Search Results (234)

We present the cloning and heterologous expression of the α-amylase gene amyBL159 from a thermophilic strain Bacillus licheniformis MGMM159, which was isolated from wastewater sediments self-heated to 70 °C. The gene was successfully cloned into the pET22b and pHT01 vectors, expressed and AmyBL159_Ec and AmyBL159_Bs recombinant α-amylases were purified from Escherichia coli BL21(DE3)pLys and Bacillus subtilis 168 strains, respectively. The AmyBL159_Ec enzyme was most active in the range of 75–95 °C, while AmyBL159_Bs showed maximum activity at temperatures from 45 to 75 °C. AmyBL159_Bs was shown to be more thermostable. Both enzymes were active over a broad pH range of 4.0–12.0. It was shown that Mn²⁺ ions enhanced the activity of both enzymes (up to 163% for AmyBL159_Ec and 142% for AmyBL159_Bs). These results highlight the importance of choosing an expression system for modulating the functional characteristics of recombinant α-amylase. The obtained AmyBL159_Ec and AmyBL159_Bs enzymes are promising for biotechnological applications under extreme conditions. The structure of the α-amylase was generated using the AlphaFold 3 web service. A structure–function analysis of this enzyme and previously studied α-amylases from B. licheniformis identified significant amino acid substitutions at positions 134(133) and 210(209) of the amino acid chain which may contribute to enhanced enzyme thermostability. Full article

Diabetic foot ulcers (DFUs) and other chronic wounds are major global health challenges, often complicated by infections and delayed healing due to excessive collagen accumulation. Microbial collagenases offer an enzymatic alternative to surgical debridement by selectively degrading collagen and potentially limiting microbial colonization. In this study, an isolated and characterized thermostable collagenase from Streptomyces scabies from rhizospheric soil in Al-Lith thermal springs, Saudi Arabia, is investigated. Identification was confirmed via 16S rRNA sequencing, and enzyme production was optimized on gelatin agar. Partial purification was achieved through ammonium sulfate precipitation and dialysis, and molecular weight (~25 kDa) was determined by Sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Activity was assessed under varying temperatures, pH, substrates, and metal ions, while antibacterial potential was tested against Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The collagenase exhibited optimal activity at 80 °C and pH 9, stability under thermophilic and alkaline conditions, activation by Fe²⁺, and notable antibacterial effects at higher concentrations. These results demonstrate that S. scabies collagenase exhibits selective antibacterial activity in vitro, suggesting its potential as an enzymatic tool for further evaluation in diabetic foot debridement and infection control. Full article

Proteases play key roles in many industrial processes and account for the majority of global enzyme sales. Geobacillus isolates from extreme environments such as marine hydrothermal vents are capable of producing high yields of proteases with thermophilic properties. Many proteases produced by Geobacillus species have been extensively studied, some of which have been purified and characterized. In addition, the high thermal stability largely depends on structural stability. Based on X-ray crystallography, several factors have been found to affect the structural stability of the thermostable proteases of Geobacillus. Moreover, the thermostable proteases of Geobacillus have a wide range of biotechnological applications, such as in detergent, food, bioremediation, leather-processing and textile industries. Therefore, this review focusses on the thermostable proteases of Geobacillus, including their characteristics, structural stability mechanisms and biotechnological applications. It will help the development of utilizing thermostable protease resources and enhancing their suitability for use in various industrial applications. Full article

Cytochrome P450 enzymes (CYPs) are versatile biocatalysts capable of performing selective oxidation reactions valuable for industrial and pharmaceutical applications. However, their catalytic efficiency is often constrained by dependence on costly electron donors, the requirement for redox partners, and uncoupling reactions that divert reducing power toward reactive oxygen species. Improving electron transfer efficiency through optimized redox partner interactions is therefore critical for developing effective CYP-based biocatalysts. In this study, we investigated the interaction between CYP119, a thermophilic CYP from Sulfolobus acidocaldarius, and putidaredoxin (Pdx), the redox partner of P450cam. Using rational design and computational modeling with PyRosetta 3, 14 CYP119 variants were modeled and analyzed by docking simulations on the Rosie Docking Server. Structural analysis identified three key mutations (N34E, D77R, and N34E/D77R) for site-directed mutagenesis. These mutations (N34E, D77R, and N34E/D77R) enhanced Pdx binding affinity by 20-, 3-, and 12-fold, respectively, without affecting substrate binding. Catalytic assays using lauric acid and indirect assays to monitor electron transfer revealed that, despite improved complex formation, the N34E variant showed reduced electron transfer efficiency compared to D77R. These findings highlight the delicate balance between redox partner binding affinity and catalytic turnover, emphasizing that fine-tuning electron transfer interfaces are essential for engineering efficient CYP biocatalysts. Full article

Plastic pollution, particularly from polyethylene terephthalate (PET), poses significant environmental concerns due to ecosystem persistence and extensive packaging use. Conventional recycling methods face inefficiencies, high costs, and limited scalability, necessitating sustainable alternatives. Biodegradation via PET hydrolases offers promising eco-friendly solutions, although most natural PET-degrading enzymes are thermophilic and require energy-intensive high temperatures. In contrast, psychrophilic enzymes function efficiently at extremely low temperatures but often lack stability under moderate conditions. Therefore, this study aimed to enhance the ability of the Mors1 enzyme from Moraxella TA144 to operate effectively under mesophilic conditions, which is closer to the optimal conditions for environmental application. Three strategic hydrophobic substitutions (K93I, E221I, and R235F) were introduced in loop regions, generating the mutant variant Mors1^MUT. Comparative characterization revealed that Mors1^MUT retained 98% of its activity at pH 9 and displayed greater resilience across both acidic and alkaline conditions than did the wild-type enzyme. Thermal stability assays revealed that Mors1^MUT preserved 61% of its activity at 40 °C and 14% at 50 °C, whereas the wild-type enzyme was fully inactivated at these temperatures. The enzymatic hydrolysis of PET films significantly improved with Mors1^MUT. Gravimetric analysis revealed weight losses of 0.83% for Mors1^WT and 3.46% for Mors1^MUT after a 12-day incubation period. This corresponds to a 4.16-fold increase in hydrolysis efficiency, confirming the enhanced catalytic performance of the mutant variant. The improvement was further validated by scanning electron microscopy (SEM), atomic force microscopy (AFM), and attenuated total reflectance–Fourier transform infrared (ATR-FTIR) analysis. Optimization of the reaction parameters through response surface methodology (enzyme load, time, pH, temperature, and agitation) confirmed increased PET hydrolysis under mild mesophilic conditions. These findings establish Mors1^MUT as a robust mesophilic PETase with enhanced catalytic efficiency and thermal stability, representing a promising candidate for sustainable PET degradation under environmentally relevant conditions. Full article

Thermophilic microorganisms represent an untapped reservoir of thermostable biocatalysts and stress-resilient biomolecules for industrial biotechnology. Aeribacillus pallidus, a Gram-positive moderate thermophile, has attracted attention for its enzymatic versatility and environmental adaptability, yet its genomic potential remains underexplored. Here, we present a comparative genomic analysis of 13 A. pallidus strains to uncover conserved and strain-specific traits relevant to biotechnology. Genomes ranged from 3.24 to 4.98 Mb, with GC content largely conserved (~39%) except for GS3372 (57.4%), indicating possible horizontal gene transfer. All strains encoded complete central metabolic pathways, while carbohydrate-active enzyme profiling revealed abundant glycoside hydrolases and glycosyltransferases, with GS3372 and MHI3390 enriched for lignocellulose-degrading enzymes. Secondary metabolite mining identified diverse biosynthetic gene clusters, including terpenes, sactipeptides, and bacteriocins, with PI8, W-12, and 8m3 exhibiting the greatest biosynthetic diversity. A core set of heat shock and universal stress proteins underscored robust thermotolerance. Phylogenomic and pan-genome analyses revealed high intraspecific diversity and an open pan-genome structure. Collectively, these findings position A. pallidus as a promising thermophilic chassis organism for sustainable applications, including biomass conversion, biofuel production, bioremediation, and the synthesis of heat-stable antimicrobial agents. Full article

Inorganic pyrophosphatase (PPase) is an enzyme that catalyzes the hydrolysis of pyrophosphate (PPi) into two phosphates. Ton1914, a thermophilic inorganic pyrophosphatase derived from Thermococcus onnurineus NA1, has good thermal stability and an extremely high optimum temperature and has been shown to reduce pyrophosphate inhibition. In this study, eight sites were selected based on sequence alignment and software calculations, and multiple single mutants were successfully constructed. After saturation and superposition mutations, six superior mutants were obtained. The enzyme activities of E97Y, D101K and L42F were increased 2.57-, 2.47- and 2.15-fold, respectively, while those of L42F/E97Y, L42F/D101K and E97Y/D101K were increased 2.60-, 2.63- and 1.88-fold, respectively, relative to the wild-type enzyme. Compared to Ton1914, all mutants more effectively increased PCR product quantity, reduced the number of qPCR cycles required to reach the threshold, and improved the efficiency of gene amplification. In the UDP-Galactose (UDP-Gal) synthesis reaction, the addition of mutants could further improve yield. When Ton1914 and mutants with the same activity were added, the yield of UDP-Gal was almost identical, effectively reducing the dosage of pyrophosphatase. Overall, the mutants showed greater prospects for industrial application. Full article

Thermophilic microorganisms are among the key natural sources of thermostable enzymes, found not only in geothermal areas but also in arid environments. In this study, eight Geobacillus strains were isolated from the arid sands of Aïn Sefra (Naâma, Algeria) and characterized both phenotypically and genetically. All strains exhibited an optimal growth temperature of 70 °C, with most showing alkaliphilic pH preferences. Proteomic and molecular analyses (MALDI-TOF MS, 16S rRNA) identified Geobacillus kaustophilus as predominant, with BOX-PCR and RAPD-PCR revealing notable intraspecies diversity. All strains synthesized at least one thermostable enzyme (protease, amylase, laccase, or DNase) at their optimal temperature (70 °C), positioning them as promising candidates for biotechnological processes requiring extreme thermal conditions. Full article

The genera Parageobacillus and Geobacillus comprise thermophilic, spore-forming bacteria. The extraordinary heat resistance of their spores, together with their ability to form biofilms and produce thermostable enzymes, makes them a relevant cause of spoilage in shelf-stable, heat-treated products like dairy and canned foods. However, these same biological traits offer valuable opportunities for the food industry. In this context, the purpose of this review is to describe the challenges posed by (Para)Geobacillus spp. as food spoilage agents, while also highlighting their existing and prospective applications in the food industry. In terms of food safety, G. stearothermophilus spores are used as biological indicators in commercially available tests to detect antibiotic residues in food within a few hours. Additionally, (Para)Geobacillus can be exploited for the fermentation of agri-food residues to produce high-value compounds such as biofuels, food ingredients and technological adjuvants, and compost. Their thermostable enzymes—such as amylases, xylanases, L-arabinose isomerases, β-galactosidases, lipases, proteases, and L-asparaginases—have potential applications in food processing and ingredient production. However, several challenges persist, including limited knowledge on genetic diversity, physiology, and metabolism, as well as low yields of biomass and target compounds. These issues reinforce the need for further studies to unlock their full potential. Full article

Tryptophan is synthesized in microorganisms via a five-step enzymatic pathway originating from chorismate, which is a product of the shikimate pathway. As a biosynthetic precursor to a wide range of high-value compounds such as indole-3-acetic acid, indigo, indirubin, and violacein, this pathway has been a central target for metabolic engineering to enhance microbial production. Anthranilate phosphoribosyltransferase (AnPRT) catalyzes the second step of the pathway by transferring a phosphoribosyl group from PRPP to anthranilate, forming phosphoribosyl anthranilate (PRA). AnPRT, the sole member of class IV phosphoribosyltransferases, adopts a unique fold and functions as a homodimer. While the structural basis of AnPRT activity has been elucidated in several organisms, thermostable variants remain underexplored despite their relevance for high-temperature bioprocessing. In this study, the crystal structure of AnPRT from the thermophilic archaeon Methanocaldococcus jannaschii (MjAnPRT) was determined at a 2.16 Å resolution. The enzyme exhibits a conserved dimeric architecture and key catalytic motifs. Comparative structural analysis with mesophilic and hyper thermophilic homologs revealed that MjAnPRT possesses enhanced local stability in catalytically important regions and strengthened inter-subunit interactions. These features likely contribute to its thermostability and provide a valuable framework for the rational design of robust AnPRTs for industrial and synthetic biology applications. Full article

Thermostable cellulases and xylanases have broad acceptance in food, feed, paper and pulp, and bioconversion of lignocellulosics. Thermophilic fungi serve as an excellent source of thermostable enzymes. This study characterized four endo-β-1,4-glucanases (two glycoside hydrolase (GH) family 5 and two GH7 members) and four endo-β-1,4-xylanases (two GH10 and two GH11 members) from thermophilic fungus Thielavia terrestris, along with one GH10 endo-β-1,4-xylanase each from thermophilic fungus Chaetomium thermophilum and mesophilic fungus Chaetomium globosum. Comparative analysis was conducted against three previously reported GH10 endoxylanases: two thermostable enzymes from the thermophilic fungus Humicola insolens and thermophilic bacterium Halalkalibacterium halodurans, and one mesophilic enzyme from model fungus Neurospora crassa. The GH10 xylanase TtXyn10C (Thite_2118148; UniProt G2R8T7) from T. terrestris demonstrated high thermostability and activity, with an optimal temperature of 80–85 °C. It retained over 60% of its activity after 2 h at 70 °C, maintained approximately 30% activity after 15 min at 80 °C, and showed nearly complete stability following 1 min of exposure to 95 °C. TtXyn10C exhibited specific activity toward beechwood xylan (1130 ± 15 U/mg) that exceeded xylanases from H. insolens and H. halodurans while being comparable to N. crassa xylanase activity. Furthermore, TtXyn10C maintained stability across a pH range of 3–9 and resisted trypsin digestion, indicating its broad applicability. The study expands understanding of enzymes from thermophilic fungi. The discovery of the TtXyn10C offers a new model for investigating the high activity-stability trade-off and structure-activity relationships critical for industrial enzymes. Full article

The growing demand for efficient sustainable biocatalysts for industrial applications has driven the exploration of extremozymes from extremophiles, particularly those thriving in geothermal environments. This study aimed to isolate and characterize extracellular amylase-producing thermophilic bacteria from the Nelumwewa geothermal spring in Sri Lanka, an underexplored ecosystem. Among the isolated thermophilic bacterial strains, NW2 isolates exhibited a prominent extracellular amylase activity. Molecular characterization by 16S rRNA gene sequencing confirmed the close phylogenetic relationship between NW2 and Thermaerobacillus caldiproteolyticus, which is well-known for thermostable proteases. Biochemical assays revealed optimal amylase activity of NW2 isolate at 60 °C and pH 8.0, with a crude enzyme activity of 0.85 U/mL. The enzyme demonstrated efficient hydrolysis of raw cassava starch, highlighting its potential for industrial applications in food, biofuel, and detergent industries. This study reports the first T. caldiproteolyticus-like strain from Sri Lanka with significant extracellular amylase activity, emphasizing the biotechnological potential of geothermal springs as sources of novel extremozymes. These findings contribute to the growing repository of thermostable enzymes, highlighting the need for further exploration of Sri Lanka’s geothermal microbial diversity for industrial biocatalysts. Full article

Tea has become one of the most popular drinks worldwide thanks to its pleasant sensory attributes and diverse health benefits. However, tannin-rich compositions have several negative effects and significantly impact the quality of tea beverages. Among various detannification methods, tannase treatment appears to be the most secure and environmentally friendly strategy. Although numerous microbial tannases have been identified and used in food processing, they are predominantly mesophilic with compromised heat tolerance, which limit their application in high-temperature tea extraction processing. Computer-assisted rational design and site-directed mutagenesis has emerged as a promising strategy in enzyme engineering to improve the thermostability of industrial enzymes. Nevertheless, relevant studies for tannase thermostability improvement remain lacking. In the present study, a novel thermophilic tannase called TanPL1 from marine fungus Penicillium longicatenatum strain SM102 was expressed in the food-grade host Yarrowia lipolytica. After purification and characterization, the thermostability of this enzyme was improved through site-directed mutagenesis guided by computer-aided rational design and molecular dynamics simulations. Then the thermostable mutant MuTanPL1 was applied in green tea processing for both polyphenol extraction and ester catechin hydrolysis. The tannase yield and specific activity values of 166.4 U/mL and 1059.3 U/mg, respectively, were achieved. The optimum pH and temperature of recombinant TanPL1 were determined to be 5.5 and 55 °C, respectively, and the enzyme exhibited high activity toward various gallic acid ester substrates. The site-directed mutagenesis method successfully generated a single-point mutant, MuTanPL1, with significantly enhanced thermostability and a higher optimum temperature of 60 °C. After 2 h of detannification by MuTanPL1, nearly all gallated catechins in green tea infusion were biotransformed. This resulted in a 202.4% and 12.1-fold increase in non-ester catechins and gallic acid levels, respectively. Meanwhile, the quality of the tea infusion was also markedly improved. Sensory evaluation and antioxidant activity assays revealed notable enhancements in these properties, while turbidity was reduced considerably. Additionally, the α-amylase inhibition activity of the tannase-treated tea infusion declined from 50.49% to 8.56%, revealing a significantly lower anti-nutritional effect. These findings suggest that the thermostable tannase MuTanPL1 holds strong application prospects in tea beverage processing. Full article

The genome of Geobacillus sp. G4, a thermophilic bacterium isolated from a geothermal field in Peru, was sequenced and analyzed to evaluate its taxonomic and biotechnological potential. This strain exhibits optimal growth at temperatures between 50 and 70 °C and at a pH range of 6.0–7.5. Phenotypic assays demonstrated extracellular enzymatic activities, including amylases, cellulases, pectinases, and xylanases, highlighting its potential for efficient polysaccharide degradation. The assembled genome comprises approximately 3.4 Mb with a G+C content of 52.59%, containing 3,490 genes, including coding sequences, rRNAs, and tRNAs. Functional annotation revealed genes associated with key metabolic pathways such as glycogen and trehalose biosynthesis, indicating adaptation to carbohydrate-rich environments. Phylogenetic analyses based on ANI and dDDH values identified Geobacillus thermoleovorans KCTC3570 as its closest relative, suggesting a strong evolutionary relationship. Additionally, the genome harbors gene clusters for secondary metabolites such as betalactone and fengycin, suggesting potential industrial and pharmaceutical applications, including bioremediation. The identification of antibiotic resistance genes, specifically those conferring glycopeptide resistance, underscores their relevance for antimicrobial resistance studies. The presence of enzymes like amylases and pullulanase further emphasizes its biotechnological potential, particularly in starch hydrolysis and biofuel production. Overall, this research highlights the significant potential of Geobacillus species as valuable sources of thermostable enzymes and biosynthetic pathways for industrial applications. Full article

Daqu, a crucial fermentation starter for Chinese Baijiu, develops distinct microbial and physicochemical profiles depending on fermentation temperature, which significantly influence enzymatic activity and flavor formation. While high-temperature (HT-Daqu, 65 °C) and medium-temperature (MT-Daqu, 60 °C) variants are known to produce different liquor aromas, systematic comparisons of their microbial and physicochemical dynamics remain limited. This study integrated physicochemical assays (moisture, starch, acidity, enzymatic activity) with 16S rRNA and ITS (Internal Transcribed Spacer) sequencing to analyze HT-Daqu (HQ1–HQ3) and MT-Daqu (MQ1–MQ3) from Sichuan breweries. Results revealed that HT-Daqu exhibited significantly lower moisture (p < 0.05) and starch content (p < 0.05) but higher acidity (p < 0.05) compared to MT-Daqu. Enzymatic activities were generally reduced in HT-Daqu, except for neutral protease. Microbial profiling revealed distinct microbial dynamics between HT-Daqu and MT-Daqu: HT-Daqu harbored thermophilic Bacillus (40–60% relative abundance) with reduced fungal diversity, while MT-Daqu prioritized fungal consortia—Aspergillus dominated MQ1 (78%) and Saccharomyces transiently peaked in MQ2 (35%)—which correlated with enhanced saccharification enzyme activities and esterification potential. Alpha-diversity indices confirmed higher bacterial diversity in HT-Daqu and greater fungal richness in MT-Daqu. Correlation networks highlighted temperature-driven linkages, such as Bacillus positively associating with acidity. These findings elucidate the trade-offs between microbial stress adaptation and metabolic efficiency under different thermal regimes, providing actionable insights for optimizing Daqu production through targeted microbial management and temperature control to enhance liquor quality. Full article