Utilization of low-stability variants in protein evolutionary engineering.

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.

Evaluation of temperature, pH and nutrient conditions in bacterial growth and extracellular hydrolytic activities of two Alicyclobacillus spp. strains.

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 DO600) at pH 3 and Alicyclobacillus sp. PA2 (0.725 DO600) at pH 5. Inducible extracellular extremozyme activities were determined for ß-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.

Stereoselective Directed Cationic Cascades Enabled by Molecular Anchoring in Terpene Cyclases.

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 %).

Release of Soybean Isoflavones by Using a ß-Glucosidase from Alicyclobacillus herbarius.

ß-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 ß-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.

Targeting the vanillic acid decarboxylase gene for Alicyclobacillus acidoterrestris quantification and guaiacol assessment in apple juices using real time PCR.

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 × 101 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 R2 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.

Production of isofloridoside from galactose and glycerol using α-galactosidase from Alicyclobacillus hesperidum.

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.

Purification and Characterization of a Novel ß-Galactosidase From the Thermoacidophile Alicyclobacillus vulcanalis.

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 ß-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 Km 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 ß-galactosidase.

Optimized immobilization of endoglucanase Cel9A onto glutaraldehyde activated chitosan nanoparticles by response surface methodology: The study of kinetic behaviors.

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 Km (from 3.703 to 12.195 [mg/ml]), which make it suitable to use in some industries such as detergents.

A novel thermal Cas12b from a hot spring bacterium with high target mismatch tolerance and robust DNA cleavage efficiency.

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 (Kcat/Km (s-1 M-1) = 8.80 × 1011), which is significantly higher than that of AacCas12b (Kcat/Km (s-1 M-1) = 7.56 × 108) 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.

Production of l-ribose from l-arabinose by co-expression of l-arabinose isomerase and d-lyxose isomerase in Escherichia coli.

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 Co2+ 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.

Efficient production of gluten hydrolase Kuma030 in E. coli by hot acid treatment without chromatography.

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.

Linear enzyme cascade for the production of (-)-iso-isopulegol.

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.

The structure of the AliC GH13 α-amylase from Alicyclobacillus sp. reveals the accommodation of starch branching points in the α-amylase family.

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 Šresolution. 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.

Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents.

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.

Ig-like Domain in Endoglucanase Cel9A from Alicyclobacillus acidocaldarius Makes Dependent the Enzyme Stability on Calcium.

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.

Structural and Biochemical Characterization of AaL, a Quorum Quenching Lactonase with Unusual Kinetic Properties.

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-ß-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 (kcat/KM > 105 M-1.s-1). Additionally, AaL exhibits unusually low KM 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.

Alicyclobacillus acidocaldarius Squalene-Hopene Cyclase: The Critical Role of Steric Bulk at Ala306 and the First Enzymatic Synthesis of Epoxydammarane from 2,3-Oxidosqualene.

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,ß-hydroxyhopenes and 3α,ß-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.

Comparison of Alicyclobacillus acidocaldarius o-Succinylbenzoate Synthase to Its Promiscuous N-Succinylamino Acid Racemase/ o-Succinylbenzoate Synthase Relatives.

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 × 102 M-1 s-1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects kcat, 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.

Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes.

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-ß-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a ß-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-ß-D-xylosidic linkages of xylans and the hydrolysis of (1-4)-ß-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.

Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants.

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.