Efficient pathway-driven scyllo-inositol production from myo-inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control.

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.

A novel univariate interpolation and bivariate regression hybrid method application to biodegradation of bisphenol A diglycidyl ether using laccases from Geobacillus stearothermophilus and Geobacillus thermoparafinivorans strains.

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.

Functional and structural characterization of a thermostable flavin reductase from Geobacillus mahadii Geo-05.

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.

Recombinant Expression and Characterization of a Novel Thermo-Alkaline Lipase with Increased Solvent Stability from the Antarctic Thermophilic Bacterium Geobacillus sp. ID17.

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 (kcat/Km) 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.

Optimization and Characterization of an Ultra-Thermostable, Acidophilic, Cellulase-Free Xylanase from a New Obligate Thermophilic Geobacillus thermoleovorans AKNT10 and its Application in Saccharification of Wheat Bran.

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.

Artificial Manganese Metalloenzymes with Laccase-like Activity: Design, Synthesis, and Characterization.

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.

Propitious catalytic response of immobilized α-amylase from G. thermoleovorans in modified APTES-Fe3O4 NPs for industrial bio-processing.

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)-Fe3O4 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, Zn2+ and Cu2+ 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.

Enzymatic modification lowers syneresis in corn starch gels during freeze-thaw cycles through 1,4-α-glucan branching enzyme.

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.

Computer-aided rational design strategy based on protein surface charge to improve the thermal stability of a novel esterase from Geobacillus jurassicus.

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 (DsCα-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 (T1/2) 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.

Designing a Specific Pretreatment on Corn Starch to Facilitate Enzymatic Rearrangement of Glycosidic Bonds for Efficiently Reducing Starch Digestibility.

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.

Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase.

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 (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) 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.

A Shuttle-Vector System Allows Heterologous Gene Expression in the Thermophilic Methanogen Methanothermobacter thermautotrophicus ΔH.

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 ß-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 ß-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.

High-Throughput Solid-Phase Assay for Substrate Profiling and Directed Evolution of Transketolase.

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.

Functional Studies of α-Riboside Activation by the α-Ribazole Kinase (CblS) from Geobacillus kaustophilus.

We report the initial characterization of the α-ribazole (α-R) kinase enzyme of Geobacillus kaustophilus (GkCblS), 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 Km 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 ß-ribazole (ß-R). Purified GkCblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). GkCblS did not phosphorylate ß-N-linked glycosides like ß-adenosine or ß-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 GkCblS 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 GkCblS has specificity for α-N-glycosidic bonds, but not for the base in α-ribosides.

Structure elucidation and docking analysis of 5M mutant of T1 lipase Geobacillus zalihae.

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.

Electrocatalytic evidence of the diversity of the oxygen reaction in the bacterial bd oxidase from different organisms.

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.

Identification and Heterologous Production of a Lipase from Geobacillus kaustophilus DSM 7263T and Tailoring Its N-Terminal by a His-Tag Epitope.

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. Km and kcat 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.

Pd-Oxazolone complexes conjugated to an engineered enzyme: improving fluorescence and catalytic properties.

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.

A thermostable Fe/Mn SOD of Geobacillus sp. PCH100 isolated from glacial soil of Indian trans-Himalaya exhibits activity in the presence of common inhibitors.

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 Fe2+ and Mn2+ 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.

Structural and functional characterization of a putative carbonic anhydrase from Geobacillus kaustophilus reveals its cambialistic function.

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 CO2 to HCO3- when compared with Cam. Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES) and 25Mg and 67Zn 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.