Thermostable Enzymes Research Articles

A meta-omics approach to explore the biofuel-producing enzyme potential from extreme environmental conditions

Geothermally warmed spring water contaminated with decomposed leaf biomass are unique hot spring ecosystems , are expected to support the recycling of various nutrients and to host lignocellulose degrading thermostable enzymes, genes and bacteria. An attempt is made in the present study to explore CAZymes in a carbohydrate-contaminated unique environment at Deulajhari spring through a multi-omics approach using an indigenous consortium developed from the spring sediment. Co-assembly of shotgun metagenome and metatranscriptome libraries from the Deulajhari hot spring consortia sample recovered seventeen refined, high-quality near-complete genomes. The predominant recovery of thermophilic aerobic chemo-heterotrophic Meiothermus and Rhodothermus has been observed through genome reconstruction. The reported production of an array of enzymes, including xylanase, β-xylosidase, endoglucanase and polysaccharide deacetylase, establishes their lignocellulose-degrading ability. The unexpected strong positive correlation between predominating Meiothermus and less prevailing members of Acetobacteraceae, unclassified Gaiellaceae, unclassified Burkholderiaceae and Tepidimonas of the consortium signifies their unexplored role in biomass degradation. Furthermore, the synergistic involvement of the diverse enzymes represented by a vast gene repertoire is responsible for the degradation of complex plant polysaccharides by the group of bacteria. The novelty of the present study stems from the identification of a diverse range of potential lignocellulose-digesting enzymes expressed by the bacterial consortium. This finding emphasizes the significant potential of these enzymes in facilitating industrial-scale production of biofuel, making it a notable contribution to the field.

Moderately Thermophilic Bacteria from Jordanian Hot Springs as Possible Sources of Thermostable Enzymes and Leukemia Cytotoxic Agents

Moderately Thermophilic Bacteria from Jordanian Hot Springs as Possible Sources of Thermostable Enzymes and Leukemia Cytotoxic Agents

Biosynthesis of Lacto-N-biose I from starch and N-acetylglucosamine via an in vitro synthetic enzymatic biosystem

Human milk oligosaccharides (HMOs) are very distinctive components in human milk and are beneficial for infant health. Lacto-N-biose I (LNB) is the core structural unit of HMOs, which could be used for the synthesis of other HMOs. In this study, an ATP-free in vitro synthetic enzymatic biosystem contained four thermostable enzymes (alpha-glucan phosphorylase from Thermococcus kodakarensis, UDP-glucose-hexose-1-phosphate uridylyltransferase from Thermotoga maritima, UDP-glucose 4-epimerase from T. maritima, lacto-N-biose phosphorylase from Clostridium thermobutyricum) were constructed for the biosynthesis of LNB from starch and N-acetylglucosamine (GlcNAc). Under the optimal conditions, 0.75 g/L and 2.23 g/L LNB was produced from 1.1 g/L and 4.4 g/L GlcNAc and excess starch, with the molar yield of 39% and 29% based on the GlcNAc concentration, respectively, confirming the feasibility of this in vitro synthetic enzymatic biosystem for LNB synthesis and shedding light on the biosynthesis of other HMOs using LNB as the core structural unit from low cost polysaccharides.

The genus Anoxybacillus: an emerging and versatile source of valuable biotechnological products.

Thermophilic and alkaliphilic microorganisms are unique organisms that possess remarkable survival strategies, enabling them to thrive on a diverse range of substrates. Anoxybacillus, a genus of thermophilic and alkaliphilic bacteria, encompasses 24 species and 2 subspecies. In recent years, extensive research has unveiled the diverse array of thermostable enzymes within this relatively new genus, holding significant potential for industrial and environmental applications. The biomass of Anoxybacillus has demonstrated promising results in bioremediation techniques, while the recently discovered metabolites have exhibited potential in medicinal experiments. This review aims to provide an overview of the key experimental findings related to the biotechnological applications utilizing bacteria from the Anoxybacillus genus.

Geobacillus Bacteriophages from Compost Heaps: Representatives of Three New Genera within Thermophilic Siphoviruses.

We report a detailed characterization of five thermophilic bacteriophages (phages) that were isolated from compost heaps in Vilnius, Lithuania using Geobacillus thermodenitrificans strains as the hosts for phage propagation. The efficiency of plating experiments revealed that phages formed plaques from 45 to 80 °C. Furthermore, most of the phages formed plaques surrounded by halo zones, indicating the presence of phage-encoded bacterial exopolysaccharide (EPS)-degrading depolymerases. Transmission Electron Microscopy (TEM) analysis revealed that all phages were siphoviruses characterized by an isometric head (from ~63 nm to ~67 nm in diameter) and a non-contractile flexible tail (from ~137 nm to ~150 nm in length). The genome sequencing resulted in genomes ranging from 38,161 to 39,016 bp. Comparative genomic and phylogenetic analysis revealed that all the isolated phages had no close relatives to date, and potentially represent three new genera within siphoviruses. The results of this study not only improve our knowledge about poorly explored thermophilic bacteriophages but also give new insights for further investigation of thermophilic and/or thermostable enzymes of bacterial viruses.

Linker-assisted engineering of chimeric xylanase-phytase for improved thermal tolerance of feed enzymes

Biological enzymes are multifunctional macromolecules that can perform hundreds of reactions simultaneously. An enzyme must possess specific characteristics to meet industrial needs, such as stability over a wide pH and temperature range and high specific activity. A phytase and xylanase mixture is generally added to poultry feed to improve the bird’s health and productivity. Despite this, animal farmers have noticed no difference in productivity, and a leading cause is the high temperature at which feed is pulverized, which inactivates enzymes. A thermo-stable enzyme system can overcome these hitches. Commonly, coatings and immobilization reduce losses caused by physical-chemical factors in feed processing and digestion. To this end, we engineered the multifunctional xylanase-phytase domains on a single polypeptide fused by a helical linker. First, the ideal linker sequence was chosen by computing each selected linker’s root mean square deviation (RMSD). The selected helical linker provides sufficient structural flexibility for substrate binding and product release evaluated by molecular docking and molecular dynamic simulation studies. Furthermore, a domain-domain interaction has stabilized the bridging partners, attaining the thermal optima for xylanase and phytase at 90 °C. Even at the above-optimal temperature (100 °C), the recombinant PLX was relatively stable and retained 64.2% and 59.2% activity for xylanase and phytase, respectively, when surveyed for ten hours. So far, to this date, this is the highest degree of thermostability achieved by any recombinant phytase or xylanase. Communicated by Ramaswamy H. Sarma

Isolation and Identification of Thermophilic Bacteria from Lahendong Hot Spring, North Sulawesi

Thermophilic bacteria are a group of procaryotic organisms that can grow in high temperature around 450C until 900C. Thermophilic bacteria are able to produce lipase thermostable enzyme. Lipase thermostable enzymes produced from thermophilic bacteria are able to catalyze the hydrolysis of triglycerides into glycerol and free fatty acids and the synthesis of esters in organic solvents. Lipase thermostable enzymes are potential to be used in industrial production. Lipase enzymes have been used for the production of laundry detergents as additives or to replace chemical detergents because of their environmental friendliness. This research was aimed to isolates and identified themophilic bacteria from Lahendong hot springs, North Sulawesi and to conduct the morphological characterization, biochemical test, and molecular identification with 16S rRNA. Results showed that isolates obtained from the- Lahendong hot spring have high similarities with Geobacillus kaustophilus, Bacillus cereus, and Geobacillus lituanicus. The three isolates are able to produce lipase thermostbale enzyme.

Enhancing the thermostability of Streptomyces cyaneofuscatus strain Ms1 tyrosinase by multi-factors rational design and molecular dynamics simulations.

This study presents a multi-factor rational design strategy combined with molecular dynamics simulation to improve the thermostability of Streptomyces cyaneofuscatus strain Ms1 tyrosinase. Candidate mutation sites were identified using Discovery Studio and FoldX software, and the double mutant G124W/G137W was obtained. The mutant was heterogeneously expressed in Escherichia coli strain Rosetta2 (DE3), and its thermostability was verified. Results indicate that the rational design method, combined with molecular dynamics simulation and protein energy calculation, improved the enzyme's thermostability more accurately and effectively. The double mutant G124W/G137W had an optimum temperature of 60°C, about 5.0°C higher than that of the wild-type TYRwt, and its activity was 171.06% higher than the wild-type TYRwt. Its thermostability was enhanced, 42.78% higher than the wild-type at 50°C. These findings suggest that the rational design strategy applied in this study can facilitate the application of industrial enzymes in the pharmaceutical industry.

Improved thermostability of type I pullulanase from Bacillus thermoliquefaciens by error-prone PCR

Pullulanase (PulB) is a starch-debranching enzyme. In order to improve its catalytic performance, random mutagenesis was performed on the pullulanase gene derived from Bacillus thermoliquefaciens. Two rounds of error-prone PCR were carried out. Mutant T252S was screened in the first round of error-prone library, which had the highest catalytic activity. During the second round of mutations, mutant enzyme G250P/T252S/G253T/N255K was screened, which had further improved catalytic activity and the best thermostability. Compared with the parent enzyme, the specific activity of mutant enzyme G250P/T252S/G253T/N255K increased by 1.9 times, Km decreased by 22.7 %, kcat increased by 28.7 %, and kcat/Km increased by 68.4 %. The thermostability of the mutant enzyme improved significantly, showing that the half-life at 60 °C was extended to 7.5 h, which was 87.5 % higher than that of the parent enzyme. The mutation sites in these two rounds were concentrated in the 250–255 regions, indicating that this region was an important region affecting the catalytic activity and Thermostability. The reasons for the change of enzymtic properties was also preliminarily analyzed through three-dimensional simulation.

Correction to: the effect of temperature on the activity and stability of the thermostable enzyme caffeine dehydrogenase from Pichia manshurica CD1 Rubia Parvin

Correction to: the effect of temperature on the activity and stability of the thermostable enzyme caffeine dehydrogenase from Pichia manshurica CD1 Rubia Parvin

Design and Analytical Evaluation of a Rapid Plasma Screening Assay for Circulating Human Papillomavirus DNA via Thermostable Enzyme Chemistries.

Infection with oncogenic strains of human papillomavirus (HPV), such as HPV-16 and HPV-18, can lead to malignant progression and tumorigenesis. As an adjunct to traditional invasive tissue sampling methods, the use of modern thermostable enzyme chemistries can aid in the development of innovative assay workflows to extract and detect circulating HPV DNA (cHPV-DNA) in liquid biopsies. In this work, we first successfully generated a model system to replicate fragmented cHPV-DNA in human plasma. Using this model system, we designed a novel thermostable enzyme chemistry-based cHPV-DNA assay for rapid clinical HPV screening and robustly evaluated its analytical assay performance. Our findings demonstrated that the use of thermostable enzymes provided faster cHPV-DNA extraction and amplification, leading to an overall three-fold improvement in overall assay time as compared to the current standard assay workflow and achieving clinically relevant levels of analytical specificity, sensitivity, and precision for accurate cHPV-DNA detection with excellent 100% sensitivity and specificity in contrived human plasma specimens. In summary, we have devised a rapid laboratory workflow to facilitate the emerging use of liquid biopsies for minimally invasive, rapid, and scalable HPV DNA testing. With facile assay modifications, our thermostable enzyme-based cHPV-DNA assay can be utilized for the detection of other clinically high-risk HPV genotypes.

Enhanced enzyme thermostability of a family I.3 lipase LipSR1 by T118A mutation at the calcium-binding site.

The lipase gene lipSR1 isolated from oil-contaminated soil exhibits high hydrolytic activity for short-chain fatty acid substrates. A single calcium ion is required to anchor the lid of LipSR1 in an open conformation by coordination with two aspartate residues and three other residues in the lid. The lid of LipSR1 is anchored by Ca2+, which is coordinated by side-chain carboxyl oxygens of Asp153 and Asp157, carbonyl oxygens of Thr118 and Ser144, and the side chain of Gln120. D157A, D153R, Q120A, S144A, and T118A mutants were produced by site-directed mutagenesis in this study. Analyses of hydrolytic activity and thermostability showed that the properties of D157A, D153R, Q120A, and S144A were almost lost, suggesting that Asp157, Asp153, Gln120, and Ser144 are important residues for LipSR1. However, the catalytic performance of T118A was clearly maintained. Moreover, the thermostability of mutant T118A was higher than that of wild-type LipSR1. These results indicated that mutation of threonine at position 118 improved the stability of the enzyme at high temperature.

The effect of temperature on the activity and stability of the thermostable enzyme caffeine dehydrogenase from Pichia manshurica CD1

The effect of temperature on the activity and stability of the thermostable enzyme caffeine dehydrogenase from Pichia manshurica CD1

Catalytic and molecular properties of alkaliphilic and thermotolerant β-etherase from Altererythrobacter sp. B11.

Phenylpropanone monomers, including guaiacyl hydroxypropanone, are important precursors for the synthesis of various chemicals. The monomers are obtained in a three-step cascade reaction catalyzed by a group of enzymes in the β-etherase system that cleaves the β-O-4 bond, the major bond in lignin. In this study, one of the β-etherase of the glutathione-S-transferase superfamily, AbLigF2, was discovered in genus Altererythrobacter, and the recombinant etherase was characterized. The enzyme showed maximal activity at 45°C, maintained 30% of its activity after 2h at 50°C, and was the most thermostable among the previously reported enzymes. Moreover, N13, S14, and S115, located near the thiol group of glutathione, had a significant effect on the maximum reaction rate of enzyme activity. This study suggests that AbLigF2 has the potential to serve as a thermostable enzyme for lignin utilization and provides insights into its catalytic mechanism.

Optimization of Biodiesel Production from Sludge of Mask Waste and Used Cooking Oil Using Lipase as Biocatalyst

Mask waste and used cooking oil contain hydrocarbons and free fatty acids. the combination of these ingredients with pretreatment heating in the microwave and the help of lipase enzymes makes the resulting sludge has the potential to be converted into biodiesel with a maximum yield when compared to making biodiesel from other materials and methods. The results of this study indicate that pre-treated sludge from mask waste and used cooking oil can be converted into biodiesel through a methanol transesterification reaction with the help of thermostable lipase enzymes from Pseudomonas. As for the optimum conditions, the highest biodiesel yield was produced at a temperature of 60℃, the best reaction time was 25 minutes, the volume of sludge was 10 mL and the optimum pH was at pH 8. The flame test on the biodiesel produced had been carried out, most of which had bright blue, red flames and orange. There are also some results that the flame is dim and even the test results are not lit.

A Bibliometric Analysis: Current Perspectives and Potential Trends of Enzyme Thermostability from 1991-2022.

Thermostability is considered a crucial parameter to evaluate the viability of enzymes in industrial applications. Over the past 31years, many studies have been reported on the thermostability of enzymes. However, there is no systematic bibliometric analysis of publications on the thermostability of enzymes. In this study, 16,035 publications related to the thermostability of enzymes were searched and collected, showing an increasing annual trend. China contributed the most publications, while the United States had the highest citation count. International Journal of Biological Macromolecules is the most productive journal in the research field. Moreover, Chinese acad sci and Khosro Khajeh are the most active institutions and prolific authors in the field, respectively. Analysis of references with the strongest citation bursts and keyword co-occurrences, magnetic nanoparticles, metal-organic frameworks, molecular dynamics, and rational design are current hot spots and significant future research directions. This study is the first comprehensive bibliometric analysis summarizing trends and developments in enzyme thermostability research. Our findings could provide scholars with an understanding of the fundamental knowledge framework of the field and identify recent potential hotspots and research trends that could facilitate the discovery of collaboration opportunities.

Isolation, characterization and identification of cellulase (Endo-β-1,4-glucanase) producing bacteria from diverse locations


 The objective of the study was to isolate cellulase producing bacteria from natural sources and identify the bacterial isolates that produce thermostable enzyme. Bacteria were isolated from samples collected from different locations like mango leaf litter, termite gut, composting yard (municipal solid waste, compost, and soil), soil from decaying paper waste, decaying wood, and saline coastal belt soil by enrichment in broth containing 1% cellulose and subsequent culturing on plates and the purified cultures stored in slants of the solidified agar medium of the same composition as enrichment medium. Primary screening of the isolates for cellulase (Endo β-1,4-glucanase) production was done by growing them on Carboxy Methyl Cellulose (CMC) agar medium and screening using indicator dye Congo red. Secondary screening of the positive isolates showing cellulolytic Index > 4 was done by crude cellulase enzyme extraction and assay using 1% CMC in 1N citrate buffer (pH 5.0) at different pH values (4.8- 7.0) and temperatures (35- 70℃). Cellulase activity (EA) in U/mL was calculated based on the absorbance change per min. which depends on the amount of reducing sugar produced by the enzyme catalysed conversion of CMC to reducing sugar. Isolates were characterized morphologically and biochemically and six isolates (Gram -ve: T1, MSW, C1, S2 and Gram +ve: W2, P) having significantly higher EA were selected for DNA extraction. DNA was quantified and the DNA of four isolates (MSW, S2, W2, P) were selected for sequence analysis. The 16S rDNA sequencing was carried out through PCR amplification and Blast using NCBI blast similarity search tool resulted in the identification of MSW, S2, W2 as E.coli, Sulfitobacter pontiacus, Bacillus subtilis respectively and P as Bacillus cereus and Rhodococcus erythropolis strain e1. Isolates showed significantly higher cellulase activity (EA) at pH 6.2 and at varying temperatures. Bacterial isolates P and C2 showed significantly higher activity at 40℃, MSW at 60℃ and others at 50℃ (EA ranging from 0.0054 – 0.0604 U/mL). Isolates W2, P, S2 had EA ranging from 0.006 - 0.03 U/mL at 70℃. This study showed that the cellulase producers could be grown at different temperatures (40 - 70℃) on cellulose based substrate. In this study the substrate specificity indicates that the crude enzyme is an endo- β-1,4-glucanase that plays a prominent role in initiating and sustaining the hydrolytic process and randomly cleaves cellulose into glucose and olygomeric polysaccharides. Hence by selecting samples with cellulosic substance from different locations with extreme environmental conditions like high temperature, high salinity it is possible to isolate cellulolytic bacteria producing cellulase enzymes that are stable at higher processing temperatures and other harsh conditions which is of importance in industrial applications.
 Keywords: Endo-β-1,4-glucanase, Cellulolytic index, Cellulase activity, Thermostable

Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.

Tuning Thermostability and Catalytic Efficiency of Aflatoxin-Degrading Enzyme by Error-prone PCR.

In our previous work, a recombinant aflatoxin-degrading enzyme derived from Myxococcus fulvus (MADE) was reported. However, the low thermal stability of the enzyme had limitations for its use in industrial applications. In this study, we obtained an improved variant of recombinant MADE (rMADE) with enhanced thermostability and catalytic activity using error-prone PCR. Firstly, we constructed a mutant library containing over 5000 individual mutants. Three mutants with T50 values higher than the wild-type rMADE by 16.5°C (rMADE-1124), 6.5°C (rMADE-1795), and 9.8°C (rMADE-2848) were screened by a high-throughput screening method. Additionally, the catalytic activity of rMADE-1795 and rMADE-2848 was improved by 81.5% and 67.7%, respectively, compared to the wild-type. Moreover, structural analysis revealed that replacement of acidic amino acids with basic amino acids by a mutation (D114H) in rMADE-2848 increased the polar interactions with surrounding residues and resulted in a threefold increase in the t1/2 value of the enzyme and made it more thermaltolerate. KEY POINTS: • Mutant libraries construction of a new aflatoxins degrading enzyme by error-prone PCR. • D114H/N295D mutant improved enzyme activity and thermostability. • The first reported enhanced thermostability of aflatoxins degrading enzyme better for its application.

Evaluating Machine Learning Models on Predicting Change in Enzyme Thermostability

Enzymes are efficient catalysts for biological reactions and can potentially be designed to speed up non-biological reactions, such as reactions in industrial processes. However, physically experimenting with new protein designs is time consuming, and an efficient method to predict protein stability is needed. Our research problem is finding the best machine learning model to predict the change in enzyme thermostability after a single point mutation in the amino acid sequence. We trained several machine learning models and found that the XGBoost model had the best performance with an R2 score of 0.593 (R2 score is a metric where higher is better and a perfect model would have a score of 1).