974sig with double signal peptides, reaching 2000 U/mL. SDS-PAGE estimated the molecular weight of Amy1974 α-amylase to be 54.6 kDa, which is consistent with the theoretical molecular mass calculations. However, the estimated molecular weight of Amy3500 α-amylase was significantly lower upon exiting the producer cells. Ca2⁺ ions exhibit a modest activating effect on the activities of Amy1974 and Amy3500 amylases, likely due to their tight binding to the protein scaffold. Both enzymes exhibited broad activity peaks between 45 and 70 °C, with a maximum at 65 °C. The Amy1974 and Amy3500 α-amylases demonstrated broad pH optima and pH-dependent thermostability, with optimum pH values at 6.5 and 5.8, and thermal stability peaks at pH 7.6 and 5.9, respectively. Both α-amylases displayed high relative activity against various starches, including corn amylopectin and potato amylose, while showing comparatively lower activity towards α-, β-, and γ-cyclodextrins. The Amy1974 amylase is effective in converting starch into dextrins of varying lengths, while Amy3500 primarily converts starch into glucose. These characteristics make them promising candidate enzymes for industrial applications.

The development of sustainable and environmentally friendly industrial processes is becoming very crucial and demanding for the rapid implementation of innovative bio-based technologies. Natural extreme environments harbor the potential for discovering and utilizing highly specific and efficient biocatalysts that are adapted to harsh conditions. This review focuses on extremophilic microorganisms and their enzymes (extremozymes) from various hot springs, shallow marine vents, and other geothermal habitats in Europe and the Caucasus region. These hot environments have been partially investigated and analyzed for microbial diversity and enzymology. Hotspots like Iceland, Italy, and the Azores harbor unique microorganisms, including bacteria and archaea. The latest results demonstrate a great potential for the discovery of new microbial species and unique enzymes that can be explored for the development of Circular Bioeconomy.Different screening approaches have been used to discover enzymes that are active at extremes of temperature (up 120 °C), pH (0.1 to 11), high salt concentration (up to 30%) as well as activity in the presence of solvents (up to 99%). The majority of published enzymes were revealed from bacterial or archaeal isolates by traditional activity-based screening techniques. However, the latest developments in molecular biology, bioinformatics, and genomics have revolutionized life science technologies. Post-genomic era has contributed to the discovery of millions of sequences coding for a huge number of biocatalysts. Both strategies, activity- and sequence-based screening approaches, are complementary and contribute to the discovery of unique enzymes that have not been extensively utilized so far.

Side streams of the dairy industry are a suitable nutrient source for cultivating microorganisms, producing enzymes, and high-value chemical compounds. The heterotrophic Escherichia coli and chemolithoautotroph Ralstonia eutropha are of major biotechnological interest. R. eutropha is a model organism for producing O2-tolerant [NiFe]-hydrogenases (Hyds) (biocatalysts), and E. coli has found widespread use as an expression platform for producing recombinant proteins, molecular hydrogen (H2), and other valuable products. Aiming at developing suitable cultivation media from side streams of the dairy industry, the pre-treatment (filtration, dilution, and pH adjustment) of cheese (sweet) whey (SW) and curd (acid) whey (AW), with and without the use of ß-glucosidase, has been performed. Growth parameters (oxidation–reduction potential (ORP), pH changes, specific growth rate, biomass formation) of E. coli BW25113 and R. eutropha H16 type strains were monitored during cultivation on filtered and non-filtered SW and AW at 37 °C, pH 7.5 and 30 °C, pH 7.0, respectively. Along with microbial growth, measurements of pH and ORP indicated good fermentative growth. Compared to growth on fructose-nitrogen minimal salt medium (control), a maximum cell yield (OD600 4.0) and H2-oxidizing Hyd activity were achieved in the stationary growth phase for R. eutropha. Hyd-3-dependent H2 production by E. coli utilizing whey as a growth substrate was demonstrated. Moreover, good biomass production and prolonged H2 yields of ~ 5 mmol/L and cumulative H2 ~ 94 mL g/L dry whey (DW) (ß-glucosidase-treated) were observed during the cultivation of the engineered E. coli strain. These results open new avenues for effective whey treatment using thermostable β-glucosidase and confirm whey as an economically viable commodity for biomass and biocatalyst production.Key points• Archaeal thermostable β-glucosidase isolated from the metagenome of a hydrothermal spring was used for lactose hydrolysis in whey.• Hydrogenase enzyme activity was induced during the growth of Ralstonia eutropha H16 on whey.• Enhanced biomass and H2production was shown in a genetically modified strain of Escherichia coli.

Laminarinase, an enzyme with a specific affinity for laminarin, a complex polysaccharide found in the cell walls of brown algae and select marine organisms, was investigated in this study. We cloned and characterised a gene encoding a putative glycoside hydrolase family 16 (GH16) laminarinase from the Jermuk hot spring metagenome by heterologous expression in Escherichia coli. The resulting product, named Jermuk-LamM, represents a novel endo-1,3-beta-D-glucanase (EC 3.2.1.39) with only 48.1 % amino acid sequence similarity to previously characterised GH16 family members catalogued in the NCBI database. To date, this stands as the sole described endo-1,3-beta-D-glucanase within the Marinimicrobia phylum. Jermuk-LamM, identified as an acidic laminarinase, exhibits robust enzymatic activity at pH 5.0 and a temperature of 55 0C, maintaining its function for a duration of at least 7 hours. Notably, this enzyme effectively catalyses the hydrolysis of both soluble and insoluble (1,3)-beta-D-glucans, as well as (1,3;1,4)-beta-D-glucans, displaying a pronounced preference for laminarin. The specificity of Jermuk-LamM lies in its cleavage of 1,3-beta-D-glucosidic linkages, yielding monosaccharides, disaccharides, and oligosaccharides. These breakdown products hold the potential for conversion into energy carriers, including alcohols, methane, and hydrogen. The enzyme's exceptional specific activities, coupled with its resistance to various additives, render Jermuk-LamM a promising candidate for various industrial applications, encompassing the realms of biofuel and pharmaceutical production.Competing Interest StatementThe authors have declared no competing interest.

N-Carbamoyl-β-Alanine Amidohydrolase (CβAA) constitute one of the most important groups of industrially relevant enzymes used in production of optically pure amino acids and derivatives. In this study, a N-carbamoyl-β-alanine amidohydrolase encoding gene from Rhizobium radiobacter MDC 8606 was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme (RrCβAA) showed a specific activity of 14 U/mg using N-carbamoyl-β-alanine as a substrate with an optimum activity of 55°C at pH 8.0. In this work, we report also the first prokaryotic N-carbamoyl-β-alanine amidohydrolases structure at a resolution of 2.0 Å. A discontinuous catalytic domain and a dimerization domain attached through a flexible hinge region at the domain interface has been revealed. We have found that the ligand is interacting with a conserved glutamic acid (Glu131), histidine (H385) and arginine (Arg291) residues. Studies let us to explain the preference on the enzyme for linear carbamoyl substrates as large carbamoyl substrates cannot fit in the active site of the enzyme. This work envisages the use of RrCβAA from the Rhizobium radiobacter MDC 8606 for the industrial production of L-α-, L-β-, and L-γ – amino acids. The structural analysis provides new insights on enzyme–substrate interaction, which shed light on engineering of N-carbamoyl-β-alanine amidohydrolases for high catalytic activity and broad substrate specificity.