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Thermophilic Geobacillus kaustophilus HTA426 genome possesses a monoacylglycerol lipase (MAGL) gene. MAGLs can synthesize emulsifiers for use in the food and pharmaceutical industries from fatty acids and glycerol. They can also be used to analyze monoacylglycerol (MAG) levels in serum and food. The MAGL gene from strain HTA426 was artificially synthesized and heterologously expressed in Escherichia coli BL21(DE3). The recombinant His-tag fused MAGL (GkMAGL) was purified using a Ni 2+ -affinity column. The purified enzyme showed a temperature optimum at 65 °C and was stable up to 75 °C after 30 min incubation. In addition, the enzyme exhibited a pH optimum of 7.5 and was stable from pH 5.0 to 11.0. The enzyme hydrolyzed monoacylglycerols and showed the highest activity toward 1-monolauroylglycerol. The enzyme was stable in the presence of various organic solvents and detergents. The addition of Triton X-100 significantly increased GkMAGL activity. The thermal stability of the enzyme was higher than that of thermostable MAGL from Geobacillus sp. 12AMOR1 (12AMOR1_MAGL). Circular dichroism spectral analysis showed that the conformational stability of the GkMAGL was higher than that of 12AMOR1_MAGL at higher temperatures. These results indicate that the GkMAGL has useful features that can be used for various biotechnological applications.

Geobacillus thermoglucosidasius NCIMB 11955 possesses advantages, such as high-temperature tolerance, rapid growth rate, and low contamination risk. Additionally, it features efficient gene editing tools, making it one of the most promising next-generation cell factories. However, as a non-model microorganism, a lack of metabolic information significantly hampers the construction of high-precision metabolic flux models. Here, we propose a BioIntelliModel (BIM) strategy based on artificial intelligence technology for the automated construction of enzyme-constrained models. 1). BIM utilises the Contrastive Learning Enabled Enzyme Annotation (CLEAN) prediction tool to analyse the entire genome sequence of G. thermoglucosidasius NCIMB 11955, uncovering potential functional proteins in non-model strains. 2). The MetaPatchM module of BIM automates the repair of the metabolic network model. 3). The Tianjin University of Science and Technology-k cat (TUST-k cat ) module predicts the k cat values of enzymes within the model. 4). The Enzyme-insert procedure constructs an enzyme-constrained model and performs a global scan to address overconstraint issues. Enzymatic data were automatically integrated into the metabolic flux model, creating an enzyme-constrained model, ec_G-ther11955. To validate model accuracy, we used both the p-thermo and ec_G-ther11955 models to predict riboflavin production strategies. The ec_G-ther11955 model demonstrated significantly higher accuracy. To further verify its efficacy, we employed ec_G-ther11955 to guide the rational design of L-valine-producing strains. Using the Optimisation Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions (OptForce), Predictive Knockout Targeting (PKT), and Flux Scanning based on Enforced Objective Flux (FSEOF) algorithms, we identified 24 knockout and overexpression targets, achieving an accuracy rate of 87.5%. Ultimately, this led to an increase of 664.04% in L-valine titre. This study provides a novel strategy for rapidly constructing non-model strain models and demonstrates the tremendous potential of artificial intelligence in metabolic engineering., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Histidine ammonia-lyase (HAL) plays a pivotal role in the non-oxidative deamination of L-histidine to produce trans -urocanic, a crucial process in amino acid metabolism. This study examines the cloning, purification, and biochemical characterization of a novel HAL from Geobacillus kaustophilus ( Gk HAL) and eight active site mutants to assess their effects on substrate binding, catalysis, thermostability, and secondary structure. The Gk HAL enzyme was successfully overexpressed and purified to homogeneity. Its primary sequence displayed 40.7% to 43.7% similarity with other known HALs and shared the same oligomeric structure in solution. Kinetic assays showed that Gk HAL has optimal activity at 85 °C and pH 8.5, with high thermal stability even after preincubation at high temperatures. Mutations at Y52, H82, N194, and E411 resulted in a complete loss of catalytic activity, underscoring their essential role in enzyme function, while mutations at residues Q274, R280, and F325 did not abolish activity but did reduce catalytic efficiency. Notably, mutants R280K and F325Y displayed novel activity with L-histidinamide, expanding the substrate specificity of HAL enzymes. Circular dichroism (CD) analysis showed minor secondary structure changes in the mutants but no significant effect on global Gk HAL folding. These findings suggest that Gk HAL could be a promising candidate for potential biotechnological applications.

Flavin reductases play a vital role in catalyzing the reduction of flavin through NADH or NADPH oxidation. The gene encoding flavin reductase from the thermophilic bacterium Geobacillus mahadii Geo-05 (GMHpaC) was cloned, overexpressed in Escherichia coli BL21 (DE3) pLysS, and purified to homogeneity. The purified recombinant GMHpaC (Class II) contains chromogenic cofactors, evidenced by maximal absorbance peaks at 370 nm and 460 nm. GMHpaC stands out as the most thermostable and pH-tolerant flavin reductase reported to date, retaining up to 95 % catalytic activity after incubation at 70 °C for 30 min and maintaining over 80 % activity within a pH range of 2-12 for 30 min. Furthermore, GMHpaC's catalytic activity increases by 52 % with FMN as a co-factor compared to FAD and riboflavin. GMHpaC, coupled with 4-hydroxyphenylacetate-3-monooxygenase (GMHpaB) from G. mahadii Geo-05, enhances the hydroxylation of 4-hydroxyphenylacetate (HPA) by 85 %. The modeled structure of GMHpaC reveals relatively conserved flavin and NADH binding sites. Modeling and docking studies shed light on structural features and amino acid substitutions that determine GMHpaC's co-factor specificity. The remarkable thermostability, high catalytic activity, and general stability exhibited by GMHpaC position it as a promising enzyme candidate for various industrial applications., Competing Interests: Declaration of competing interest The author declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Microbial xylanases are enzymes of great importance due to their wide industrial applications, especially in the degradation of lignocellulosic biomass into fermentable sugars. This study aimed to describe the production optimization and partial characterization of an ultra-thermostable, acidophilic, cellulase-free xylanase from an obligate thermophilic eubacterium Geobacillus thermoleovorans strain-AKNT10 (Ac.No. LT158229) isolated from a hot-spring of Puga Valley located at an altitude of 4419 m in Ladakh, India. The optimization of cultural conditions improved enzyme yield by 10.49-fold under submerged fermentation. The addition of 1% (w/v) xylose induced the enzyme synthesis by ~ 165 and 371% when supplemented in the fermentation medium containing wheat bran (WB) 1 and 3%, respectively. The supplementation of sucrose reduced the xylanase production by ~ 25%. Results of partial characterization exhibited that xylanase was optimally active at pH 6.0 and 100 °C. Enzyme retained > 75%, > 83%, and > 84% of activity at 4 °C for 28 days, 100 °C for 60 min, and pHs 3-8 for 60 min, respectively. An outstanding property of AKNT10-xylanase, was the retention of > 71% residual activity at extreme conditions (121 °C and 15 psi pressure) for 15 min. Enzymatic saccharification showed that enzyme was also capable to liberate maximum reducing sugars within 4-8 h under optimized conditions thus it could be a potential candidate for the bioconversion of lignocellulosic biomass as well as other industrial purposes. To the best of our knowledge, this is the first report on such an ultra-thermo-pressure-tolerant xylanase optimally active at pH 6 and 100 °C from the genus Geobacillus., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli-Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo -inositol into scyllo -inositol at 30°C. In a scaled-up reaction, 10 g of myo -inositol was converted to 1.8 g of scyllo -inositol, which was further purified to yield 970 mg of pure powder. Notably, myo -inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification., Importance: Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo -inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo -inositol, holding potential pharmaceutical significance as scyllo -inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems., Competing Interests: The authors declare no conflict of interest.

Lipases are enzymes that hydrolyze long-chain carboxylic esters, and in the presence of organic solvents, they catalyze organic synthesis reactions. However, the use of solvents in these processes often results in enzyme denaturation, leading to a reduction in enzymatic activity. Consequently, there is significant interest in identifying new lipases that are resistant to denaturing conditions, with extremozymes emerging as promising candidates for this purpose. Lip7, a lipase from Geobacillus sp. ID17, a thermophilic microorganism isolated from Deception Island, Antarctica, was recombinantly expressed in E. coli C41 (DE3) in functional soluble form. Its purification was achieved with 96% purity and 23% yield. Enzymatic characterization revealed Lip7 to be a thermo-alkaline enzyme, reaching a maximum rate of 3350 U mg -1 at 50 °C and pH 11.0, using p-nitrophenyl laurate substrate. Notably, its kinetics displayed a sigmoidal behavior, with a higher kinetic efficiency ( k cat / K m ) for substrates of 12-carbon atom chain. In terms of thermal stability, Lip7 demonstrates stability up to 60 °C at pH 8.0 and up to 50 °C at pH 11.0. Remarkably, it showed high stability in the presence of organic solvents, and under certain conditions even exhibited enzymatic activation, reaching up to 2.5-fold and 1.35-fold after incubation in 50% v / v ethanol and 70% v / v isopropanol, respectively. Lip7 represents one of the first lipases from the bacterial subfamily I.5 and genus Geobacillus with activity and stability at pH 11.0. Its compatibility with organic solvents makes it a compelling candidate for future research in biocatalysis and various biotechnological applications.

Laccase is an oxidase of great industrial interest due to its ability to catalyze oxidation processes of phenols and persistent organic pollutants. However, it is susceptible to denaturation at high temperatures, sensitive to pH, and unstable in the presence of high concentrations of solvents, which is a issue for industrial use. To solve this problem, this work develops the synthesis in an aqueous medium of a new Mn metalloenzyme with laccase oxidase mimetic catalytic activity. Geobacillus thermocatenulatus lipase (GTL) was used as a scaffold enzyme, mixed with a manganese salt at 50 °C in an aqueous medium. This leads to the in situ formation of manganese(IV) oxide nanowires that interact with the enzyme, yielding a GTL-Mn bionanohybrid. On the other hand, its oxidative activity was evaluated using the ABTS assay, obtaining a catalytic efficiency 300 times higher than that of Trametes versicolor laccase. This new Mn metalloenzyme was 2 times more stable at 40 °C, 3 times more stable in the presence of 10% acetonitrile, and 10 times more stable in 20% acetonitrile than Novozym 51003 laccase. Furthermore, the site-selective immobilized GTL-Mn showed a much higher stability than the soluble form. The oxidase-like activity of this Mn metalloenzyme was successfully demonstrated against other substrates, such as l-DOPA or phloridzin, in oligomerization reactions.

Bisphenol A diglycidyl ether (BADGE), a derivative of the well-known endocrine disruptor Bisphenol A (BPA), is a potential threat to long-term environmental health due to its prevalence as a micropollutant. This study addresses the previously unexplored area of BADGE toxicity and removal. We investigated, for the first time, the biodegradation potential of laccase isolated from Geobacillus thermophilic bacteria against BADGE. The laccase-mediated degradation process was optimized using a combination of response surface methodology (RSM) and machine learning models. Degradation of BADGE was analyzed by various techniques, including UV-Vis spectrophotometry, high-performance liquid chromatography (HPLC), Fourier transform infrared (FTIR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS). Laccase from Geobacillus stearothermophilus strain MB600 achieved a degradation rate of 93.28% within 30 min, while laccase from Geobacillus thermoparafinivorans strain MB606 reached 94% degradation within 90 min. RSM analysis predicted the optimal degradation conditions to be 60 min reaction time, 80°C temperature, and pH 4.5. Furthermore, CB-Dock simulations revealed good binding interactions between laccase enzymes and BADGE, with an initial binding mode selected for a cavity size of 263 and a Vina score of -5.5, which confirmed the observed biodegradation potential of laccase. These findings highlight the biocatalytic potential of laccases derived from thermophilic Geobacillus strains, notably MB600, for enzymatic decontamination of BADGE-contaminated environments., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: Although Geobacillus are significant thermophilic bacteria source, there are no reports of thermostable esterase gene in Geobacillus jurassicus or rational design strategies to increase the thermal stability of esterases., Results: Gene gju768 showed a highest similarity of 15.20% to esterases from Geobacillus sp. with detail enzymatic properties. Using a combination of Gibbs Unfolding Free Energy (∆∆G) calculator and the distance from the mutation site to the catalytic site (Ds Cα-Cα ) to screen suitable mutation sites with elimination of negative surface charge, the mutants (D24N, E221Q, and E253Q) displayed stable mutants with higher thermal stability than the wild-type (WT). Mutant E253Q exhibited the best thermal stability, with a half-life (T 1/2 ) at 65 °C of 32.4 min, which was 1.8-fold of the WT (17.9 min)., Conclusion: Cloning of gene gju768 and rational design based on surface charge engineering contributed to the identification of thermostable esterase from Geobacillus sp. and the exploration of evolutionary strategies for thermal stability., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The current research in the food industry regarding enzymatic modification to enhance the freeze-thaw (FT) stability of starch is limited. The present study aimed to investigate the FT stability of normal corn starch (NCS) modified using 1,4-α-glucan branching enzyme (GBE) derived from Geobacillus thermoglucosidans STB02. Comprehensive analyses, including syneresis, scanning electron microscopy, and low-field nuclear magnetic resonance, collectively demonstrated the enhanced FT stability of GBE-modified corn starch (GT-NCS-30) in comparison to its native form. Its syneresis was 66.4 % lower than that of NCS after three FT cycles. Notably, GBE treatment induced changes in the pasting properties and thermal resistance of corn starch, while simultaneously enhancing the mechanical strength of the starch gel. Moreover, X-ray diffractograms and microstructural assessments of freeze-thawed gels indicated that GBE treatment effectively hindered the association of corn starch molecules, particularly amylose retrogradation. The enhanced FT stability of GBE-modified starch can be attributed to alterations in the starch structure induced by GBE. This investigation establishes a foundation for further exploration into the influence of GBE treatment on the FT stability of starch and provides a theoretical basis for further research in this area., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Investigation of a novel thermophilic esterase gene from Geobacillus subterraneus DSMZ 13552 indicated a high amino acid sequence similarity of 25.9% to a reported esterase from Geobacillus sp. A strategy that integrated computer-aided rational design tools was developed to select mutation sites. Six mutants were selected from four criteria based on the simulated saturation mutation (including 19 amino acid residues) results. Of these, the mutants Q78Y and G119A were found to retain 87% and 27% activity after incubation at 70 °C for 20 min, compared with the 19% activity for the wild type. Subsequently, a double-point mutant (Q78Y/G119A) was obtained and identified with optimal temperature increase from 65 to 70 °C and a 41.51% decrease in K m . The obtained T 1/2 values of 42.2 min (70 °C) and 16.9 min (75 °C) for Q78Y/G119A showed increases of 340% and 412% compared with that in the wild type. Q78Y/G119A was then employed as a biocatalyst to synthesize cinnamyl acetate, for which the conversion rate reached 99.40% with 0.3 M cinnamyl alcohol at 60 °C. The results validated the enhanced enzymatic properties of the mutant and indicated better prospects for industrial application as compared to that in the wild type. This study reported a method by which an enzyme could evolve to achieve enhanced thermostability, thereby increasing its potential for industrial applications, which could also be expanded to other esterases., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Challenges in enzyme and product recovery are currently intriguing in modern biotechnology. Coping enzyme stability, shelf life and efficiency, nanomaterials-based immobilization were epitomized of industrial practice. Herein, a α-amylase from Geobacillus thermoleovorans was purified and bound effectively on to a modified 3-Aminopropyltriethoxysilane (APTES)-Fe 3 O 4 nanoparticle. It was revealed that the carrier-bound enzyme catalysis (pH 8 and 60 °C) was significant in contrast to the free enzyme (pH 7.5 and 55 °C). Furthermore, Zn 2+ and Cu 2+ were shown to cause inhibitory effects in both enzyme states. Unlike chloroform, toluene, benzene, and butanol, minimal effects were observed with ethanol, acetone, and hexane. The bound enzyme retained 27.4 % of its initial activity after being stored for 36 days. In addition, the reusability of the bound enzyme showed a gradual decline in activity after the first cycle; however, after 13 cycles, its residual activity at 53 % was observed. These data proved significant enough to use this enzyme for industrial starch and analogous substrate bio-processing., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gurumurthy DM reports administrative support, equipment, drugs, or supplies, travel, and writing assistance were provided by Amity University Noida. Neelagund SE reports a relationship with Kuvempu University that includes: non-financial support. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bacterial 1,4-α-glucan branching enzymes (GBEs) provide a viable strategy for glycosidic bond rearrangement in starch and regulation of its digestion rate. However, the exponential increase in paste viscosity during starch gelatinization has a detrimental effect on the catalytic action of GBEs, thereby limiting productivity and product performance. Here, we designed an enzymatic treatment on corn starch granules by the GBE from Rhodothermus obamensis STB05 (Ro-GBE) prior to the glycosidic bond rearrangement of gelatinized starch catalyzed using the GBE from Geobacillus thermoglucosidans STB02 (Gt-GBE). Specifically, a moderate amount of Ro-GBE was required for the pretreatment stage. The dual GBE modification process enabled the treatment of more concentrated starch slurry (up to 20%, w/w) and effectively reduced starch digestibility. The resulting product contained a rapidly digestible starch fraction of 66.0%, which was 11.4% lower than that observed in the single Gt-GBE-modified product. The mechanistic investigation showed that the Ro-GBE treatment promoted swelling and gelatinization of starch granules, reduced starch paste viscosity, and increased the mobility of water molecules in the starch paste. It also created a preferable substrate for Gt-GBE. These changes improved the transglycosylation efficiency of Gt-GBE. These findings provide useful guidance for designing an efficient process to regulate starch digestibility.

Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of molecular hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis). Yet, a genetic system for these model microbes was missing despite intensive work for four decades. Here, we report the successful implementation of genetic tools for Methanothermobacter thermautotrophicus ΔH. We developed shuttle vectors that replicated in Escherichia coli and M. thermautotrophicus ΔH. For M. thermautotrophicus ΔH, a thermostable neomycin resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The shuttle-vector DNA was transferred from E. coli into M. thermautotrophicus ΔH via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus ΔH, we demonstrated heterologous gene expression of a thermostable β-galactosidase-encoding gene ( bgaB ) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assay, we found significantly different β-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus ΔH on formate as the sole growth substrate, while this was not possible for the empty-vector control. IMPORTANCE The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus , we set the cornerstone to engineer the microbe for optimized methane production but also for production of high-value platform chemicals in power-to-x processes.

Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH 4 ) 2 SO 4 (40% w / v ) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U 10 (5 6 ) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.

Thiamine diphosphate-dependent enzymes, and specifically transketolases, form one of the most important families of biocatalytic tools for enantioselective carbon-carbon bond formation yielding various hydroxyketones of biological interest. To enable substrate profiling of transketolases for acceptance of different donors and acceptors, a simple, direct colorimetric assay based on pH reaction variation was developed to establish a high-throughput solid-phase assay. This assay reduces the screening effort in the directed evolution of transketolases, as only active variants are selected for further analysis. Transketolase activity is detected as bicarbonate anions released from the α-ketoacid donor substrate, which causes the pH to rise. A pH indicator, bromothymol blue, which changes color from yellow to blue in alkaline conditions, was used to directly detect, with the naked eye, clones expressing active transketolase variants, obviating enzyme extraction., (© 2021 Wiley-VCH GmbH.)

Cytochrome bd oxidase is a bacterial terminal oxygen reductase that was suggested to enable adaptation to different environments and to confer resistance to stress conditions. An electrocatalytic study of the cyt bd oxidases from Escherichia coli, Corynebacterium glutamicum and Geobacillus thermodenitrificans gives evidence for a different reactivity towards oxygen. An inversion of the redox potential values of the three hemes is found when comparing the enzymes from different bacteria. This inversion can be correlated with different protonated glutamic acids as evidenced by reaction induced FTIR spectroscopy. The influence of the microenvironment of the hemes on the reactivity towards oxygen is discussed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We report the initial characterization of the α-ribazole (α-R) kinase enzyme of Geobacillus kaustophilus ( Gk CblS), which converts α-R to α-R-phosphate (α-RP) during the synthesis of cobamides. We implemented a continuous spectrophotometric assay to obtain kinetic parameters for several potential substrates and to study the specificity of the enzyme for α-N-linked ribosides. The apparent K m values for α-R and ATP were 358 and 297 μM, respectively. We also report methods for synthesizing and quantifying non-commercially available α-ribosides and β-ribazole (β-R). Purified Gk CblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). Gk CblS did not phosphorylate β-N-linked glycosides like β-adenosine or β-R. Expression of G. kaustophilus cblS + in a Salmonella enterica subsp. enterica sv Typhimurium LT2 ( S. enterica ) strain lacking the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyl transferase (CobT) enzyme resulted in the activation of various benzimidazole α-ribosides, and the synthesis of benzimidazolyl cobamides to levels that supported robust growth. Notably, α-Ado did not support growth under similar conditions, in spite of the fact that Gk CblS phosphorylated α-Ado in vitro . When α-Ado was provided at a very high concentration, growth was observed. This result suggested that in S. enterica α-Ado transport may be inefficient. We conclude that Gk CblS has specificity for α-N-glycosidic bonds, but not for the base in α-ribosides.

Lipases are versatile biocatalysts with many biotechnological applications and the necessity of screening, production and characterization of new lipases from diverse microbial strains to meet industrial needs is constantly emerging. In this study, the lipase gene (gklip) from a thermophilic bacterium, Geobacillus kaustophilus DSM 7263  T was cloned into the pET28a ( +) vector with N-terminal 6xHis-tag. The recombinant gklip gene was heterologously expressed in host E. coli BL21 (DE3) cells and purified by Ni-NTA affinity chromatography. Histidine tag was removed from the purified 6xHistag-Gklip enzyme with thrombin enzyme and the molecular mass was determined to be approximately 43 kDa by SDS-PAGE. Gklip showed optimal activity at pH 8.0 and 50 °C. The specific hydrolytic activities against substrates were significantly increased by the removal of the His-tag. K m and k cat values of Gklip against p-nitrophenyl palmitate (pNPP, 4-nitrophenyl palmitate) as the target substrate were found to be as 1.22 mM and 417.1 min -1 , respectively. Removing His-tag changed the substrate preference of the enzyme leading to maximum lipolytic activity towards C10 and C12 lipids. Similarly, the activity against coconut oil that containing 62% medium-chain fatty acids was significantly higher than other oils. Furthermore, preservation of activity in the presence of inhibitors, organic solvents support the effect of lid structure of the enzyme.

5M mutant lipase was derived through cumulative mutagenesis of amino acid residues (D43E/T118N/E226D/E250L/N304E) of T1 lipase from Geobacillus zalihae. A previous study revealed that cumulative mutations in 5M mutant lipase resulted in decreased thermostability compared to wild-type T1 lipase. Multiple amino acids substitution might cause structural destabilization due to negative cooperation. Hence, the three-dimensional structure of 5M mutant lipase was elucidated to determine the evolution in structural elements caused by amino acids substitution. A suitable crystal for X-ray diffraction was obtained from an optimized formulation containing 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.4 and 0.2 M sodium chloride with 2.5 mg/mL protein concentration. The three-dimensional structure of 5M mutant lipase was solved at 2.64 Å with two molecules per asymmetric unit. The detailed analysis of the structure revealed that there was a decrease in the number of molecular interactions, including hydrogen bonds and ion interactions, which are important in maintaining the stability of lipase. This study facilitates understanding of and highlights the importance of hydrogen bonds and ion interactions towards protein stability. Substrate specificity and docking analysis on the open structure of 5M mutant lipase revealed changes in substrate preference. The molecular dynamics simulation of 5M-substrates complexes validated the substrate preference of 5M lipase towards long-chain p-nitrophenyl-esters., Competing Interests: The authors have declared that no competing interests exist.

Superoxide dismutases are the enzymes involved in dismutation of superoxide radicals into oxygen and hydrogen peroxide. The present work reports a thermostable Fe/Mn SOD of Geobacillus sp. strain PCH100 (GsSOD) isolated from glacial soil. Purified recombinant GsSOD is a dimeric protein of ~57 kDa that exhibited highest activity at a temperature of 10 °C and pH of 7.8. Maximum enzyme velocity and Michaelis constant of the GsSOD were 1098.90 units/mg and 0.62 μM, respectively. At 80 °C, thermal inactivation rate constant and half-life of GsSOD were 3.33 × 10 -3  min -1 and 208 min, respectively. Interestingly, GsSOD tolerated a temperature of 100 °C and 130 °C up to 15 min and 5 min, respectively. Circular dichroism and differential scanning calorimetry confirmed thermostable nature of GsSOD. Apoenzyme of GsSOD regained enzymatic activity in the presence of Fe 2+ and Mn 2+ as metal ion cofactors. GsSOD was stable under varying concentrations of chemicals, namely ethylenediaminetetraacetic acid, potassium cyanide, hydrogen peroxide, chloroform-ethanol, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, Tween-20, Triton X-100, urea, and guanidine hydrochloride. The enzyme exhibited >70% activity in presence of 10 mM metal ions. Owing to its thermostable nature and resistance to chemical inhibitors, GsSOD is a potential enzyme for industrial applications., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Proteases account for approximately 60% of the enzyme market in the world, and they are used in various industrial applications including the detergent industry. In this study, production and characterization of a novel serine protease of thermophilic Geobacillus sp. GS53 from Balçova geothermal region, İzmir, Turkey, were performed. The thermostable protease was purified through ammonium sulfate precipitation and anion-exchange chromatography. The results showed that the protease had 137.8 U mg -1 of specific activity and optimally worked at 55 o C and pH 8. It was also active in a broad pH (4-10) and temperature (25-75 °C) ranges. The protease was highly stable at 85 °C and demonstrated relative stability at pH 4, 7, and 10. Also, the enzyme had high stability against organic solvents and surfactants; enzyme relative activity did not decrease below 81% upon preincubation for 10 min. Ca 2+ , Cu 2+ , and Zn 2+ ions slightly induced protease activity. The protease was highly specific to casein, skim milk, Hammerstein casein, and BSA substrates. These results revealed that the protease might have a potential effect in a variety of industrial fields, especially the detergent industry, because of its high thermostability and stability to surfactants.

Cardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of mitochondria. CL can increase the activity of integral membrane proteins, in particular components of respiratory pathways. We here report that CL activated detergent-solubilized cytochrome bd, a terminal oxidase from Escherichia coli. CL enhanced the oxygen consumption activity ~ twofold and decreased the apparent K M value for ubiquinol-1 as substrate from 95 µM to 35 µM. Activation by CL was also observed for cytochrome bd from two Gram-positive species, Geobacillus thermodenitrificans and Corynebacterium glutamicum, and for cytochrome bo 3 from E. coli. Taken together, CL can enhance the activity of detergent-solubilized cytochrome bd and cytochrome bo 3 .

Carbonic anhydrases (CA) are the most ubiquitous ancient zinc metalloenzymes known. Here we report the structural and functional analysis of a hypothetical protein GK2848 from Geobacillus kaustophilus. The analysis revealed that it belongs to the γ-class of CA (termed as Cag). Only a limited number of γ-class CA's have been characterized till date. Interestingly Cag contains magnesium at its active site instead of a traditional zinc ion. Based on the structural and sequence comparison with similar γ-CA's the putative active site residues of Cag were identified. This analysis revealed that an important catalytic residue and a proton shuttle residue (Glu62 and Glu84 respectively) of Cam (previously characterized γ-CA from Methanosarcina thermophila) are absent in Cag, however certain other active site residues are conserved both in Cag and Cam. This suggests that Cag uses a different set of residues for the reversible hydration of CO 2 to HCO 3 - when compared with Cam. Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES) and 25 Mg and 67 Zn NMR studies on Cag and its mutants revealed that either Mg or Zn can occupy the active site which suggests the cambialistic nature of the enzyme., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Different Pd-complexes containing orthometallated push-pull oxazolones were inserted by supramolecular Pd-amino acid coordination on two genetically engineered modified variants of the thermoalkalophilic Geobacillus thermocatenolatus lipase (GTL). Pd-lipase conjugation was performed on the solid phase in the previously immobilized form of GTL under mild conditions, and soluble conjugated Pd-GTL complexes were obtained by simply desorbing by washing with an acetonitrile aqueous solution. Three different Pd complexes were incorporated into two different genetically modified enzyme variants, one containing all the natural cysteine residues changed to serine residues, and another variant including an additional Cys mutation directly in the catalytic serine (Ser114Cys). The new Pd-enzyme conjugates were fluorescent even at ppm concentrations, while under the same conditions free Pd complexes did not show fluorescence at all. The Pd conjugation with the enzyme extremely increases the catalytic profile of the corresponding Pd complex from 200 to almost 1000-fold in the hydrogenation of arenes in aqueous media, achieving in the case of GTL conjugated with orthopalladated 4a an outstanding TOF value of 27 428 min -1 . Also the applicability of GTL-C114 conjugated with orthopalladated 4b in a site-selective C-H activation reaction under mild conditions has been demonstrated. Therefore, the Pd incorporation into the enzyme produces a highly stable conjugate, and improves remarkably the catalytic activity and selectivity, as well as the fluorescence intensity, of the Pd complexes.

Two putative genes, lip29 and est29 , encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli . The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDSPAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p -nitrophenyl palmitate as a substrate was observed at 50°C and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and in silico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.

In this study lipolytic biocatalysts GD-95RM, GDEst-95 and GDEst-lip were immobilized by encapsulation in calcium alginate beads . All three immobilized biocatalysts demonstrated significantly increased thermal stability at 60-70 °C temperatures and the activity of GD-95RM lipase increased by 50% at 70-80 °C following the immobilization. Moreover, encapsulated GDEst-95 esterase retained higher than 50% lipolytic activity after 3 months of incubation with butanol (25%) and ethanol (50%); GDEst-lip enzyme possessed 50% activity after 2 months of treatment with ethanol (25%) and methanol (25%); and GD-95RM lipase displayed higher that 50% activity after two-week incubation with methanol (50%). All three immobilized enzymes displayed long-term storage capability (>50% activity) at least until 3 months at 4 °C. It was also detected that immobilized GD-95RM and GDEst-lip can perform flow hydrolysis of both avocado oil and p-NP dodecanoate in prototype packed-bed column reactor. The analysis of continuous transesterification of avocado or sunflower oil with ethanol or methanol as substrates confirmed that encapsulated GD-95RM and GDEst-lip enzymes is a useful approach to produce fatty acid alkyl esters., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, the most effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLip-lip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Luo Han Guo fruit extract ( Siraitia grosvenorii ), mainly composed of mogroside V (50%), could be considered a suitable alternative to free sugars; however, its commercial applications are limited by its unpleasant off-notes. In the present work, a central composite design method was employed to optimize the transglycosylation of a mogroside extract using cyclodextrin glucosyltransferases (CGTases) from three different bacteriological sources ( Paenibacillus macerans , Geobacillus sp., and Thermoanaerobacter sp.) considering various experimental parameters such as maltodextrin and mogroside concentration, temperature, time of reaction, enzymatic activity, and pH. Product structures were determined by liquid chromatography coupled to a diode-array detector (LC-DAD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sensory analysis of glucosylated mogrosides showed an improvement in flavor attributes relevant to licorice flavor and aftereffect. Consequently, an optimum methodology was developed to produce new modified mogrosides more suitable when formulating food products as free sugar substitutes.

A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD 600 ) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD 600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD 600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl 2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this study, the heterologous expression and biochemical characterization of a thermostable α-amylase from Geobacillus sp. GS33 was investigated. The recombinant α-amylase was overexpressed in Escherichia coli BL21 (λDE) and purified via anion exchange and size-exclusion chromatography. The purified α-amylase had a molecular weight of about 60 kDa, and was active in a broad range of pH 3-10 and temperature (40-90 °C) with maximum activity at pH 7-8 and 60 °C. The enzyme retained 50% residual activity at 65 °C, but only 20% at 85 °C after 16 h. At pH 9 and pH 7, the residual activity at 65 °C was 50% and 30%, respectively. The enzyme was remarkably activated by Co 2+ , Ca 2+ , Mg 2+ , PMSF, DTT, and Triton X-100, but partially inhibited by Cu 2+ , methanol, hexane, ethanol, acetone, SDS, and Tween 20. A molecular phylogeny analysis showed that the enzyme's amino acid sequence had the closest connection with an α-amylase from Geobacillus thermoleovorans subsp. stromboliensis nov. 3D-structure-based amino acid sequence alignments revealed that the three catalytic residues (D217, E246, D314) and the four Ca 2+ ion coordination residues (N143, E177, D186, H221) were conserved in α-amylase from Geobacillus sp. GS33. The temperature stability and neutral pH optimum suggest that the enzyme may be useful for industrial applications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aspartate aminotransferase catalyzes the transfer of an amino group from l-aspartate to α-oxoglutarate. A gene encoding aspartate aminotransferase, AST Gt , from Geobacillus thermopakistaniensis was cloned and expressed in Escherichia coli. The purified recombinant AST Gt exhibited highest activity at 65 °C and pH 7.0. The activity was dependent on pyridoxal phosphate but not on any metal ions. Stoichiometry of purified AST Gt demonstrated that 0.1 pyridoxal phosphate was attached per subunit of the enzyme. Determination of molecular weight by gel filtration chromatography indicated that AST Gt existed in a dimeric form in solution. Thermostability experiments showed no significant change in activity even after 16 h incubation at 65 °C. AST Gt exhibited apparent V max and K m values of 120 μmol min -1 mg -1 and 1.5 mM, respectively, against l-aspartate. Substrate specificity experiments indicated the highest relative activity against aspartate (100%) followed by tyrosine (27%) and proline (16%). To the best of our knowledge, this is the first report on cloning and characterization of an AST from genus Geobacillus., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Spore photoproduct lyase is a radical S -adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S] 1+ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S-C5' bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5' of the 5'-deoxyadenosyl radical (5'-dAdo • ). During catalysis, homolytic cleavage of the Fe-C5' bond liberates 5'-dAdo • for reaction with substrate, but here, we use Ω formation without substrate to determine the thermal stability of Ω. The reaction of Geobacillus thermodenitrificans SPL ( Gt SPL) with SAM forms Ω within ∼15 ms after mixing. By monitoring the decay of Ω through rapid freeze-quench trapping at progressively longer times we find an ambient temperature decay time of the Ω Fe-C5' bond of τ ≈ 5-6 s, likely shortened by enzymatic activation as is the case with the Co-C5' bond of B 12 . We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5'-dAdo • radical liberated by Ω. In the absence of substrate, Ω undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val • ; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile • or Cys • radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.

With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant Bacillus caldovelox arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. With the expressions of ASS and ASL genes conferring resistance to L-arginine deiminase (ADI) which is undergoing Phase III clinical trial, BCA-M offers the advantage of a broader spectrum of susceptible cancer cells. Mechanistic studies showed that BCA-M inhibited the growth of human cervical cancer cells by inducing apoptosis and cell cycle arrest at S and/or G 2 /M phases. Our results also displayed that autophagy served as a protective mechanism, while the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination to the autophagy inhibitor, chloroquine (CQ), on human cervical cancer cells.

The genus Geobacillus , belonging to the phylum Firmicutes, is one of the most important genera and comprises thermophilic bacteria. The genus Geobacillus was erected with the taxonomic reclassification of various Bacillus species. Taxonomic studies of Geobacillus remain in progress. However, there is no comprehensive review of the characteristic features, taxonomic status and study of various applications of this interesting genus. The main aim of this review is to give a comprehensive account of the genus Geobacillus . At present the genus acomprises 25 taxa, 14 validly published (with correct name), nine validly published (with synonyms) and two not validly published species. We describe only validly published species of the genera Geobacillus and Parageobacillus . Vegetative cells of Geobacillus species are Gram-strain-positive or -variable, rod-shaped, motile, endospore-forming, aerobic or facultatively anaerobic, obligately thermophilic and chemo-organotrophic. Growth occurs in the pH range 6.08.5 and a temperature of 37-75 °C. The major cellular fatty acids are iso-C15:o, iso-C16:0 and iso-C17:o. The main menaquinone type is MK-7. The G-+C content of the DNA ranges between 48.2 and 58 mol%. The genus Geobacillus is widely distributed in nature, being mostly found in many extreme locations such as hot springs, hydrothermal vents, marine trenches, hay composts, etc. Geobacillus species have been widely exploited in various industrial and biotechnological applications, and thus are promising candidates for further studies in the future.

A putative laccase gene (lacG) from Geobacillus sp. JS12 was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli BL21 (DE3) cells, and the protein was primarily found in inclusion bodies. The resulting insoluble proteins were solubilized with 6 M guanidine HCl and refolded using an on-column refolding procedure. Ni-chelation affinity chromatography found the laccase to be a 30 kDa monomeric protein. Spectrophotometry and electron paramagnetic resonance (EPR) analysis indicated LacG as a multi-copper oxidase, with the usual laccase copper sites, Type 1, 2, and 3 Cu(II). The optimum pH for enzymatic activity was 3.0, 6.0, and 6.5 with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The recombinant protein displayed high thermostability, with a heat inactivation half-life of approximately 2 h at 95 °C, and an optimum temperature of 80 °C with 2,6-DMP. Catalytic efficiency (k cat /K m ) showed that guaiacol and 2,6-DMP were highly oxidized by the enzyme. The enzymatic reaction was significantly enhanced by Co 2+ and Mn 2+ , while activity was strongly inhibited in the presence of Fe 2+ , Zn 2+ , and thiol compounds. LacG decolorized 43% of Congo red and 14% of Malachite green, and the addition of ABTS as a redox mediator dramatically increased the dye decolorization efficiency., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pullulanase type I (PulA) is a debranching enzyme that specifically cleaves α-1,6-glycosidic linkages in pullulan. Pullulan has not only diverse applications in food industry but also has immune-stimulatory effects on B and T cells, and found to enhance the production of various anti-inflammatory cytokines in human. Moreover, pullulan has been suggested as a possible anti-cancer drug delivery agent without adjuvant due to its unique structure. The process of pullulan degradation is unresolved due to imprecise pullulanase structural characteristics. Therefore, the present study aimed to understand the structural and functional characteristics of pullulanase enzyme from Geobacillus thermopakistaniensis MAS1 strain using various computational approaches. The physio-chemical topographies and secondary structure of GT&#95;PulA were explored using ProPram, InterPro and SMART. Various tools like I-TASSER, ModRefiner, RAMPAGE, PROCHECK and MOE 2009.10 were used to construct and verify the 3D structural model. The structural elucidation confirmed the significant domains, i.e., CBM48, CBM2, and TIM barrel having catalytically active residues, and conserved region YNGWDP. CBM2 domain along with TIM barrel has a capacity to bind different ligands and proved favorable for multiple substrate catalyses. These structural properties can have a potential effect on enhancing enzymatic activity of GT&#95;PulA enzyme.

A comparative structure analysis between space- and an Earth-grown T1 recombinant lipase from Geobacillus zalihae had shown changes in the formation of hydrogen bonds and ion-pair interactions. Using the space-grown T1 lipase validated structure having incorporated said interactions, the recombinant T1 lipase was re-engineered to determine the changes brought by these interactions to the structure and stability of lipase. To understand the effects of mutation on T1 recombinant lipase, five mutants were developed from the structure of space-grown T1 lipase and biochemically characterized. The results demonstrate an increase in melting temperature up to 77.4 °C and 76.0 °C in E226D and D43E, respectively. Moreover, the mutated lipases D43E and E226D had additional hydrogen bonds and ion-pair interactions in their structures due to the improvement of stability, as observed in a longer half-life and an increased melting temperature. The biophysical study revealed differences in β-Sheet percentage between less stable (T118N) and other mutants. As a conclusion, the comparative analysis of the tertiary structure and specific residues associated with ion-pair interactions and hydrogen bonds could be significant in revealing the thermostability of an enzyme with industrial importance.

The 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02 (GtGBE, EC 2.4.1.18) does not possess the thermostability required by modified starch industry. To increase its thermostability, a rational design strategy was used to introduce additional salt bridges into GtGBE. The strategy involved in mutation of individual residues to form "local" two-residue salt bridges. Accordingly, five of local salt bridges (Q231R-D227, Q231K-D227, T339E-K335, T339D-K335, and I571D-R569 mutants) were separately introduced into GtGBE. The half-times of these mutants at 60 °C were 17% to 51% longer than that of wild-type. Subsequently, these two-residue salt bridges were extended to form salt bridge networks (Q231R/K-D227-D131H, T339D/E-K335-I291H, and I571D-R569-R617H mutants). Among these mutants, except I571D-R569-R617H, the half-times of Q231R/K-D227-D131H, T339D/E-K335-I291H mutants at 60 °C were 15%, 17%, 21% and 17% longer than those of the corresponding two-residue salt bridges, respectively. The results showed that design and introduction of salt bridges improves enzyme thermostability in GtGBE., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A mannose-6-phosphate isomerase (MPI) from Geobacillus thermodenitrificans was expressed and successfully encapsulated into the Saccharomyces cerevisiae spores. Our results demonstrated that compared to the free enzyme, the MPI triple mutant encapsulated in osw2 Δ spores exhibited much preferred enzymatic properties, such as enhanced catalytic activity, excellent reusability, thermostability, and tolerance to various harsh conditions. In combination with an l-arabinose isomerase (AI) also from G. thermodenitrificans , this technique of spore encapsulation was applied for producing a high-value rare sugar l-ribose from biomass-derived l-arabinose. Using a 10 mL reaction system, 350 mg of l-ribose was produced from 1 g of l-arabinose with a conversion yield of 35% by repeatedly reacting with 200 mg of AI-encapsulated spores and 300 mg of MPI-encapsulated spores. This study provides a very useful and concise approach for the synthesis of rare sugars and other useful compounds.

L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser 161 ->Cys 161 ). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys 161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.

In this work, we present a generalizable directed computational evolution protocol to effectively reduce the sequence space to be explored in rational enzyme design. The protocol involves in silico mutation modeling and substrate docking to rapidly identify mutagenesis hotspots that may enhance an enzyme's substrate binding and overall catalysis. By applying this protocol to a quorum-quenching Geobacillus kaustophilus lactonase, GKL, we generated 1,881 single mutants and docked high-energy intermediates of nine acyl homoserine lactones onto them. We found that Phe28 and Tyr99 were two hotspots that produced most of the predicted top 20 mutants. Of the 180 enzyme-substrate combinations (top 20 mutants × 9 substrates), 51 (28%) exhibited enhanced substrate binding and 22 (12%) had better overall activity when compared with wild-type GKL. X-ray crystallographic studies of Y99C and Y99P provided rationalized explanations for the enhancement in enzyme function and corroborated the utility of the protocol., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In this study three lipases GD-28, GD-95 and GD-66 (all 43 kDa in size), isolated from Geobacillus spp. were subjected to directed evolution experiments to yield a new synthetic lipolytic enzyme. This new lipase, obtained by DNA shuffling and epPCR, was named GDlip43 (also 43 kDa in size). It demonstrated increased thermoactivity, thermostability, an ability to hydrolyze short and long acyl chain p-NP esters and was activated by different organic solvents. Different activity of GDlip43 raised the hypothesis of new candidate amino acids which could be important for the activity of Geobacillus lipases. Based on the sequence alignment of parental and GDlip43 lipase, three candidate amino acids were selected. The importance of these amino acids, localized at positions 153, 154 and 247 (all of which are distant from the catalytic center of Geobacillus lipases) was investigated using site-directed mutagenesis. Directed evolution experiments also yielded another new lipase - GDlip30 (30 kDa in size). This low molecular mass derivative of GDlip43 had clearly detectable lipolytic activity (40 U/mg) and is the smallest currently known active Geobacillus lipase variant., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bacillus thermocatenulatus lipase (BTL2), a member of the isolated lipase family known as thermoalkalophilic lipases, carries potential for industrial applications owing to its ability to catalyze versatile reactions under extreme conditions. This study investigates the molecular effects of distinct solvents on the stability of BTL2 at different temperatures, aiming to contribute to lipase use in industrial applications. Initially, molecular dynamic (MD) simulations were carried out to address for the molecular impacts of distinct solvents on the structural stability of BTL2 at different temperatures. Two lipase conformations representing the active and inactive forms were simulated in 5 solvents including water, ethanol, methanol, cyclohexane, and toluene. Low temperature simulations showed that polar solvents led to enhanced lid fluctuations compared with non-polar solvents reflecting a more dynamic equilibrium between active and inactive lipase conformations in polar solvents including water, while the overall structure of the lipase in both forms became more rigid in non-polar solvents than they were in polar solvent. Notably, the native lipase fold was maintained in non-polar solvents even at high temperatures, indicating an enhancement of lipase's thermostability in non-polar organic solvents. Next, we conducted experiments for which BTL2 was expressed in a heterologous host and purified to homogeneity, and its thermostability in different solvents was assessed. Parallel to the computational findings, experimental results suggested that non-polar organic solvents contributed to BTL2's thermostability at concentrations as high as 70% (v/v). Altogether, this study provides beneficial insights to the lipase use under extreme conditions. Graphical Abstract.

Esterases are widely used in the food industry. Here, a new thermophilic bacterium, Geobacillus thermodenitrificans PS01, was isolated and the esterase-encoding gene est1 was cloned, sequenced, and recombinant expressed in Escherichia coli Tuner (DE3). The highest activity of recombinant Est1 was detected at pH 8.0, and 40 °C and the extreme stability was observed at pH 6-9 over 30 days at 4 °C. In particular, Est1 can hydrolyze short- to medium-chain (C2-C10) triglycerides and p-nitrophenyl esters (C2-C12) and was not inhibited by most metal ions. Kinetic parameters of p-nitrophenyl butyrate hydrolysis under optimal conditions were determined: K m , 22.76 μM; k cat , 10,415 s -1 ; and k cat /K m , 457.53 μM -1  s -1 . The outstanding specification of Est1 indicates its potential for use in industrial applications.

Enrichment of omega-3 fatty acids (ɷ-3 FAs) in natural oils is important to realize their health benefits. Lipases are promising catalysts to perform this enrichment, however, fatty acid specificity of lipases is poor. We attempted to improve the fatty acid selectivity of a lipase from Geobacillus thermoleovorans (GTL) by two approaches. In a semi-rational approach, amino acid positions critical for binding were identified by docking the substrate to the GTL and best substitutes at these positions were identified by site saturation mutagenesis followed by screening to obtain a variant of GTL (CM-GTL). In the second approach based on rational design, a variant of GTL was designed (DM-GTL) wherein the active site was narrowed by incorporating two heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky ɷ-3 FAs. The affinities DM-GTL with designed substrates were evaluated in silico. Both, CM-GTL and DM-GTL have shown excellent ability to discriminate against the ɷ-3 FAs during hydrolysis of oils. Engineering the binding pocket of an enzyme of a complex substrate, such as a triglyceride, by incorporating the information on substrate structure and computationally derived binding modes, has resulted in designing two efficient lipase variants with improved substrate selectivity., Competing Interests: The authors have declared that no competing interests exist.

By sequence comparison, the majority of 1,4-α-glucan-branching enzymes (GBEs) consist of an N-terminal carbohydrate-binding domain, a TIM-barrel catalytic domain, and a C-terminal all-beta domain. Among these structures, the GBE from Geobacillus thermoglucosidans STB02 uniquely has a highly charged 26-amino-acid C-terminal extension, whose functional roles are the least understood. In this research, the functional significance of the C-terminal domain in GBE from G. thermoglucosidans STB02 and its extension were assessed using a C-terminal deletion analysis. Mutants lacking of more than 7 residues of the C-terminal all-beta domain could not be detected in lysates of their Escherichia coli expression strains, suggesting that an intact all-beta domain is required for structural stability. In contrast, truncation of the C-terminal extension resulted in greater stability and solubility than the wild type, as well as a lower sensitivity to the presence of added metal ions. Comparison of this mutant with the wild type suggests that the interaction of metal ions with the C-terminal extension influences performance of this enzyme.

The enzyme Urocanate Hydratase (UH) participates in the catabolic pathway of L-histidine. Trypanosoma cruzi Urocanate Hydratase (TcUH) is identified as a therapeutic molecular target in the WHO/TDR Targets Database. We report the 3D structure determination and number of features of TcUH, and compared it to other few available bacterial UH structures. Each monomer presents two domains and one NAD + molecule. Superpositions revealed differences in the relative orientation of domains within monomers, such that TcUH monomer A resembles Urocanate Hydratase from Geobacillus kaustophilus (GkUH) (open conformation), while monomer C resembles Urocanate Hydratase from Pseudomonas putida (PpUH) and Urocanate Hydratase from Bacillus subtilis (BsUH) (closed conformations). We use the structure of TcUH to make considerations about 3 non-deleterious and 2 deleterious mutations found in human UHs: non-deleterious mutations could be accommodated without large displacements or interaction interruptions, whereas deleterious mutations in one case might disrupt an α-helix (as previously suggested) and in the other case, besides disrupting the enzyme interaction with the substrate, might interfere with interdomain movement., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2019. Published by Elsevier B.V.)