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.

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.

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.

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.

Structural and functional analyses of an L-asparaginase from Geobacillus thermopakistaniensis.

Genome sequence of Geobacillus thermopakistaniensis contains an open reading frame annotated as a type II L-asparaginase (ASNaseGt). Critical structural analysis disclosed that ASNaseGt might be a type I L-asparaginase. In order to determine whether it is a type I or type II L-asparaginase, we have performed the structural-functional characterization of the recombinant protein as well as analyzed the localization of ASNaseGt in G. thermopakistaniensis. ASNaseGt exhibited optimal activity at 52 °C and pH 9.5. There was a > 3-fold increase in activity in the presence of ß-mercaptoethanol. Apparent Vmax and Km values were 2735 U/mg and 0.35 mM, respectively. ASNaseGt displayed high thermostability with >80 % residual activity even after 6 h of incubation at 55 °C. Recombinant ASNaseGt existed in oligomeric form. Addition of ß-mercaptoethanol lowered the degree of oligomerization and displayed that tetrameric form was the most active, with a specific activity of 4300 U/mg. Under physiological conditions, ASNaseGt displayed >50 % of the optimal activity. Localization studies in G. thermopakistaniensis revealed that ASNaseGt is a cytosolic protein. Structural and functional characterization, and localization in G. thermopakistaniensis displayed that ASNaseGt is not a type II but a type I L-asparaginase.

A novel alkane monooxygenase evolved from a broken piece of ribonucleotide reductase in Geobacillus kaustophilus HTA426 isolated from Mariana Trench.

We have accidentally found that a thermophilic Geobacillus kaustophilus HTA426 is capable of degrading alkanes although it has no alkane oxygenating enzyme genes. Our experimental results revealed that a putative ribonucleotide reductase small subunit GkR2loxI (GK2771) gene encodes a novel heterodinuclear Mn-Fe alkane monooxygenase/hydroxylase. GkR2loxI protein can perform two-electron oxidations similar to homonuclear diiron bacterial multicomponent soluble methane monooxygenases. This finding not only answers a long-standing question about the substrate of the R2lox protein clade, but also expands our understanding of the vast diversity and new evolutionary lineage of the bacterial alkane monooxygenase/hydroxylase family.

Similarities of Geobacillus bacteria based on their profiles of antimicrobial susceptibility in milk samples.

The genus Geobacillus is composed of thermophilic bacteria that exhibit diverse biotechnological potentialities. Specifically, Geobacillus stearothermophilus is included as a test bacterium in commercial microbiological inhibition methods, although it exhibits limited sensitivity to aminoglycosides, macrolides, and quinolones. Therefore, this article evaluates the antibiotic susceptibility profiles of five test bacteria (G. stearothermophilus subsp. calidolactis C953, Geobacillus thermocatenulatus LMG 19007, Geobacillus thermoleovorans LMG 9823, Geobacillus kaustophilus DSM 7263 and Geobacillus vulcani 13174). For that purpose, the minimum inhibitory concentrations (MICs) of 21 antibiotics were determined in milk samples for five test bacteria using the radial diffusion microbiological inhibition method. Subsequently, the similarities between bacteria and antibiotics were analyzed using cluster analysis. The dendrogram of this multivariate analysis shows an association between a group formed by G. thermocatenulatus and G. stearothermophilus and another by G. thermoleovorans, G. kaustophilus and G. vulcani. Finally, future microbiological methods could be developed in microtiter plates using G. thermocatenulatus as test bacterium, as it exhibits similar sensitivities to G. stearothermophilus. Conversely, G. vulcani, G. thermoleovorans and G. kaustophilus show higher MICs than G. thermocatenulatus.

Sequence identification and in silico characterization of novel thermophilic lipases from Geobacillus species.

Microbial lipases are utilized in various biotechnological areas, including pharmaceuticals, food, biodiesel, and detergents. In this study, we cloned and sequenced Lip21 and Lip33 genes from Geobacillus sp. GS21 and Geobacillus sp. GS33, then we in silico and experimentally analyzed the encoded lipases. For this purpose, Lip21 and Lip33 were cloned, sequenced, and their amino acid sequences were investigated for determination of biophysicochemical characteristics, evolutionary relationships, and sequence similarities. 3D models were built and computationally affirmed by various bioinformatics tools, and enzyme-ligand interactions were investigated by docking analysis using six ligands. Biophysicochemical property of Lip21 and Lip33 was also determined experimentally and the results demonstrated that they had similar isoelectric point (pI) (6.21) and Tm (75.5°C) values as Tm was revealed by denatured protein analysis of the circular dichroism spectrum and pI was obtained by isoelectric focusing. Phylogeny analysis indicated that Lip21 and Lip33 were the closest to lipases from Geobacillus sp. SBS-4S and Geobacillus thermoleovorans, respectively. Alignment analysis demonstrated that S144-D348-H389 was catalytic triad residues in Lip21 and Lip33, and enzymes possessed a conserved Gly-X-Ser-X-Gly motif containing catalytic serine. 3D structure analysis indicated that Lip21 and Lip33 highly resembled each other and they were α/ß hydrolase-fold enzymes with large lid domains. BANΔIT analysis results showed that Lip21 and Lip33 had higher thermal stability, compared to other thermostable Geobacillus lipases. Docking results revealed that Lip21- and Lip33-docked complexes possessed common residues (H112, K115, Q162, E163, and S141) that interacted with the substrates, except paranitrophenyl (pNP)-C10 and pNP-C12, indicating that these residues might have a significant action on medium and short-chain fatty acid esters. Thus, Lip21 and Lip33 can be potential candidates for different industrial applications.

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

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

Recombinant expression and characterization of a new laccase, bioinformatically identified, from the Antarctic thermophilic bacterium Geobacillus sp. ID17.

Geobacillus sp. ID17 is a gram-positive thermophilic bacterium isolated from Deception Island, Antarctica, which has shown to exhibit remarkable laccase activity in crude extract at high temperatures. A bioinformatic search using local databases led to the identification of three putative multicopper oxidase sequences in the genome of this microorganism. Sequence analysis revealed that one of those sequences contains the four-essential copper-binding sites present in other well characterized laccases. The gene encoding this sequence was cloned and overexpressed in Escherichia coli, partially purified and preliminary biochemically characterized. The resulting recombinant enzyme was recovered in active and soluble form, exhibiting optimum copper-dependent laccase activity at 55 °C, pH 6.5 with syringaldazine substrate, retaining over 60% of its activity after 1 h at 55 and 60 °C. In addition, this thermophilic enzyme is not affected by common inhibitors SDS, NaCl and L-cysteine. Furthermore, biodecolorization assays revealed that this laccase is capable of degrading 60% of malachite green, 54% of Congo red, and 52% of Remazol Brilliant Blue R, after 6 h at 55 °C with aid of ABTS as redox mediator. The observed properties of this enzyme and the relatively straightforward overexpression and partial purification of it could be of great interest for future biotechnology applications.

Anoxybacillus: an overview of a versatile genus with recent biotechnological applications.

The Bacillaceae family members are considered to be a good source of microbial factories for biotechnological processes. In contrast to Bacillus and Geobacillus, Anoxybacillus, which would be thermophilic and spore-forming group of bacteria, is a relatively new genus firstly proposed in the year of 2000. The development of thermostable microbial enzymes, waste management and bioremediation processes would be a crucial parameter in the industrial sectors. There has been increasing interest in Anoxybacillus strains for biotechnological applications. Therefore, various Anoxybacillus strains isolated from different habitats have been explored and identified for biotechnological and industrial purposes such as enzyme production, bioremediation and biodegradation of toxic compounds. Certain strains have ability to produce exopolysaccharides possessing biological activities including antimicrobial, antioxidant and anticancer. This current review provides past and recent discoveries regarding Anoxybacillus strains and their potential biotechnological applications in enzyme industry, environmental processes and medicine.

Recombinase polymerase amplification using novel thermostable strand-displacing DNA polymerases from Aeribacillus pallidus and Geobacillus zalihae.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), and strand-displacing DNA polymerase (Pol). Component instability and the need to store commercial kits in a deep freezer until use are some limitations of RPA. In a previous study, Bacillus stearothermophilus Pol (Bst-Pol) was used as a thermostable strand-displacing DNA polymerase in RPA. Here, we attempted to optimize the lyophilization conditions for RPA with newly isolated thermostable DNA polymerases for storage at room temperature. We isolated novel two thermostable strand-displacing DNA polymerases, one from a thermophilic bacterium Aeribacillus pallidus (H1) and the other from Geobacillus zalihae (C1), and evaluated their performances in RPA reaction. Urease subunit ß (UreB) DNA from Ureaplasma parvum serovar 3 was used as a model target for evaluation. The RPA reaction with H1-Pol or C1-Pol was performed at 41 °C with the in vitro synthesized standard UreB DNA. The minimal initial copy numbers of standard DNA from which the amplified products were observed were 600, 600, and 6000 copies for RPA with H1-Pol, C1-Pol, and Bst-Pol, respectively. Optimization was carried out using RPA components, showing that the lyophilized RPA reagents containing H1-Pol exhibited the same performance as the corresponding liquid RPA reagents. In addition, lyophilized RPA reagents with H1-Pol showed almost the same activity after two weeks of storage at room temperature as the freshly prepared liquid RPA reagents. These results suggest that lyophilized RPA reagents with H1-Pol are preferable to liquid RPA reagents for onsite use.

Structural and functional analyses of GH51 alpha-L-arabinofuranosidase of Geobacillus vulcani GS90 reveal crucial residues for catalytic activity and thermostability.

Alpha-L-arabinofuranosidase (Abf) is of big interest in various industrial areas. Directed evolution is a powerful strategy to identify significant residues underlying Abf properties. Here, six active variants from GH51 Abf of Geobacillus vulcani GS90 (GvAbf) by directed evolution were overproduced, extracted, and analyzed at biochemical and structural levels. According to the activity and thermostability results, the most-active and the least-active variants were found as GvAbf51 and GvAbf52, respectively. GvAbf63 variant was more active than parent GvAbf by 20% and less active than GvAbf51. Also, the highest thermostability belonged to GvAbf52 with 80% residual activity after 1 h. Comparative sequence and structure analyses revealed that GvAbf51 possessed L307S displacement. Thus, this study suggested that L307 residue may be critical for GvAbf activity. GvAbf63 had H30D, Q90H, and L307S displacements, and H30 was covalently bound to E29 catalytic residue. Thus, H30D may decrease the positive effect of L307S on GvAbf63 activity, preventing E29 action. Besides, GvAbf52 possessed S215N, L307S, H473P, and G476C substitutions and S215 was close to E175 (acid-base residue). S215N may partially disrupt E175 action. Overall effect of all substitutions in GvAbf52 may result in the formation of the C-C bond between C171 and C213 by becoming closer to each other.

Crystal Structure of 4,6-α-Glucanotransferase GtfC-ΔC from Thermophilic Geobacillus 12AMOR1: Starch Transglycosylation in Non-Permuted GH70 Enzymes.

GtfC-type 4,6-α-glucanotransferase (α-GT) enzymes from Glycoside Hydrolase Family 70 (GH70) are of interest for the modification of starch into low-glycemic index food ingredients. Compared to the related GH70 GtfB-type α-GTs, found exclusively in lactic acid bacteria (LAB), GtfCs occur in non-LAB, share low sequence identity, lack circular permutation of the catalytic domain, and feature a single-segment auxiliary domain IV and auxiliary C-terminal domains. Despite these differences, the first crystal structure of a GtfC, GbGtfC-ΔC from Geobacillus 12AMOR1, and the first one representing a non-permuted GH70 enzyme, reveals high structural similarity in the core domains with most GtfBs, featuring a similar tunneled active site. We propose that GtfC (and related GtfD) enzymes evolved from starch-degrading α-amylases from GH13 by acquiring α-1,6 transglycosylation capabilities, before the events that resulted in circular permutation of the catalytic domain observed in other GH70 enzymes (glucansucrases, GtfB-type α-GTs). AlphaFold modeling and sequence alignments suggest that the GbGtfC structure represents the GtfC subfamily, although it has a so far unique alternating α-1,4/α-1,6 product specificity, likely determined by residues near acceptor binding subsites +1/+2.

New Platform for Screening Genetic Libraries at Elevated Temperatures: Biological and Genomic Information and Genetic Tools of Geobacillus thermodenitrificans K1041.

Geobacillus thermodenitrificans K1041 is an unusual thermophile that is highly transformable via electroporation, making it a promising host for screening genetic libraries at elevated temperatures. In this study, we determined its biological properties, draft genome sequence, and effective vectors and also optimized the electroporation procedures in an effort to enhance its utilization. The organism exhibited swarming motility but not detectable endospore formation, and growth was rapid at 60°C under neutral and relatively low-salt conditions. Although the cells showed negligible acceptance of shuttle plasmids from general strains of Escherichia coli, methylation-controlled plasmids from dam mutant strains were efficiently accepted, suggesting circumvention of a restriction-modification system in G. thermodenitrificans K1041. We optimized the electroporation procedure to achieve efficiencies of 103 to 105 CFU/µg for five types of plasmids, which exhibited the different copy numbers and segregational stabilities in G. thermodenitrificans K1041. Some sets of plasmids were compatible. Moreover, we observed substantial plasmid-directed production of heterologous proteins in the intracellular or extracellular environments. Our successful construction of a library of promoter mutants using K1041 cells as hosts and subsequent screening at elevated temperatures to identify improved promoters revealed that G. thermodenitrificans K1041 was practical as a library host. The draft genomic sequence of the organism contained 3,384 coding genes, including resA and mcrB genes, which are involved in restriction-modification systems. Further examination revealed that in-frame deletions of resA increased transformation efficiencies, but mcrB deletion had no effect. The ΔresA mutant exhibited transformation efficiencies of >105 CFU/µg for some plasmids. IMPORTANCE Geobacillus thermodenitrificans K1041 has yet to be fully characterized. Although it is transformable via electroporation, it rarely accepts Escherichia coli-derived plasmids. This study clarified the biological and genomic properties of G. thermodenitrificans K1041. Additionally, we developed an electroporation procedure resulting in efficient acceptance of E. coli-derived plasmids. This procedure produced transformants using small amounts of plasmids immediately after the ligation reaction. Thus, G. thermodenitrificans K1041 was identified as a host for screening promoter mutants at elevated temperatures. Furthermore, because this strain efficiently produced heterologous proteins, it could serve as a host for screening thermostable proteins encoded in random mutant libraries or metagenomes. We also generated a ΔresA mutant that exhibited transformation efficiencies of >105 CFU/µg, which were highest in cases of electroporation-based transformation of Geobacillus spp. with E. coli-derived plasmids. Our findings provide a new platform for screening diverse genetic libraries at elevated temperatures.

Biochemical characterization and detection of antitumor activity of l-asparaginase from thermophilic Geobacillus kaustophilus DSM 7263T.

L-asparaginases, which are oncolytic enzymes, have been used in clinical applications for many years. These enzymes are also important in food processing industry due to their potential in acrylamide-mitigation. In this study, the gene for l-asparaginase (GkASN) from a thermophilic bacterium, Geobacillus kaustophilus, was cloned and expressed in E. coli Rosetta™2 (DE3) cells utilizing the pET-22b(+) vector. The 6xHis-tag attached enzyme was purified and analyzed both biochemically and structurally. The molecular mass of GkASN was determined as ∼36 kDa by SDS-PAGE, Western Blotting, and MALDI-TOF MS analyses. Optimum temperature and pH for the enzyme was determined as 55 °C and 8.5, respectively. The enzyme retained 89% of its thermal stability at 37 °C and 75% at 55 °C after 6 h of incubation. The enzyme activity was inhibited in the presence of Cu2+, Fe3+, Zn2+, and EDTA, while the activity was enhanced in the presence of Mn2+, Mg2+, and thiol group protective agents such as 2-mercaptoethanol and DTT. The structural modeling analysis demonstrated that the catalytic residues of the enzyme were partially similar to other asparaginases. The therapeutic potential of GkASN was tested on hepatocellular carcinoma cells, a solid cancer type with high mortality rate and rapidly increasing incidence in recent years. We showed that the GkASN-induced asparagine deficiency effectively reduced the metastatic synergy in HCC SNU387 cells on a xCELLigence system with differentiated epithelial Hep3B and poorly differentiated metastatic mesenchymal HCC SNU387 cells.

Role of C-terminal domain in a manganese-catalase from Geobacillus thermopakistaniensis.

Catalases catalyze the decomposition of hydrogen peroxide into water and oxygen. We have characterized two manganese-catalases from Geobacillus thermopakistaniensis, CatGt and Cat-IIGt, which exhibited significant variation in their sequence, structure and properties. There was only 23% sequence identity between the two. The striking structural difference was the presence of an extended C-terminal domain in CatGt. Molecular modelling and docking studies revealed that deletion of the C-terminal domain removes non-specific binding, which results in increased substrate affinity. To verify experimentally, a C-terminal truncated version of CatGt, named as CatGt-ΔC, was produced in Escherichia coli and effects of deletion were analyzed. There was no significant difference in optimal pH, optimal temperature and substrate specificity of CatGt and CatGt-ΔC. However, Km value was reduced from 259 to 157 mM and CatGt-ΔC exhibited ∼1.5-fold higher catalytic efficiency as compared to CatGt. Furthermore, removal of the C-terminal domain converted the tetrameric nature to monomeric, and reduced the thermostability of the truncated protein. These results demonstrate that C-terminal domain of CatGt might have little role in maintaining enzyme function but provides additional structural stability to the protein, which is a desired property for industrial applications.

Efficient monoacylglycerol synthesis by carboxylesterase EstGtA2 from Geobacillus thermodenitrificans in a solvent-free two-phase system.

The present study investigated high-yield monoacylglycerol (MAG) synthesis by bacterial lipolytic enzymes in a solvent-free two-phase system. Esterification by monoacylglycerol lipase from Bacillus sp. H-257 (H257) required a high glycerol/fatty acid molar ratio for efficient MAG synthesis. Screening of H257 homologues revealed that carboxylesterase derived from Geobacillus thermodenitrificans, EstGtA2, exhibited a higher esterification rate than H257. Moreover, neutralizing the pH of the acidic reaction solution by adding potassium hydroxide (KOH) solution further increased the esterification rate. The esterification rate by EstGtA2 reached 75% under conditions of equivalent molar amounts of glycerol and fatty acid, and the MAG rate (MAG/total glyceride) was 97%. The neutralized pH of the reaction solution likely affected the thermal stability of EstGtA2 during the esterification reaction. Screening for thermal-tolerant variants revealed that the EstGtA2S26I variant was stable at 75 °C for 30 min, a condition under which wild-type EstGtA2 was completely inactivated. The esterification rate by the EstGtA2S26I variant reached 90%, and the MAG rate was 96%. The addition of alkali and the use of a thermal-tolerant enzyme were important for obtaining high-yield MAG in a solvent-free two-phase system utilizing EstGtA2.

Production Optimization and Biochemical Characterization of Cellulase from Geobacillus sp. KP43 Isolated from Hot Spring Water of Nepal.

This study is aimed at isolating and identifying a thermophilic cellulolytic bacterium from hot spring water and characterizing thermostable cellulase produced by the isolate. Enrichment and culture of water sample was used for isolation of bacterial strains and an isolate with highest cellulase activity was chosen for the production, partial purification, and biochemical characterization of the enzyme. Different staining techniques, enzymatic tests, and 16s ribosomal DNA (16s rDNA) gene sequencing were used for the identification of the isolate. The cellulase producing isolate was Gram positive, motile, and sporulated rod-shaped bacterium growing optimally between 55°C and 65°C. Based on partial 16s rDNA sequence analysis, the isolate was identified as Geobacillus sp. and was designated as Geobacillus sp. KP43. The cellulase enzyme production condition was optimized, and the product was partially purified and biochemically characterized. Optimum cellulase production was observed in 1% carboxymethyl cellulose (CMC) at 55°C. The molecular weight of the enzyme was found to be approximately 66 kDa on 12% SDS-PAGE analysis. Biochemical characterization of partially purified enzyme revealed the temperature optimum of 70°C and activity over a wide pH range. Further, cellulase activity was markedly stimulated by metal ion Fe2+. Apart from cellulases, the isolate also depicted good xylanase, cellobiase, and amylase activities. Thermophilic growth with a variety of extracellular enzyme activities at elevated temperature as well as in a wide pH range showed that the isolated bacteria, Geobacillus sp. KP43, can withstand the harsh environmental condition, which makes this organism suitable for enzyme production for various biotechnological and industrial applications.