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α-Amylases (E.C 3.2.1.1) hydrolyse starch into smaller moieties such as maltose and glucose by breaking α-1,4-glycosidic linkages. The application of α-amylases in various industries has made the large-scale productions of these enzymes crucial. Thermostable α-amylase that catalyses starch degradation at the temperatures higher than 50 °C is favourable in harsh industrial applications. Due to ease in genetic manipulation and bulk production, this enzyme is most preferably produced by microorganisms. Bacillus sp. and Escherichia coli are commonly used microbial expression hosts for α-amylases (30 to 205 kDa in molecular weight). These amylases can be purified using ultrafiltration, salt precipitation, dialysis, and column chromatography. Recently, affinity column chromatography has shown the most promising result where the recovery rate was 38 to 60% and purification up to 13.2-fold. Microbial thermostable α-amylases have the optimum temperature and pH ranging from 50 °C to 100 °C and 5.0 to 10.5, respectively. These enzymes have high specificity towards potato starch, wheat starch, amylose, and amylopectin. EDTA (1 mM) gave the highest inhibitory effect (79%), but Ca2+ (5 mM) was the most effective co-factor with 155%. This review provides insight regarding thermostable α-amylases obtained from microbial sources for industrial applications.

The bioconversion of lignocellulose into monosaccharides is critical for ensuring the continual manufacturing of biofuels and value-added bioproducts. Enzymatic degradation, which has a high yield, low energy consumption, and enhanced selectivity, could be the most efficient and environmentally friendly technique for converting complex lignocellulose polymers to fermentable monosaccharides, and it is expected to make cellulases and xylanases the most demanded industrial enzymes. The widespread nature of thermophilic microorganisms allows them to proliferate on a variety of substrates and release substantial quantities of cellulases and xylanases, which makes them a great source of thermostable enzymes. The most significant breakthrough of lignocellulolytic enzymes lies in lignocellulose-deconstruction by enzymatic depolymerization of holocellulose into simple monosaccharides. However, commercially valuable thermostable cellulases and xylanases are challenging to produce in high enough quantities. Thus, the present review aims at giving an overview of the most recent thermostable cellulases and xylanases isolated from thermophilic and hyperthermophilic microbes. The emphasis is on recent advancements in manufacturing these enzymes in other mesophilic host and enhancement of catalytic activity as well as thermostability of thermophilic cellulases and xylanases, using genetic engineering as a promising and efficient technology for its economic production. Additionally, the biotechnological applications of thermostable cellulases and xylanases of thermophiles were also discussed.

Enhancing the thermostability of cellulose-degrading enzymes is pivotal for establishing an efficient bioconversion system from cellulosic materials to value-added compounds. Here, by introducing random and saturation mutagenesis into the Thermoascus aurantiacus β-glucosidase gene, we generated a hyperthermostable mutant with five amino acid substitutions. Analysis of temperature-induced unfolding revealed the involvement of each replacement in the increased Tm value. Structural analysis showed that all replacements are located at the periphery of the catalytic pocket. D433N replacement, which had a pronounced thermostabilizing effect (ΔTm ＝ 4.5°C), introduced an additional hydrogen bond with a backbone carbonyl oxygen in a long loop structure. The mutant enzyme expressed in Kluyveromyces marxianus exhibited a Tm of 82°C and hydrolyzed cellobiose with kcat and Km values of 200 s－1 and 1.8 mM, respectively. When combined with a thermostable endoglucanase, the mutant enzyme released 20％ more glucose than wild-type enzyme from cellulosic material. The mutant enzyme is therefore a noteworthy addition to the existing repertoire of thermostable β-glucosidases.

Background The emergence of serious issues of multidrug resistance in the past few years have enforced the use of bacteriocins for combating infections. Threat posed to public health by various multidrug resistant (MDR) organisms can be resolved by discovering new antimicrobial proteins with broad spectrum of inhibition. Results In the current study, Bacteriocin (BAC-IB17) produced by Bacillus subtilis KIBGE-IB17 is found to be effective against different strains of methicillin resistant Staphylococcus aureus (MRSA). The approximate molecular mass of BAC-IB17 is 10.7 kDa. This unique bacteriocin is found to be highly thermostable and pH stable in nature. It also showed its stability against various heavy metals, organic solvents, surfactants and proteolytic enzymes. Amino acid profile of BAC-IB17 clearly showed that this protein mainly consists of non-polar and basic amino acids whereas; some acidic amino acids were also detected. Sequence of first 15 amino acid residues obtained from N-terminal sequencing of BAC-IB17 were NKPEALVDYTGVXNS. Conclusions The anti-MRSA property of purified bacteriocin may be used to prevent the spread of MRSA infections. Remarkable features of BAC-IB17 suggests its applications in various pharmaceutical and food industries as it can function under a variety of harsh environmental conditions.

Tannase is widely used in tea beverage processing because of its ability to catalyze the hydrolysis of hydrolysable tannins or gallic acid esters and effectively improve the quality of tea extracts through enzymatic extraction. A new thermophilic tannase was cloned from Aspergillus niger FJ0118 and characterized. The tannase exhibited an optimal reaction temperature of 80 °C and retained 89.6% of the initial activity after incubation at 60 °C for 2 h. The enzymatic extraction of green tea at high temperature (70 °C) for a short time (40 min) was devised on the basis of the superior thermal stability of tannase. The enzymatic reaction significantly increased the total polyphenol content of green tea extract from 137 g·kg−1 to 291 g·kg−1. The enzymatic reaction effectively degraded the ester catechins into non-ester catechins compared with the water extraction method. Results suggested that the thermally stable tannase exhibited potential applications in the enzymatic extraction of green tea beverage.

The genus Geobacillus comprises thermophilic bacilli capable of endospore formation. The members of this genus provide thermostable proteins and can be used in whole cell applications at elevated temperatures; therefore, these organisms are of biotechnological importance. While these applications have been described in previous reviews, the present paper highlights the environmental adaptations and genome diversifications of Geobacillus spp. and their applications in evolutionary-protein engineering. Despite their obligate thermophilic properties, Geobacillus spp. are widely distributed in nature. Because several isolates demonstrate remarkable properties for cell reproduction in their respective niches, they seem to exist not only as endospores but also as vegetative cells in diverse environments. This suggests their excellence in environmental adaptation via genome diversification; in fact, evidence suggests that Geobacillus spp. were derived from Bacillus spp. while diversifying their genomes via horizontal gene transfer. Moreover, when subjected to an environmental stressor, Geobacillus spp. diversify their genomes using inductive mutations and transposable elements to produce derivative cells that are adaptive to the stressor. Notably, inductive mutations in Geobacillus spp. occur more rapidly and frequently than the stress-induced mutagenesis observed in other microorganisms. Owing to this, Geobacillus spp. can efficiently generate mutant genes coding for thermostable enzyme variants from the thermolabile enzyme genes under appropriate selection pressures. This phenomenon provides a new approach to generate thermostable enzymes, termed as thermoadaptation-directed enzyme evolution, thereby expanding the biotechnological potentials of Geobacillus spp. In this review, we have discussed this approach using successful examples and major challenges yet to be addressed.

Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/μL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

Arabinofuranosidase plays an essential role in the process of hydrolysis of arabinoxylan (AX). Thermostable, versatile, and efficient arabinofuranosidase is thus of great interest for the biorefinery industry. A GH51 arabinofuranosidase, Abf51, from Hungateiclostridium clariflavum DSM 19732 was heterogeneously expressed in Escherichia coli . Abf51 was found to have an optimal pH and temperature of 6.5 and 60 °C, respectively, with very high thermostability. At the optimal working temperature (60 °C), Abf51 retained over 90% activity after a 2-day incubation and over 60% activity after a 6-day incubation. Abf51 could effectively remove the arabinofuranosyls from three kinds of AX oligosaccharides [2 3 -α- l -arabinofuranosyl-xylotriose (A 2 XX), 3 2 -α- l -arabinofuranosyl-xylobiose (A 3 X), and 2 3 3 3 -di-α- l -arabinofuranosyl-xylotriose (A 2 + 3 XX)], which characterized as either single substitution or double substitution by arabinofuranosyls on terminal xylopyranosyl units. The maximal catalytic efficiency ( K cat / K m ) was observed using p -nitrophenyl-α- l -arabinofuranoside (pNPAF) as a substrate (205.0 s −1 mM −1 ), followed by using A 3 X (22.8 s −1 mM −1 ), A 2 XX (6.9 s −1 mM −1 ), and A 2 + 3 XX (0.5 s −1 mM −1 ) as substrates. Moreover, the presence of Abf51 significantly stimulated the saccharification level of AX (18.5 g L −1 ) up to six times along with a β-xylanase as well as a β-xylosidase. Interestingly, in our survey of top thermostable arabinofuranosidases, most members were found from GH51, probably due to their owning of (β/α) 8 -barrel architectures. Our results suggested the great importance of GH51s as candidates for thermostable, versatile, and efficient arabinofuranosidases toward industry application.

The success of mRNA vaccines against SARS-CoV-2 has prompted interest in mRNA-based pharmaceuticals due to their rapid production, adaptability, and safety. Despite these advantages, the inherent instability of mRNA and its rapid degradation in vivo underscores the need for an encapsulation system for the administration and delivery of RNA-based therapeutics. Lipid nanoparticles (LNPs) have proven the most robust and safest option for in vivo applications. However, the mid- to long-term storage of mRNA-LNPs still requires sub-zero temperatures along the entire chain of supply, highlighting the need to develop alternatives to improve mRNA vaccine stability under non-freezing conditions to facilitate logistics and distribution. Lyophilization presents itself as an effective alternative to prolong the shelf life of mRNA vaccines under refrigeration conditions, although a complex optimization of the process parameters is needed to maintain the integrity of the mRNA-LNPs. Recent studies have demonstrated the feasibility of freeze-drying LNPs, showing that lyophilized mRNA-LNPs retain activity and stability. However, long-term functional data remain limited. Herein, we focus on obtaining an optimized lyophilizable mRNA-LNP formulation through the careful selection of an optimal buffer and cryoprotectant and by tuning freeze-drying parameters. The results demonstrate that our optimized lyophilization process maintains LNP characteristics and functionality for over a year at refrigerated temperatures, offering a viable solution to the logistical hurdles of mRNA vaccine distribution.

Thermal stability is one of the most desirable characteristics in the search for novel lipases. The search for thermophilic microorganisms for synthesising functional enzyme biocatalysts with the ability to withstand high temperature, and capacity to maintain their native state in extreme conditions opens up new opportunities for their biotechnological applications. Thermophilic organisms are one of the most favoured organisms, whose distinctive characteristics are extremely related to their cellular constituent particularly biologically active proteins. Modifications on the enzyme structure are critical in optimizing the stability of enzyme to thermophilic conditions. Thermostable lipases are one of the most favourable enzymes used in food industries, pharmaceutical field, and actively been studied as potential biocatalyst in biodiesel production and other biotechnology application. Particularly, there is a trade-off between the use of enzymes in high concentration of organic solvents and product generation. Enhancement of the enzyme stability needs to be achieved for them to maintain their enzymatic activity regardless the environment. Various approaches on protein modification applied since decades ago conveyed a better understanding on how to improve the enzymatic properties in thermophilic bacteria. In fact, preliminary approach using advanced computational analysis is practically conducted before any modification is being performed experimentally. Apart from that, isolation of novel extremozymes from various microorganisms are offering great frontier in explaining the crucial native interaction within the molecules which could help in protein engineering. In this review, the thermostability prospect of lipases and the utility of protein engineering insights into achieving functional industrial usefulness at their high temperature habitat are highlighted. Similarly, the underlying thermodynamic and structural basis that defines the forces that stabilize these thermostable lipase is discussed.Key points• The dynamics of lipases contributes to their non-covalent interactions and structural stability.• Thermostability can be enhanced by well-established genetic tools for improved kinetic efficiency.• Molecular dynamics greatly provides structure-function insights on thermodynamics of lipase.