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Antibodies and antibody mimics are extensively used in the pharmaceutical industry, where stringent safety standards are required. Implementing heat sterilization during or after the manufacturing process could help prevent contamination by viruses and bacteria. However, conventional antibodies and antibody mimics are not suitable for heat sterilization because they irreversibly denature at high temperatures. In this study, we focused on the refolding property of the cold shock protein from the hyperthermophile Thermotoga maritima (TmCSP), which denatures at elevated temperatures but regains its native structure upon re-cooling. We designed and constructed a mutant library of TmCSP in which amino acid residues in its three surface loops were diversified. From the library, mutant TmCSPs that bind to each of eight target proteins were selected by phage and yeast surface display methods. We confirmed that the secondary structure and binding affinity of all the selected mutants were restored after heat treatment followed by cooling. Additionally, freeze-drying did not impair their binding affinity. The crystal structure of a mutant TmCSP in complex with its target, the esterase from Alicyclobacillus acidocaldarius, revealed specific interactions between them. These results clearly demonstrate the feasibility of creating heat-sterilizable antibody mimics using TmCSP as a scaffold., (© 2024 The Protein Society.)

Urethanase is a promising biocatalyst for degrading carcinogen ethyl carbamate (EC) in fermented foods. However, their vulnerability to high ethanol and/or salt and acidic conditions severely limits their applications. In this study, a novel urethanase from Alicyclobacillus pomorum ( Ap UH) was successfully discovered using a database search. Ap UH shares 49.4% sequence identity with the reported amino acid sequences. It belongs to the Amidase Signature family and has a conserved "K-S-S" catalytic triad and the characteristic "GGSS" motif. The purified enzyme overexpressed in Escherichia coli exhibits a high EC affinity ( K m , 0.306 mM) and broad pH tolerance (pH 4.0-9.0), with an optimum pH 7.0. Enzyme activity remained at 58% in 12% (w/v) NaCl, and 80% in 10% (v/v) ethanol or after 1 h treatment with the same ethanol solution at 37 °C. Ap UH has no hydrolytic activity toward urea. Under 30 °C, the purified enzyme (200 U/L) degraded about 15.4 and 43.1% of the EC in soy sauce samples (pH 5.0, 6.0), respectively, in 5 h. Furthermore, the enzyme also showed high activity toward the class 2A carcinogen acrylamide in foods. These attractive properties indicate their potential applications in the food industry.

Evolutionary engineering involves repeated mutations and screening and is widely used to modify protein functions. However, it is important to diversify evolutionary pathways to eliminate the bias and limitations of the variants by using traditionally unselected variants. In this study, we focused on low-stability variants that are commonly excluded from evolutionary processes and tested a method that included an additional restabilization step. The esterase from the thermophilic bacterium Alicyclobacillus acidocaldarius was used as a model protein, and its activity at its optimum temperature of 65 °C was improved by evolutionary experiments using random mutations by error-prone PCR. After restabilization using low-stability variants with low-temperature (37 °C) activity, several re-stabilizing variants were obtained from a large number of variant libraries. Some of the restabilized variants achieved by removing the destabilizing mutations showed higher activity than that of the wild-type protein. This implies that low-stability variants with low-temperature activity can be re-evolved for future use. This method will enable further diversification of evolutionary pathways., 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 study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Extremophile bacteria have developed the metabolic machinery for living in extreme temperatures, pH, and high-salt content. Two novel bacterium strains Alicyclobacillus sp. PA1 and Alicyclobacillus sp. PA2, were isolated from crater lake El Chichon in Chiapas, Mexico. Phylogenetic tree analysis based on the 16SrRNA gene sequence revealed that the strain Alicyclobacillus sp. PA1 and Alicyclobacillus sp. PA2 were closely related to Alicyclobacillus species (98% identity and 94.73% identity, respectively). Both strains were Gram variable, and colonies were circular, smooth and creamy. Electron microscopy showed than Alicyclobacillus sp. PA1 has a daisy-like form and Alicyclobacillus sp. PA2 is a regular rod. Both strains can use diverse carbohydrates and triglycerides as carbon source and they also can use organic and inorganic nitrogen source. But, the two strains can grow without any carbon or nitrogen sources in the culture medium. Temperature, pH and nutrition condition affect bacterial growth. Maximum growth was produced at 65 °C for Alicyclobacillus sp. PA1 (0.732 DO 600 ) at pH 3 and Alicyclobacillus sp. PA2 (0.725 DO 600 ) at pH 5. Inducible extracellular extremozyme activities were determined for β-galactosidase (Alicyclobacillus sp. PA1: 88.07 ± 0.252 U/mg, Alicyclobacillus sp. PA2: 51.57 ± 0.308 U/mg), cellulose (Alicyclobacillus sp. PA1: 141.20 ± 0.585 U/mg, Alicyclobacillus sp. PA2: 51.57 ± 0.308 U/mg), lipase (Alicyclobacillus sp. PA1: 138.25 ± 0.600 U/mg, Alicyclobacillus sp. PA2: 175.75 ± 1.387 U/mg), xylanase (Alicyclobacillus sp. PA1: 174.72 ± 1.746 U/mg, Alicyclobacillus sp. PA2: 172.69 ± 0.855U/mg), and protease (Alicyclobacillus sp. PA1: 15.12 ± 0.121 U/mg, Alicyclobacillus sp. PA2: 15.33 ± 0.284 U/mg). These results provide new insights on extreme enzymatic production on Alicyclobacillus species., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cascade reactions appeared as a cutting-edge strategy to streamline the assembly of complex structural scaffolds from naturally available precursors in an atom-, as well as time, labor- and cost-efficient way. We herein report a strategy to control cationic cyclization cascades by exploiting the ability of anchoring dynamic substrates in the active site of terpene cyclases via designed hydrogen bonding. Thereby, it is possible to induce "directed" cyclizations in contrast to established "non-stop" cyclizations (99:1) and predestinate cascade termination at otherwise catalytically barely accessible intermediates. As a result, we are able to provide efficient access to naturally widely occurring apocarotenoids, value-added flavors and fragrances in gram-scale by replacing multi-stage synthetic routes to a single step with unprecedented selectivity (>99.5 % ee) and high yields (up to 89 %)., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

β-Glucosidases are used in the food industry to hydrolyse glycosidic bonds in complex sugars, with enzymes sourced from extremophiles better able to tolerate the process conditions. In this work, a novel β-glycosidase from the acidophilic organism Alicyclobacillus herbarius was cloned and heterologously expressed in Escherichia coli BL21(DE3). AheGH1 was stable over a broad range of pH values (5-11) and temperatures (4-55 °C). The enzyme exhibited excellent tolerance to fructose and good tolerance to glucose, retaining 65 % activity in the presence of 10 % (w/v) glucose. It also tolerated organic solvents, some of which appeared to have a stimulating effect, in particular ethanol with a 1.7-fold increase in activity at 10 % (v/v). The enzyme was then applied for the cleavage of isoflavone from isoflavone glucosides in an ethanolic extract of soy flour, to produce soy isoflavones, which constitute a valuable food supplement, full conversion was achieved within 15 min at 30 °C., (© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Alicyclobacillus spp. has recently received much attention due to its implication in the spoilage of pasteurized fruit juices, which is characterized by the formation of guaiacol. Previous researches indicate that not all Alicyclobacillus spp. are able to produce guaiacol. The aim of this study was to identify possible differences in the vanillic acid decarboxylase gene involved in guaiacol biosynthesis and then develop specific detection methods for guaiacol producing Alicyclobacillus. Agarose gel electrophoresis results showed that the partial vdcC gene was present in all the guaiacol producing Alicyclobacillus, but absent in non-guaicaol producing strains apart from A. fastidiosus DSM 17978. On the basis of the vdcC gene sequence, a primer pair specific to A. acidoterrestris was designed; then a SYBR Green I real time PCR was established for the direct quantification of A. acidoterrestris in apple juice, and the detection limit was 2.6 × 10 1  CFU/mL. The developed real time PCR system was used to detect A. acidoterrestris in 36 artificially contaminated apple juice samples and guaiacol production in the sample was also analyzed by GC-MS. The Gompertz model was employed to describe the relationship between A. acidoterrestris cell concentration and guaiacol content, and the value of R 2 was 0.854. This work provides an alternative to conventional methods of guaiacol quantification and A. acidoterrestris detection and could be very useful for the early recognition of A. acidoterrestris contamination in fruit juices., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Thermoacidophiles are microorganisms capable of optimum growth under a combination of high temperature and low pH. These microorganisms are a rich source of thermo- and acid- active/stable glycosyl hydrolases. Such enzymes could find use as novel biocatalysts in industrial processes, as operation at elevated temperature can increase substrate solubility, decrease viscosity, and reduce the risk of microbial contamination. We report the purification and characterization of an intracellular β-galactosidase from the thermoacidophile Alicyclobacillus vulcanalis DSM 16176. The enzyme was purified 110-fold, with a 5.89% yield. Denatured (83.7 kDa) and native (179 kDa) molecular masses were determined by SDS-PAGE and gel filtration, respectively, and suggest the enzyme functions as a homodimer. LC-MS/MS analysis confirmed identity, and bioinformatic analysis indicates the enzyme to be a member of the glycosyl hydrolase family 42 (GH42). Highest activity was measured at 70 °C and pH 6.0. The K m on the substrates ONPG and lactose were 5 and 258 mM, respectively. This enzyme is thermostable, retaining 76, 50, and 42% relative activity after 30, 60, and 120 min, respectively, at 70 °C. This property could lend its use to high-temperature industrial processes requiring a thermo-active β-galactosidase.

Immobilization of enzyme onto nanoparticles such as chitosan can have biotechnological importance. In this study, chitosan nanoparticles (ChNPs) were prepared by Ionic gelation method and Endoglucanase Cel9A from Alicyclobacillus acidocaldariius (AaCel9A) immobilized on the nanoparticles. The FTIR results showed that the enzymes were immobilized on the ChNPs. The dynamic light scattering and scanning electron microscope (SEM) results illustrated that the AaCel9A-ChNPs approximately had 40 nm diameters. For optimizing enzyme immobilization, response surface methodology was employed using different variables (pH, enzyme immobilization time, and enzyme to ChNPs ratio [E/Cs]). The results showed that the high immobilization efficiency was achieved in pH 7, E/Cs of 0.4 in 2.63 hr. The enzyme activity results showed that, immobilization increased optimum pH for activity (from 6.5 to 7.5) and the enzyme K m (from 3.703 to 12.195 [mg/ml]), which make it suitable to use in some industries such as detergents., (© 2020 American Institute of Chemical Engineers.)

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas), such as Cas9 and Cpf1, are RNA-guided endonucleases that target and degrade nucleic acids, providing powerful genomic editing and molecular diagnostic tools. Cas12b enzymes are distinct effectors; however, their features and catalytic boundaries require further characterization. We identified BrCas12b from the thermophile bacterium Brevibacillus sp. SYSU G02855 as a novel ortholog of cas12b. Biochemical analyses revealed that BrCas12b is a dual-RNA-guided endonuclease with higher optimum reaction temperature than that of other reported members of Cas12b. The seed sequence of BrCas12b is only 4 nt in length, indicating that it has greater target mismatch tolerance than that of previously reported Cas effectors; however, it contains a compensatory effect at the position of the cleavage site. Using fluorescence-based detection method to evaluate target cleavage efficiency, we showed that BrCas12b has robust enzymatic cleavage activity (K cat /K m (s - 1  M - 1 ) = 8.80 × 10 11 ), which is significantly higher than that of AacCas12b (K cat /K m (s - 1  M - 1 ) = 7.56 × 10 8 ) from Alicyclobacillus acidoterrestris. The results increase our understanding of the catalytic mechanism of Cas12b family members and suggest that BrCas12b might be useful in the application of genomic editing and molecular diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Isofloridoside (D-isofloridoside and L-isofloridoside) is the main photosynthetic product in red algae. Here, given the importance of isofloridoside, a potentially effective method to produce isofloridoside from galactose and glycerol using whole-cell biocatalysts harboring α-galactosidase was developed. α-Galactosidase-encoding genes from Alicyclobacillus hesperidum, Lactobacillus plantarum, and Bifidobacterium adolescentis were cloned and the proteins were overproduced in Escherichia coli. The α-galactosidase from A. hesperidum (AHGLA) was chosen to synthesize isofloridoside. The effects of reaction pH, temperature, and substrate concentration were investigated. In the optimum biotransformation conditions, the final isofloridoside concentration reached 0.45 M (galactose conversion 23 %). The reaction mixtures were purified using activated charcoal and calcined Celite, and the purified product was identified as a mixture of D- and L-isofloridoside by liquid chromatography-mass spectrometry and nuclear magnetic resonance. This study provides a possible feasible method for the biosynthesis of isofloridoside from low-cost glycerol and galactose., (Copyright © 2019 Elsevier Inc. All rights reserved.)

l-Ribose is an important pharmaceutical intermediate that is used in the synthesis of numerous antiviral and anticancer drugs. However, it is a non-natural and expensive rare sugar. Recently, the enzymatic synthesis of l-ribose has attracted considerable attention owing to its considerable advantages over chemical approaches. In this work, a new strategy was developed for the production of l-ribose from the inexpensive starting material l-arabinose. The l-arabinose isomerase (l-AIase) gene from Alicyclobacillus hesperidum and the d-lyxose isomerase (d-LIase) gene from Thermoflavimicrobium dichotomicum were cloned and co-expressed in Escherichia coli, resulting in recombinant cells harboring the vector pCDFDuet-Alhe-LAI/Thdi-DLI. The co-expression system exhibited optimal activity at a temperature of 70 °C and pH 6.0, and the addition of Co 2+ enhanced the catalytic activity by 27.8-fold. The system containing 50 g L -1 of recombinant cells were relatively stable up to 55 °C. The co-expression system (50 g L -1 of recombinant cells) afforded 20.9, 39.7, and 50.3 g L -1 of l-ribose from initial l-arabinose concentrations of 100, 300, and 500 g L -1 , corresponding to conversion rate of 20.9%, 13.2%, and 10.0%, respectively. Overall, this study provides a viable approach for producing l-ribose from l-arabinose under slightly acidic conditions using a co-expression system harboring l-AIase and d-LIase genes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Kumamolisin from Alicyclobacillus sendaiensis strain NTAP-1 is a serine protease with collagenase activity. After molecular engineering, a kumamolisin mutant, named Kuma030, was obtained with high proteolytic activity against gluten, which might cause celiac disease. Kuma030 exhibited its potential application in industrial and medicine, while challenges remained of its large-scale purification and production. In the studies here, we successfully overexpressed the Kuma030 in E. coli BL21 (DE3) by anchoring a SUMO (Small Ubiquitin-like Modifier) fusion protein at its N-terminal end. In addition, a fast protein purification procedure was developed according to the acidophilic and thermophilic properties of Alicyclobacillus sendaiensis. After a simple acid treatment followed by a heat treatment, a total of 9.9 mg functional Kuma030 was quickly obtained form 1 L LB media culture. This purified Kuma030 was confirmed to be functional to cleave the PQ sequences in a designed protein substrate, and the gluten in actual food samples, such as whole wheat bread and beer, in a fast manner. Our studies provided an efficient strategy for the overexpression and purification of functional Kuma030 in E. coli, which might expand its broad practical applications., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Biocatalysis has developed enormously in the last decade and now offers solutions for the sustainable production of chiral and highly functionalised asset molecules. Products generated by enzymatic transformations are already being used in the food, feed, chemical, pharmaceutical and cosmetic industry, and the accessible compound panoply is expected to expand even further. In particular, the combination of stereo-selective enzymes in linear cascade reactions is an elegant strategy toward enantiomeric pure compounds, as it reduces the number of isolation and purification steps and avoids accumulation of potentially unstable intermediates. Here, we present the set-up of an enzyme cascade to selectively convert citral to (-)-iso-isopulegol by combining an ene reductase and a squalene hopene cyclase. In the initial reaction step, the ene reductase YqjM from Bacillus subtilis selectively transforms citral to (S)-citronellal, which is subsequently cyclised exclusively to (-)-iso-isopulegol by a mutant of the squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC). With this approach, we can convert citral to an enantiopure precursor for isomenthol derivatives.

α-Amylases are glycoside hydrolases that break the α-1,4 bonds in starch and related glycans. The degradation of starch is rendered difficult by the presence of varying degrees of α-1,6 branch points and their possible accommodation within the active centre of α-amylase enzymes. Given the myriad industrial uses for starch and thus also for α-amylase-catalysed starch degradation and modification, there is considerable interest in how different α-amylases might accommodate these branches, thus impacting on the potential processing of highly branched post-hydrolysis remnants (known as limit dextrins) and societal applications. Here, it was sought to probe the branch-point accommodation of the Alicyclobacillus sp. CAZy family GH13 α-amylase AliC, prompted by the observation of a molecule of glucose in a position that may represent a branch point in an acarbose complex solved at 2.1 Å resolution. Limit digest analysis by two-dimensional NMR using both pullulan (a regular linear polysaccharide of α-1,4, α-1,4, α-1,6 repeating trisaccharides) and amylopectin starch showed how the Alicyclobacillus sp. enzyme could accept α-1,6 branches in at least the -2, +1 and +2 subsites, consistent with the three-dimensional structures with glucosyl moieties in the +1 and +2 subsites and the solvent-exposure of the -2 subsite 6-hydroxyl group. Together, the work provides a rare insight into branch-point acceptance in these industrial catalysts., (open access.)

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.

The acyclic molecule squalene (1) is cyclized into 6,6,6,6,5-fused pentacyclic hopene (2) and hopanol (3; ca. 5:1) through the action of Alicyclobacillus acidocaldarius squalene-hopene cyclase (AaSHC). The polycyclization reaction proceeds with regio- and stereochemical specificity under precise enzymatic control. This pentacyclic hopane skeleton is generated by folding 1 into an all-chair conformation. The Ala306 residue in AaSHC is conserved in known squalene-hopene cyclases (SHCs); however, increasing the steric bulk (A306T and A306V) led to the accumulation of 6,6,6,5-fused tetracyclic scaffolds possessing 20R stereochemistry in high yield (94 % for A306V). The production of the 20R configuration indicated that 1 had been folded in a chair-chair-chair-boat conformation; in contrast, the normal chair-chair-chair-chair conformation affords the tetracycle with 20S stereochemistry, but the yield produced by the A306V mutant was very low (6 %). Consequently, bulk at position 306 significantly affects the stereochemical fate during the polycyclization reaction. The SHC also accepts (3R) and (3S)-2,3-oxidosqualenes (OXSQs) to generate 3α,β-hydroxyhopenes and 3α,β-hydroxyhopanols through polycyclization initiated at the epoxide ring. However, the Val and Thr mutants generated epoxydammarane scaffolds from (3R)-OXSQ; this indicated that the polycyclization cascade started in these instances at the terminal double bond position. This work is the first to report the polycyclization of oxidosqualene starting at the terminal double bond., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) has an Ig-like domain and the enzyme stability is dependent to calcium. In this study the effect of calcium on the structure and stability of the wild-type enzyme and the truncated form (the wild-type enzyme without Ig-like domain, AaCel9AΔN) was investigated. Fluorescence quenching results indicated that calcium increased and decreased the rigidity of the wild-type and truncated enzymes, respectively. RMSF results indicated that AaCel9A has two flexible regions (regions A and B) and deleting the Ig-like domain increased the truncated enzyme stability by decreasing the flexibility of region B probably through increasing the hydrogen bonds. Calcium contact map analysis showed that deleting the Ig-like domain decreased the calcium contacting residues and their calcium binding affinities, especially, in region B which has a role in calcium binding site in AaCel9A. Metal depletion and activity recovering as well as stability results showed that the structure and stability of the wild-type and truncated enzymes are completely dependent on and independent of calcium, respectively. Finally, one can conclude that the deletion of Ig-like domain makes AaCel9AΔN independent of calcium via decreasing the flexibility of region B through increasing the hydrogen bonds. This suggests a new role for the Ig-like domain which makes AaCel9A structure dependent on calcium.

Quorum quenching lactonases are enzymes that are capable of disrupting bacterial signaling based on acyl homoserine lactones (AHL) via their enzymatic degradation. In particular, lactonases have therefore been demonstrated to inhibit bacterial behaviors that depend on these chemicals, such as the formation of biofilms or the expression of virulence factors. Here we characterized biochemically and structurally a novel representative from the metallo-β-lactamase superfamily, named AaL that was isolated from the thermoacidophilic bacterium Alicyclobacillus acidoterrestris. AaL is a potent quorum quenching enzyme as demonstrated by its ability to inhibit the biofilm formation of Acinetobacter baumannii. Kinetic studies demonstrate that AaL is both a proficient and a broad spectrum enzyme, being capable of hydrolyzing a wide range of lactones with high rates (k cat /K M  > 10 5  M -1 .s -1 ). Additionally, AaL exhibits unusually low K M values, ranging from 10 to 80 µM. Analysis of AaL structures bound to phosphate, glycerol, and C6-AHL reveals a unique hydrophobic patch (W26, F87 and I237), involved in substrate binding, possibly accounting for the enzyme's high specificity. Identifying the specificity determinants will aid the development of highly specific quorum quenching enzymes as potential therapeutics.

Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/ o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 10 2 M -1 s -1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects k cat , by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

Background: The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-D-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-D-xylans to remove successive D-xylose residues from the non-reducing termini., Results: We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation., Conclusion: Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.

Squalene-hopene cyclases (SHCs) catalyze the polycyclization of squalene into a mixture of hopene and hopanol. Recently, amino-acid residues lining the catalytic cavity of the SHC from Alicyclobacillus acidocaldarius were replaced by small and large hydrophobic amino acids. The alteration of leucine 607 to phenylalanine resulted in increased enzymatic activity towards the formation of an intermolecular farnesyl-farnesyl ether product from farnesol. Furthermore, the addition of small-chain alcohols acting as nucleophiles led to the formation of non-natural ether-linked terpenoids and, thus, to significant alteration of the product pattern relative to that obtained with the wild type. It is proposed that the mutation of leucine at position 607 may facilitate premature quenching of the intermediate by small alcohol nucleophiles. This mutagenesis-based study opens the field for further intermolecular bond-forming reactions and the generation of non-natural products., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases-mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a β-galactosidase from Alicyclobacillus acidocaldarius in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

C2c1 is a type V-B CRISPR-Cas system dual-RNA-guided DNA endonuclease. Here, we report the crystal structure of Alicyclobacillus acidoterrestris C2c1 in complex with a chimeric single-molecule guide RNA (sgRNA). AacC2c1 exhibits a bi-lobed architecture consisting of a REC and NUC lobe. The sgRNA scaffold forms a tetra-helical structure, distinct from previous predictions. The crRNA is located in the central channel of C2c1, and the tracrRNA resides in an external surface groove. Although AacC2c1 lacks a PAM-interacting domain, our analysis revealed that the PAM duplex has a similar binding position found in Cpf1. Importantly, C2c1-sgRNA system is highly sensitive to single-nucleotide mismatches between guide RNA and target DNA. The resulting reduction in off-target cleavage renders C2c1 a valuable addition to the current arsenal of genome-editing tools. Together, our findings indicate that sgRNA assembly is achieved through a mechanism distinct from that reported previously for Cas9 or Cpf1 endonucleases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a "locked" conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: An endoglucanase gene (gluE1) was cloned from a thermalacidophilus (Alicyclobacillus tengchongensis CGMCC1504) isolated from a hot spring, and the sequence and biochemical characterization of enzyme were analyzed., Methods: The full-length gluE1 was obtained based on genome sequencing, analysis of amino acid sequence of GluE1. gluE1 was ligated into pEASY-E2 vector and expressed in Escherichia coli BL21 (DE3) cells. GluE1 was purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography, and then the enzyme characterizations were determined., Results: The 1020 bp full-length gluE1 (50.5% GC content) encodes a 339 residues polypeptide (GluE1: 40.45 kDa). GluE1 showed the highest identity of 97% with endoglucanase in public databases, and <60% identities with other endoglucanase. GluE1 efficiently hydrolyzed CMC-Na, soluble starch and barley-β-glucan, which showed apparent optimal at pH 6.5 and 55℃. GluE1 was stable and active (>60%) at pH 5.0-10.0, and had a high stability at 37℃; and it exhibited Km, Vmax and kcat values of 8.58 mg/mL, 416.67 U/mg and 280.90 s-1 respectively. GluE1 was strongly inhibited by Ag+, Hg2+ and SDS, partial promoted by β-Mercaptoethanol, Pb2+, Mg2+, Ca2+ and Na+, 30% NaCl still retains more than 64% of the activity. The residual enzyme activity kept 93% after pre-incubation of the enzyme in 30% NaCl., Conclusion: Endoglucanase gene gluE1 from Alicyclobacillus was first reported, and GluE1 showed a good pH stability and strong halo-tolerant property. GluE1 might have greater potential applications.

Background: Vanillic acid decarboxylase (VAD) is the key enzyme responsible for guaiacol production in Alicyclobacillus acidoterrestris; however, information related to this enzyme is currently unavailable. The aim of this study is to characterise the VAD from A. acidoterrestris., Results: Specific activity of VAD in vanillic acid-induced A. acidoterrestris DSM 3923 cells was highest in the early stage of the log phase, and almost undetectable in the stationary and death phases. Of the four techniques used to extract VAD, sonication was found to be the most effective and recovered 3.23 U mg(-1) of VAD. Through optimisation of the crucial parameters for sonication, the recovery of VAD had more than doubled (6.81 U mg(-1) ). The crude enzyme extract was purified by ammonium sulfate precipitation and a 9.87-fold purification was obtained. The partially purified VAD exhibited optimum activity at pH 6.0-6.5, 45°C and was stable at pH 5.0-7.5, 20-45°C. The Km and Vmax values of the VAD were 0.53 mmol L(-1) and 96 U mg(-1) protein, respectively. VAD activity was stimulated by Co(2+) and Mn(2+) , but was inhibited by Ni(2+) , Cu(2+) , Ba(2+) and Fe(3+) . Cinnamic acid, ferulic acid, resveratrol, quercetin and rutin at the concentration of 1 mmol L(-1) could completely inhibit the activity of VAD., Conclusion: The present study provides the first report on the characteristics of the VAD from A. acidoterrestris, which will contribute to the development of more effective control methods to minimise A. acidoterrestris-related spoilage in fruit juices. © 2015 Society of Chemical Industry., (© 2015 Society of Chemical Industry.)

Few members of glycoside hydrolase (GH) family 113 have been characterized, and information on substrate recognition by and the catalytic mechanism of this family is extremely limited. In the present study, a novel endo-β-1,4-mannanase of GH 113, Man113A, was identified in thermoacidophilic Alicyclobacillus sp. strain A4 and found to exhibit both hydrolytic and transglycosylation activities. The enzyme had a broad substrate spectrum, showed higher activities on glucomannan than on galactomannan, and released mannobiose and mannotriose as the main hydrolysis products after an extended incubation. Compared to the only functionally characterized and structure-resolved counter part Alicyclobacillus acidocaldarius ManA (AaManA) of GH 113, Man113A showed much higher catalytic efficiency on mannooligosaccharides, in the order mannohexaose ≈ mannopentaose > mannotetraose > mannotriose, and required at least four sugar units for efficient catalysis. Homology modeling, molecular docking analysis, and site-directed mutagenesis revealed the vital roles of eight residues (Trp13, Asn90, Trp96, Arg97, Tyr196, Trp274, Tyr292, and Cys143) related to substrate recognition by and catalytic mechanism of GH 113. Comparison of the binding pockets and key residues of β-mannanases of different families indicated that members of GH 113 and GH 5 have more residues serving as stacking platforms to support -4 to -1 subsites than those of GH 26 and that the residues preceding the acid/base catalyst are quite different. Taken as a whole, this study elucidates substrate recognition by and the catalytic mechanism of GH 113 β-mannanases and distinguishes them from counterparts of other families., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The enzymatic reactions of geranylfarnesol (8) and its acetate 9, classified as sesterterpenes (C25), using squalene-hopene cyclase (SHC) were investigated. The enzymatic reaction of 8 afforded 6/6-fused bicyclic 20, 6/6/6-fused tricyclic 21, and 6/6/6/6-fused tetracyclic compounds 22 and 23 as the main products (35% yield), whereas that of 9 afforded two 6/6/6-fused tricyclic compounds 24 and 25 in a high yield (76.3%) and a small amount (5.0%) of 26 (the acetate of 22). A significantly higher conversion of 9 indicates that the arrangement of the substrate in the reaction cavity changed. The lipophilic nature and/or the bulkiness of the acetyl group may have changed its binding with SHC, thus placing the terminal double bond of 9 in the vicinity of the DXDD motif of SHC, which is responsible for the proton attack on the double bond to initiate the polycyclization reaction. The results obtained for 8 are different to some extent than those reported by Shinozaki et al. The products obtained in this study were deprotonated compounds; however, the products reported by Shinozaki et al. were hydroxylated compounds.

Structural calcium sites control protein thermostability and activity by stabilizing native folds and changing local conformations. Alicyclobacillus acidocaldarius survives in thermal-acidic conditions and produces an endoglucanase Cel9A (AaCel9A) which contains a calcium-binding site (Ser465 to Val470) near the catalytic cleft. By superimposing the Ca(2+)-free and Ca(2+)-bounded conformations of the calcium site, we found that Ca(2+) induces hydrophobic interactions between the calcium site and its nearby region by driving a conformational change. The hydrophobic interactions at the high-B-factor region could be enhanced further by replacing the surrounding polar residues with hydrophobic residues to affect enzyme thermostability and activity. Therefore, the calcium-binding residue Asp468 (whose side chain directly ligates Ca(2+)), Asp469, and Asp471 of AaCel9A were separately replaced by alanine and valine. Mutants D468A and D468V showed increased activity compared with those of the wild type with 0 mM or 10 mM Ca(2+) added, whereas the Asp469 or Asp471 substitution resulted in decreased activity. The D468A crystal structure revealed that mutation D468A triggered a conformational change similar to that induced by Ca(2+) in the wild type and developed a hydrophobic interaction network between the calcium site and the neighboring hydrophobic region (Ala113 to Ala117). Mutations D468V and D468A increased 4.5°C and 5.9°C, respectively, in melting temperature, and enzyme half-life at 75°C increased approximately 13 times. Structural comparisons between AaCel9A and other endoglucanases of the GH9 family suggested that the stability of the regions corresponding to the AaCel9A calcium site plays an important role in GH9 endoglucanase catalysis at high temperature., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Alicyclobacillus acidocaldarius, a thermoacidophilic bacterium, has a repertoire of thermo- and acid-stable enzymes that deconstruct lignocellulosic compounds. The work presented here describes the ability of A. acidocaldarius to reduce the concentration of the phenolic compounds: phenol, ferulic acid, ρ-coumaric acid and sinapinic acid during growth conditions. The extent and rate of the removal of these compounds were significantly increased by the presence of micro-molar copper concentrations, suggesting activity by copper oxidases that have been identified in the genome of A. acidocaldarius. Substrate removal kinetics was first order for phenol, ferulic acid, ρ-coumaric acid and sinapinic acid in the presence of 50 μM copper sulfate. In addition, laccase enzyme assays of cellular protein fractions suggested significant activity on a lignin analog between the temperatures of 45 and 90 °C. This work shows the potential for A. acidocaldarius to degrade phenolic compounds, demonstrating potential relevance to biofuel production and other industrial processes.

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) is a monomeric enzyme with 537 residues. This enzyme has an Ig-like domain in the N-terminus of the catalytic domain. In this study, the role of the Ig-like domain on the activity, stability, and structural rigidity of AaCel9A and the effect of calcium on enzyme activity and stability were examined by comparing a truncated enzyme with deletion of the Ig-like domain (AaCel9AΔN) to the wild-type enzyme. Our results showed that the deletion of the Ig-like domain increased the catalytic efficiency of the truncated enzyme up to threefold without any significant changes in the K m of the enzyme. Furthermore, pH and temperature optimum for activity were shifted from 6.5 to 7.5 and from 65 to 60 °C, respectively, by deletion of the Ig-like domain. The thermal stability and fluorescence quenching results indicated that the stability and rigidity of the truncated enzyme have been more than that of the wild-type enzyme. Calcium similarly increased the catalytic efficiency of the enzymes (up to 40 %) and remarkably raised the stability of the AaCel9A compared to the AaCel9AΔN. This shows that Ig-like domain has a role in the increase of the enzyme stability by calcium in the wild-type enzyme.

Alicyclobacillus acidoterrestris has recently received much attention due to its implication in the spoilage of pasteurized fruit juices, which was manifested by the production of guaiacol. Vanillic acid and vanillin have been accepted as the biochemical precursors of guaiacol in fruit juices. The purpose of this study was to try to find other precursors and elucidate details about the conversion of vanillic acid and vanillin to guaiacol by A. acidoterrestris. Four potential substrates including ferulic acid, catechol, phenylalanine and tyrosine were analyzed, but they could not be metabolized to guaiacol by all the thirty A. acidoterrestris strains tested. Resting cell studies and enzyme assays demonstrated that vanillin was reduced to vanillyl alcohol by NADPH-dependent vanillin reductase and oxidized to vanillic acid by NAD(P)(+)-dependent vanillin dehydrogenases in A. acidoterrestris DSM 3923. Vanillic acid underwent a nonoxidative decarboxylation to guaiacol. The reversible vanillic acid decarboxylase involved was oxygen insensitive and pyridine nucleotide-independent., (Copyright © 2015. Published by Elsevier B.V.)

A mutation, D478N, was obtained by an error-prone polymerase chain reaction using the L-arabinose isomerase (L-AI) gene from Alicyclobacillus hesperidum URH17-3-68 as the template. The mutated isomerase showed higher activity for D-galactose isomerization. The mutation site obtained from random mutagenesis was then introduced as a single-site mutation using site-directed mutagenesis. Single-site variants, D478N, D478Q, D478A, D478K, and D478R, were constructed. The optimum temperatures were all higher than 60 °C. D478A, D478N, and D478Q retained more than 80 % of the maximum relative activity of the wild-type L-AI at 75 °C. With the exception of the D478A variant, all variants showed decreased optimum pH values in the acidic range (6.0-6.5). All of the variant L-AIs could be significantly activated by the addition of Co(2+) and Mn(2+). D478N and D478Q showed higher catalytic efficiencies (k cat/K m) toward D-galactose than that of wild-type L-AI. In addition, the D478N and D478Q variants exhibited a much higher conversion ratio of D-galactose to D-tagatose at 6.0 than the wild-type L-AI. According to the molecular model, residue D478 was located on the surface of the enzyme and distant from the active site. It was supposed that the charged state of residue 478 may influence the optimum pH for substrate binding or isomerization.

Unlabelled: Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic β-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2 to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic β-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA., Importance: Deacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic β-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover, this study can enrich our understanding of the functions of these enzymes from this family., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The paper at hand deals with biocatalysis in bicontinuous microemulsions. The latter consist of a dynamic network of oil and water domains separated by a monolayer of surfactant molecules, i.e. the interfacial layer. A microemulsion with the composition buffer--n-octane--nonionic surfactant was tested as reaction medium for an enzyme-catalysed reaction with a focus on the conversion of hydrophobic substrates, which are difficult to convert in aqueous buffer solutions. For the study at hand, we chose to investigate the activity of the squalene-hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC) towards its natural substrate squalene in bicontinuous microemulsions. Firstly, the study revealed that the activity of AacSHC depends linearly on the enzyme concentration. Secondly, a hyperbolic curve was found for the dependence of the activity on the substrate concentration and a saturation of the AacSHC at substrate concentrations above 20mM was observed. Thirdly, the composition of the interfacial layer was found to have no significant influence on the activity or on the conformation of AacSHC. Surprisingly and unexpectedly, a distinctly enhanced selectivity towards hopene was discovered in the microemulsion. To conclude, bicontinuous microemulsions were found to be a suitable reaction medium for biocatalytic reactions with the enzyme AacSHC., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and α-glucuronidase from Alicyclobacillus acidocaldarius (GH10-XA and GH67-GA, respectively) and a α-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 °C (3.5 days). In addition, GH67-GC was the most stable α-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-α-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations <0.3 μM) and in much less reaction time (2h vs 12h) if compared to other known biotransformations catalyzed by thermophilic enzymes. In addition, remarkably, together with a thermophilic β-xylosidase, they catalyzed the production of xylose from the smart cooking pre-treated biomass of one of the most promising energy crops for second generation biorefineries. We demonstrated that search by the CAZy Data Bank of currently available genomes and detailed enzymatic characterization of recombinant enzymes allow the identification of glycoside hydrolases with novel and interesting properties and applications., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A novel endoglucanase encoding gene was cloned from Alicyclobacillus vulcanalis and expressed in E. coli. The deduced amino acid sequence showed highest identity with α-L-arabinofuranosidase-like proteins from glycoside hydrolase family 51. The recombinant enzyme was purified by affinity chromatography and characterised in terms of its potential suitability for lignocellulose hydrolysis at high temperature in the production of bioethanol. The purified enzyme displayed maximum activity at 80 °C and pH 3.6-4.5. Tween 20 was found to have a beneficial effect on enzyme activity and thermal stability. When incubated in the presence of 0.1% Tween 20, the enzyme retained full activity after 72 h at 70 °C and 78% of original activity after 72 h at 75 °C. Maximum activity was observed on carboxymethyl cellulose, and the purified enzyme also hydrolysed lichenan, barley β-glucan and xylan. The purified enzyme decreased the viscosity of carboxymethyl cellulose when assessed at 70-85 °C and was capable of releasing reducing sugars from acid-pretreated straw at 70 and 75 °C. The results indicate the potential suitability of the enzyme for industrial application in the production of cellulosic bioethanol.

The enzymatic regioselective hydrolysis of (a) acetylated mono- to tetrasaccharides of different nature, (b) of acetylated aryl glycosides and (c) of different acetylated nucleosides was studied enlarging the portfolio of substrates that can be employed by the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. The reactions were optimised to the extent that the amount of enzyme needed was lowered of two orders of magnitude with respect to the previously reported reactions, namely from 4000 to 40 U of enzyme per reaction. New additional solvents were screened and dramatic changes in regioselectivity were observed depending on the amount and type of solvent used. For example, in the presence of 10 % DMF, only two α-D-glucose products 6-OH and 4,6-OH (in a 76:24 ratio) were detected, whereas with 25 % DMF, at least four products of similar amount were observed. This versatility adds specific value to the biocatalyst making possible the design of biocatalytic reactions with different hydrophobic ester substrates. As an additional remarkable example, EST2 catalysed with a good yield and high regioselectivity the hydrolysis of p-nitrophenyl β-D-xylopyranoside triacetate producing only the monoacetylated derivative with acetyl group in 3-O-position, in 2 min. The results with nucleosides as substrates are particularly interesting. The peracetates of 3',5'-di-O-acetylthymidine are converted almost quantitatively (95 %) to the monoacetylated derivative possessing free secondary OH; this regioselectivity is complementary to hydrolysis/alcoholysis reactions catalysed by CAL-B lipase or to other microbial hydrolytic biocatalysts, generally giving products with free primary OH groups. A docking analysis was undertaken with all analysed substrates suggesting a structural interpretation of the results. In most of cases, the best pose of the selected substrate was in line with the observed regioselectivity.

Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site., (© The Authors Journal Compilation © 2015 Biochemical Society.)

An α-galactosidase gene (gal36A4) of glycosyl hydrolase family 36 was identified in the genome of Alicyclobacillus sp. A4. It contains an ORF of 2,187 bp and encodes a polypeptide of 728 amino acids with a calculated molecular mass of 82.6 kDa. Deduced Gal36A4 shows the typical GH36 organization of three domains--the N-terminal β-sheets, the catalytic (β/α)8-barrels, and the C-terminal antiparallel β-sheet. The gene product was produced in Escherichia coli and showed both hydrolysis and transglycosylation activities. The optimal pH for hydrolysis activity was 6.0, and a stable pH range of 5.0-11.0 was found. The enzyme had a temperature optimum of 60 °C. It is specific for α-1,6-glycosidic linkages and had a K m value of 1.45 mM toward pNPGal. When using melibiose as both donor and acceptor of galactose, Gal36A4 showed the transfer ratio of 23.25 % at 96 h. With respect to acceptor specificity, all tested monosaccharides, disaccharides, and oligosaccharides except for D-xylose and L-arabinose were good acceptors for transglycosylation. Thus, Gal36A4 may find diverse applications in industrial fields, especially in the food industry.

Alicycliphilus denitrificans is a versatile, ubiquitous, facultative anaerobic bacterium. Alicycliphilus denitrificans strain BC can use chlorate, nitrate, and oxygen as electron acceptor for growth. Cells display a prolonged lag-phase when transferred from nitrate to chlorate and vice versa. Furthermore, cells adapted to aerobic growth do not easily use nitrate or chlorate as electron acceptor. We further investigated these responses of strain BC by differential proteomics, transcript analysis, and enzyme activity assays. In nitrate-adapted cells transferred to chlorate and vice versa, appropriate electron acceptor reduction pathways need to be activated. In oxygen-adapted cells, adaptation to the use of chlorate or nitrate is likely difficult due to the poorly active nitrate reduction pathway and low active chlorate reduction pathway. We deduce that the Nar-type nitrate reductase of strain BC also reduces chlorate, which may result in toxic levels of chlorite if cells are transferred to chlorate. Furthermore, the activities of nitrate reductase and nitrite reductase appear to be not balanced when oxygen-adapted cells are shifted to nitrate as electron acceptor, leading to the production of a toxic amount of nitrite. These data suggest that strain BC encounters metabolic challenges in environments with fluctuations in the availability of electron acceptors. All MS data have been deposited in the ProteomeXchange with identifier PXD000258., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A carboxylesterase gene from thermophilic bacterium, Alicyclobacillus tengchongensis, was cloned and expressed in Escherichia coli BL21 (DE3). The gene coded for a 513 amino acid protein with a calculated molecular mass of 57.82 kDa. The deduced amino acid sequence had structural features highly conserved among serine hydrolases, including Ser204, Glu325, and His415 as a catalytic triad, as well as type-B carboxylesterase serine active site (FGGDPENITIGGQSAG) and type-B carboxylesterase signature 2 (EDCLYLNIWTP). The purified enzyme exhibited optimum activity with β-naphthyl acetate at 60 °C and pH 7 as well as stability at 25 °C and pH 7. One unit of the enzyme hydrolyzed 5 mg malathion l(-1) by 50 % within 25 min and 89 % within 100 min. The enzyme strongly degraded malathion and has a potential use for the detoxification of malathion residues.

PROMISCUOUS ENZYMES: The substrate promiscuity of squalene-hopene cyclases has been explored and applied in the enzyme-catalyzed synthesis of heterocyclic terpenoids. Features of this work include cyclization reactions without pyrophosphate activation, and stereospecific ring closure of substrates of varying chain length and terminal nucleophile. This provides a biocatalytic alternative to traditional chemical catalysts., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Here we report a comprehensive analysis through alanine-scanning mutagenesis of the contribution of surface ion pairs to the thermal stability of Alicyclobacillus acidocaldarius esterase 2 (EST2). We produced 16 single mutants, 4 double mutants corresponding to selected ion pairs R31/E118, E43/K102, R58/D130, D145/R148, 2 double mutants (R63A/R98A and E50A/D94A) involving residues of a large ion network on the protein surface and the double-mutant R98A/R148A meant to disrupt the R98 interactions within the said network and, contextually, the interaction between R148 and D145. The double-mutant E43A/E273K was obtained by chance. All selected residues were replaced with alanine except E91, which was mutated to a glycine and K102, which was changed to a glutamine. All 24 proteins were over-expressed in Escherichia coli, purified and characterized with respect to the main features. Structural stability data were compared with an in silico prediction of ΔΔG values. Our study of the individual factors involved in thermostability and their structural interpretation reveals that the great stability of this thermophilic protein can be explained by the contribution of a few residues at the protein surface.

Aleuritopteris ferns produce triterpenes and sesterterpenes with tricyclic cheilanthane and tetracyclic 18-episcalarane skeletons. The structural and mechanistic similarities between both classes of fern terpene suggest that their biosynthetic enzymes may be closely related. We investigate here whether a triterpene synthase is capable of recognizing geranylfarnesols as a substrate, and is able to convert them to cyclic sesterterpenes. We found that a bacterial triterpene synthase converted all-E-geranylfarnesol (1b) into three scalarane sesterterpenes with 18αH stereochemistry (5, 7 and 8), as well as mono- and tricyclic sesterterpenes (6 and 9). In addition, 2Z-geranylfarnesol (4) was converted into an 18-episcalarane derivative (10), whose skeleton can be found in sesterterpenes isolated from Aleuritopteris ferns. These results provide insight into sesterterpene biosynthesis in Aleuritopteris ferns.

To study the pH dependence of l-arabinose isomerase (AI) activity and stability, we compared homologous AIs with their chimeras. This study demonstrated that an ionizable amino acid near the catalytic site determines the optimal pH (pH(opt)) for activity, whereas the N-terminal surface R residues play an important role in determining the pH(opt) for stability.

Background: The Bacillus subtilis spore has long been used as a surface display system with potential applications in a variety of fields ranging from mucosal vaccine delivery, bioremediation and biocatalyst development. More recently, a non-recombinant approach of spore display has been proposed and heterologous proteins adsorbed on the spore surface. We used the well-characterized β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius as a model to study enzyme adsorption, to analyze whether and how spore-adsorption affects the properties of the enzyme and to improve the efficiency of the process., Results: We report that purified β-galactosidase molecules were adsorbed to purified spores of a wild type strain of B. subtilis retaining ca. 50% of their enzymatic activity. Optimal pH and temperature of the enzyme were not altered by the presence of the spore, that protected the adsorbed β-galactosidase from exposure to acidic pH conditions. A collection of mutant strains of B. subtilis lacking a single or several spore coat proteins was compared to the isogenic parental strain for the adsorption efficiency. Mutants with an altered outermost spore layer (crust) were able to adsorb 60-80% of the enzyme, while mutants with a severely altered or totally lacking outer coat adsorbed 100% of the β-galactosidase molecules present in the adsorption reaction., Conclusion: Our results indicate that the spore surface structures, the crust and the outer coat layer, have an negative effect on the adhesion of the β-galactosidase. Electrostatic forces, previously suggested as main determinants of spore adsorption, do not seem to play an essential role in the spore-β-galactosidase interaction. The analysis of mutants with altered spore surface has shown that the process of spore adsorption can be improved and has suggested that such improvement has to be based on a better understanding of the spore surface structure. Although the molecular details of spore adsorption have not been fully elucidated, the efficiency of the process and the pH-stability of the adsorbed molecules, together with the well documented robustness and safety of spores of B. subtilis, propose the spore as a novel, non-recombinant system for enzyme display.

Circular dichroism and differential scanning calorimetry measurements showed that esterase 2 from the thermophilic microorganism Alicyclobacillus acidocaldarius, EST2, and its variant in which the first 35 residues have been deleted, EST2-36 del, unfold reversibly on increasing temperature, and possess two cooperative and coupled domains [12]. Structural features of the α/β hydrolase fold of EST2, with nine α-helices packed against the central twisted β-sheet, do not allow a straightforward identification of these two cooperative and coupled domains. Molecular dynamics simulations, each one 20 ns long, have been performed at 300, 400 and 500 K, on both proteins in explicit water. Suitable analysis of MD trajectories has allowed a reliable identification of the two cooperative domains (i.e., the less stable one corresponds to external α-helices, whereas the more stable one corresponds to the central twisted β-sheet) and the attribution of the key coupling role to the last and long α-helix of EST2., (Copyright © 2011 Elsevier B.V. All rights reserved.)