Isolation and characterization of multiple-stress tolerant bacteria from radon springs.

Radon springs, characterized by their high concentrations of radon gas (Rn222), are extreme environments with unique physicochemical conditions distinct from conventional aquatic ecosystems. Our research aimed to investigate microbial life in radon springs, focusing on isolating extremophilic bacteria and assessing their resistance to adverse conditions. Our study revealed the prevalence of Actinomycetia species in the radon spring environment. We conducted various tests to evaluate the resistance of these isolates to oxidative stress, irradiation, desiccation, and metal ion content. These extremophilic bacteria showed overall higher resistance to these stresses compared to control strains. Lipidomic analysis was also employed to provide insights into the adaptive mechanisms of these bacteria which were found mainly in the correlations among individual clusters and changes in content of fatty acids (FA) as well as differences between content and type of FAs of environmental isolates and type strains.

The Deciphering of Growth-Dependent Strategies for Quorum-Sensing Networks in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is recognized as a significant cause of morbidity and mortality among nosocomial pathogens. In respiratory infections, P. aeruginosa acts not only as a single player but also collaborates with the opportunistic fungal pathogen Aspergillus fumigatus. This study introduced a QS molecule portfolio as a potential new biomarker that affects the secretion of virulence factors and biofilm formation. The quantitative levels of QS molecules, including 3-o-C12-HSL, 3-o-C8-HSL, C4-HSL, C6-HSL, HHQ, PQS, and PYO, measured using mass spectrometry in a monoculture, indicated metabolic changes during the transition from planktonic to sessile cells. In the co-cultures with A. fumigatus, the profile of abundant QS molecules was reduced to 3-o-C12-HSL, C4-HSL, PQS, and PYO. A decrease in C4-HSL by 50% to 170.6 ± 11.8 ng/mL and an increase 3-o-C12-HSL by 30% up to 784.4 ± 0.6 ng/mL were detected at the stage of the coverage of the hyphae with bacteria. Using scanning electron microscopy, we showed the morphological stages of the P. aeruginosa biofilm, such as cell aggregates, maturated biofilm, and cell dispersion. qPCR quantification of the genome equivalents of both microorganisms suggested that they exhibited an interplay strategy rather than antagonism. This is the first study demonstrating the quantitative growth-dependent appearance of QS molecule secretion in a monoculture of P. aeruginosa and a co-culture with A. fumigatus.

Killing Effect of Bacillus Velezensis FZB42 on a Xanthomonas Campestris pv. Campestris (Xcc) Strain Newly Isolated from Cabbage Brassica Oleracea Convar. Capitata (L.): A Metabolomic Study.

The potential use of Bacillus velezensis FZB42 for biological control of various phytopathogens has been documented over the past few years, but its antagonistic interactions with xanthomonads has not been studied in detail. Novel aspects in this study consist of close observation of the death of Xanthomonas campestris pv. campestris cells in a co-culture with B. velezensis FZB42, and quantification of lipopeptides and a siderophore, bacillibactin, involved in the killing process. A new robust Xcc-SU isolate tolerating high concentrations of ferric ions was used. In a co-culture with the antagonist, the population of Xcc-SU was entirely destroyed within 24-48 h, depending on the number of antagonist cells used for inoculation. No inhibitory effect of Xcc-SU on B. velezensis was observed. Bacillibactin and lipopeptides (surfactin, fengycin, and bacillomycin) were present in the co-culture and the monoculture of B. velezensis. Except for bacillibactin, the maximum contents of lipopeptides were higher in the antagonist monoculture compared with the co-culture. Scanning electron microscopy showed that the death of Xcc-SU bacteria in co-culture was caused by cell lysis, leading to an enhanced occurrence of distorted cells and cell ghosts. Analysis by mass spectrometry showed four significant compounds, bacillibactin, surfactin, fengycin, and bacillomycin D amongst a total of 24 different forms detected in the co-culture supernatant: Different forms of surfactin and fengycin with variations in their side-chain length were also detected. These results demonstrate the ability of B. velezensis FZB42 to act as a potent antagonistic strain against Xcc.

Identification of Homologous Polyprenols from Thermophilic Bacteria.

Sixteen strains of five genera of thermophilic bacteria, i.e., Alicyclobacillus, Brevibacillus, Geobacillus, Meiothermus, and Thermus, were cultivated at a temperature from 42 to 70 °C. Twelve strains were obtained from the Czech Collection of Microorganisms, while four were directly isolated and identified by 16S rRNA gene sequencing from the hot springs of the world-famous Carlsbad spa (Czech Republic). Polyprenol homologs from C40 to C65 as well as free undecaprenol (C55), undecaprenyl phosphate, and undecaprenyl diphosphate were identified by shotgun analysis and RP-HPLC/MS-ESI+ (reverse phase high-performance liquid chromatography-high-resolution positive electrospray ionization mass spectrometry). The limit of detection (50 pM) was determined for individual homologs and free polyprenols and their phosphates. Thus, it has been shown that at least some thermophilic bacteria produce not just the major C55 polyprenol as previously described, but a mixture of homologs.

Bringing SEM and MSI Closer Than Ever Before: Visualizing Aspergillus and Pseudomonas Infection in the Rat Lungs.

A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dryer. The SEM generated images with well-preserved structures, allowing for monitoring of bacterial cells and fungal hyphae in the infected tissue. Importantly, the consecutive nonfixed frozen sections were fully compatible with MALDI-MSI, providing molecular biomarker maps of Pseudomonas aeruginosa. The protocol enables bimodal image fusion in the in-house software CycloBranch, as demonstrated by SEM and MALDI-MSI.

Characteristics of microbial community of soil subjected to industrial production of antibiotics.

Ecosystems worldwide are exposed to pollutants connected to the industrial production of pharmaceuticals. The objective of this study was to study the composition and characteristics of the soil microbial communities that had been exposed to long-term selection pressure caused by the industrial production of penicillin G. Soil samples from four sites among the penicillin G production plant were analysed using 16S rRNA profiling via Illumina MiSeq platform and were compared with the control samples from four sites outside the plant. Total metagenomic DNA from the impacted soil was also used for the preparation of E. coli T1R-based fosmid library which was consequently qualitatively tested for the presence of penicillin G acylase (PGA)-encoding genes using the method of sequence homology. Analyses of alpha diversity revealed that the long-term antibiotic presence in the soil significantly increased the microbial diversity and richness in terms of Shannon diversity index (p = 0.002) and Chao estimates (p = 0.004). Principal component analysis showed that the two types of communities (on-site and control) could be separated at the phylum, class and genus level. The on-site soil was enriched in Betaproteobacteria, Deltaproteobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetia, while a significant decrease in Actinobacteria was observed. Metagenomic fosmid library revealed high hit rates in identifying PGAs (14 different genes identified) and confirmed the biotechnological potential of soils impacted by anthropogenic activity. This study offers new insights into the changes in microbial communities of soils exposed to anthropogenic activity as well as indicates that those soils may represent a hotspot for biotechnologically interesting targets.

Rhizoferrin Glycosylation in Rhizopus microsporus.

Rhizopus spp. are the most common etiological agents of mucormycosis, causing over 90% mortality in disseminated infections. The diagnosis relies on histopathology, culture, and/or polymerase chain reaction. For the first time, the glycosylation of rhizoferrin (RHF) was described in a Rhizopus microsporus clinical isolate by liquid chromatography and accurate tandem mass spectrometry. The fermentation broth lyophilizate contained 345.3 ± 13.5, 1.2 ± 0.03, and 0.03 ± 0.002 mg/g of RHF, imido-RHF, and bis-imido-RHF, respectively. Despite a considerable RHF secretion rate, we did not obtain conclusive RHF detection from a patient with disseminated mucormycosis caused by the same R. microsporus strain. We hypothesize that parallel antimycotic therapy, RHF biotransformation, and metabolism compromised the analysis. On the other hand, the full profile of posaconazole metabolites was retrieved by our in house software CycloBranch.

Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris.

To take full advantage of recombinant Pichia pastoris (Komagataella phaffii) as a production system for heterologous proteins, the complex protein secretory process should be understood and optimised by circumventing bottlenecks. Typically, little or no attention has been paid to the fate of newly synthesised protein inside the cell, or its passage through the secretory pathway, and only the secreted product is measured. However, the system's productivity (i.e. specific production rate qp), includes productivity of secreted (qp,extra) plus intracellularly accumulated (qp,intra) protein. In bioreactor cultivations with P. pastoris producing penicillin G acylase, we studied the dynamics of product formation, i.e. both the specific product secretion (qp,extra) and product retention (qp,intra) as functions of time, as well as the kinetics, i.e. productivity in relation to specific growth rate (µ). Within the time course, we distinguished (I) an initial phase with constant productivities, where the majority of product accumulated inside the cells, and qp,extra, which depended on µ in a bell-shaped manner; (II) a transition phase, in which intracellular product accumulation reached a maximum and productivities (intracellular, extracellular, overall) were changing; (III) a new phase with constant productivities, where secretion prevailed over intracellular accumulation, qp,extra was linearly related to µ and was up to three times higher than in initial phase (I), while qp,intra decreased 4-6-fold. We show that stress caused by heterologous protein production induces cellular imbalance leading to a secretory bottleneck that ultimately reaches equilibrium. This understanding may help to develop cultivation strategies for improving protein secretion from P. pastoris.Key Points• A novel concept for industrial bioprocess development.• A Relationship between biomass growth and product formation in P. pastoris.• A Three (3) phases of protein production/secretion controlled by the AOX1-promoter.• A Proof of concept in production of industrially relevant penicillin G acylase.

Effect of the anti-inflammatory drug diclofenac on lipid composition of bacterial strain Raoultella sp. KDF8.

The strain Raoultella sp. KDF8 was cultivated on three sources of carbon and energy, glycerol, ethanol and diclofenac, for periods of time ranging from 24 to 72 h. Using thin-layer chromatography, nine classes of phospholipids were detected and the amount of phosphatidylethanolamine (PtdEtn) decreased with increasing cultivation time. Conversely, the ratio of phospholipids having three or four acyls (acyl-phosphatidylglycerol (APtdGro), N-acyl-PtdEtn (NAPtdEtn) and cardiolipin (Ptd2Gro) increased during cultivation. GC-MS analysis showed that the percentage of fatty acids containing a cyclopropane ring increased almost tenfold whereas the amount of fatty acids bearing even-numbered chains dropped to less than one-third after 24 h and 72 h in cultures on glycerol and diclofenac, respectively. Shotgun analysis showed significant changes in the representation of molecular species of phospholipids. For instance, there was a 36-fold change in the ratio of 16:1/16:1/16:1-APtdGro to c17:0/c17:0/c17:0-APtdGro and a 12-fold ratio change for 16:1/16:1/16:1-NAPtdEtn to c17:0/c17:0/c17:0-NAPtdEtn; the Ptd2Gro ratio of 16:1 to c17:0 acids equalled 1750. Our results show that the bacteria overcome destabilization of the inner cytoplasmic cell membrane and a bacterial outer membrane by altering the geometric arrangement of acyl chains, i.e. switching from monounsaturated to cyclopropane fatty acids (16:1 versus c17:0).

The crystal structure of XdpB, the bacterial old yellow enzyme, in an FMN-free form.

Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal ß-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress.

Agrobacterium bohemicum sp. nov. isolated from poppy seed wastes in central Bohemia.

Two non-pathogenic strains R89-1 and R90T isolated from poppy seed (Papaver somniferum L.) wastes were phenotypically and genotypically characterized. Multilocus sequence analysis (MLSA) was conducted with six genes (atpD, glnA, gyrB, recA, rpoB, 16S rRNA). The strains represented a new species which clustered with Agrobacterium rubi NBRC 13261T and Agrobacterium skierniewicense Ch11T type strains. MLSA was further accompanied by whole-genome phylogeny, in silico DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses for both strains. ANI and dDDH values were deep below the species delineation threshold. Phenotypic features of the novel strains unequivocally allowed their differentiation from all other Agrobacterium species. Unlike other agrobacteria, the strains were salt sensitive and were able to biotransform morphine alkaloids. The dominant cellular fatty acids are 18:1 w7c, 16:0 and 12:0 aldehyde/16:1 iso I/14:0 3OH summed in feature 2 and the major respiratory quinine is Q-10 (87%). The DNA G+C content is 56mol%. Microbial community analysis indicated probable association with P. somniferum plant material. Altogether, these characteristics showed that strains R90T and R89-1 represent a new species of the genus Agrobacterium which we propose to name Agrobacterium bohemicum. The type strain of A. bohemicum is R90T (=CCM 8736T=DSM 104667T).

Potential of the strain Raoultella sp. KDF8 for removal of analgesics.

The bacterial strain KDF8 capable of growth in the presence of diclofenac and codeine analgesics was obtained after chemical mutagenesis of nature isolates from polluted soils. The strain KDF8 was identified as Raoultella sp. based on its morphology, biochemical properties, and 16S rRNA gene sequence. It was deposited in the Czech Collection of Microorganisms under the number CCM 8678. A growing culture efficiently removed diclofenac (92% removal) and partially also codeine (about 30% degradation) from culture supernatants within 72 h at 28 °C. The degradation of six analgesics by the whole cell catalyst was investigated in detail. The maximum degradation of diclofenac (91%) by the catalyst was achieved at pHINI of 7 (1 g/L diclofenac). The specific removal rate at high concentrations of diclofenac and codeine increased up to 16.5 mg/gCDW per h and 5.1 mg/gCDW per h, respectively. HPLC analysis identified 4'-hydroxydiclofenac as a major metabolite of diclofenac transformation and 14-hydroxycodeinone as codeine transformation product. The analgesics ibuprofen and ketoprofen were also removed, albeit to a lower extent of 3.2 and 2.0 mg/gCDW per h, respectively. Naproxen and mefenamic acid were not degraded.

Draft Genome Sequence of Pantoea agglomerans JM1, a Strain Isolated from Soil Polluted by Industrial Production of Beta-Lactam Antibiotics That Exhibits Valacyclovir-Like Hydrolase Activity.

Strain Pantoea agglomerans JM1 was isolated from the soil of a microbiome that had been exposed to polluting pharmaceuticals. The bacterium exhibited enzymatic activities useful for the biotransformation of beta-lactams. The genome of the strain was assembled and described, and the gene encoding valacyclovir-like hydrolase was identified.

Potential of Pichia pastoris for the production of industrial penicillin G acylase.

This study deals with the potential of Pichia pastoris X-33 for the production of penicillin G acylase (PGAA) from Achromobacter sp. CCM 4824. Synthetic gene matching the codon usage of P. pastoris was designed for intracellular and secretion-based production strategies and cloned into vectors pPICZ and pPICZα under the control of AOX1 promoter. The simple method was developed to screen Pichia transformants with the intracellularly produced enzyme. The positive correlation between acylase production and pga gene dosage for both expression systems was demonstrated in small scale experiments. In fed-batch bioreactor cultures of X-33/PENS2, an extracellular expression system, total PGAA expressed from five copies reached 14,880 U/L of an active enzyme after 142 h; however, 60% of this amount retained in the cytosol. The maximum PGAA production of 31,000 U/L was achieved intracellularly from nine integrated gene copies of X-33/PINS2 after 90 h under methanol induction. The results indicate that in both expression systems the production level of PGAA is similar but there is a limitation in secretion efficiency.

Current state and perspectives of penicillin G acylase-based biocatalyses.

In the course of more than 60-year history, penicillin G acylase (PGA) gained a unique position among enzymes used by pharmaceutical industry for production of ß-lactam antibiotics. Kinetically controlled enzymatic syntheses of cephalosporins of novel generations in which PGA catalyzes coupling of activated acyl donor with nucleophile belong among the latest large-scale applications. Contrary to rather specific roles of other enzymes involved in ß-lactam biocatalyses, the PGA seems to have the greatest potential. On the laboratory scale, other applications with industrial potential were described, e.g., directed evolution of the enzyme to meet specific demands of industrial processes or its modification into the enzyme catalyzing reactions with novel substrates. The fact that ß-lactams represent the most important group of antibiotics comprising 65 % of the world antibiotic market explains such a tremendous and continuous interest in this enzyme. Indeed, the annual consumption of PGA has recently been estimated to range from 10 to 30 million tons. The application potential of the enzyme goes beyond the ß-lactam biocatalysis due to its enantioselectivity and promiscuity: the PGA can be used for the production of achiral and chiral compounds convenient for the preparation of synthons and active pharmaceutical ingrediences, respectively. These biocatalyses, however, still wait for large-scale application.

Penicillin G acylase from Achromobacter sp. CCM 4824 : an efficient biocatalyst for syntheses of beta-lactam antibiotics under conditions employed in large-scale processes.

Penicillin G acylase from Achromobacter sp. (NPGA) was studied in the enzymatic synthesis of ß-lactam antibiotics by kinetically controlled N-acylation. When compared with penicillin acylase of Escherichia coli (PGA), the NPGA was significantly more efficient at syntheses of ampicillin and amoxicillin (higher S/H ratio and product accumulation) in the whole range of substrate concentrations. The degree of conversion of 6-aminopenicillanic acid to amoxicillin and ampicillin (160 mM 6-APA, 350 mM acyl donor methylester[Symbol: see text]HCl, pH 6.3, 25 °C, reaction time of 200 min) with immobilized NPGA equaled 96.9 % and 91.1 %, respectively. The enzyme was highly thermostable with maximum activity at 60 °C (pH 8.0) and 65 °C (pH 6.0). Activity half-life at 60 °C (pH 8.0) and at 60 °C (pH 6.0) was 24 min and 6.9 h, respectively. Immobilized NPGA exhibited long operational stability with half-life of about 2,000 cycles for synthesis of amoxicillin at conversion conditions used in large-scale processes (230 mM 6-APA, 340 mM D-4-hydroxyphenylglycine methylester[Symbol: see text]HCl, 27.5 °C, pH 6.25). We discuss our results with literature data available for related penicillin acylases in terms of their industrial potential.

Effect of the amino acid substitution in the DNA-binding domain of the Fur regulator on production of pyoverdine.

The ferric uptake regulator gene (fur), its promoter region and Fur box of pvdS gene involved in siderophore-mediated iron uptake system were sequenced in the parent strain Pseudomonas aeruginosa PAO1 and in the fur mutant FPA121 derived from the strain PAO1. We identified the gene fur 179 bearing a novel, single-point mutation that changed the amino acid residue Gln60Pro in the DNA-binding domain of the Fur protein. The synthesis of pyoverdine was studied in cultures of the strains PAO1 and FPA121 grown in iron-deplete and iron-replete (60 µmol/L FeIII) medium. The amino acid replacement in the regulatory Fur protein is responsible for the overproduction of pyoverdine in iron-deplete and iron-replete medium. No mutation was identified in the Fur box of the gene pvdS.

Whole-cell oxidation of omeprazole sulfide to enantiopure esomeprazole with Lysinibacillus sp. B71.

Production of enantiopure esomeprazole by biocatalysis is of great demand by pharmaceutical industry. A Gram-positive bacterium oxidizing omeprazole sulfide 1a (5-methoxy-2-[((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)thio]-1H-benzoimidazole) to (S)-sulfoxide esomeprazole 2a (S)-5-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl) methylsulfinyl]-3H-benzoimidazole was isolated from soil polluted with elemental sulfur. The strain exhibited the highest identity with the genus Lysinibacillus and catalyzed oxidation of 1a into enantiopure esomeprazole with conversion of 77% in a stirred bioreactor, fed-batch culture. No consecutive oxidation of (S)-sulfoxide to sulfone was observed during whole-cell catalysis. The unique characteristics of the catalyst provide a solid basis for further improvement and development of sustainable green bioprocess.

Heterologous expression of leader-less pga gene in Pichia pastoris: intracellular production of prokaryotic enzyme.

BACKGROUND: Penicillin G acylase of Escherichia coli (PGAEc) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of beta-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGAEc in this host and studied yeast production capacity and enzyme authenticity. RESULTS: Leader-less pga gene encoding PGAEcunder the control of AOX1 promoter was cloned in Pichia pastoris X-33. The intracellular overproduction of heterologous PGAEc(hPGAEc) was evaluated in a stirred 10 litre bioreactor in high-cell density, fed batch cultures using different profiles of transient phases. Under optimal conditions, the average volumetric activity of 25900 U l-1 was reached. The hPGAEc was purified, characterized and compared with the wild-type PGAEc. The alpha-subunit of the hPGAEc formed in the cytosol was processed aberrantly resulting in two forms with C- terminuses extended to the spacer peptide. The enzyme exhibited modified traits: the activity of the purified enzyme was reduced to 49%, the ratios of hydrolytic activities with cephalexin, phenylacetamide or 6-nitro-3-phenylacetylamidobenzoic acid (NIPAB) to penicillin G increased and the enzyme showed a better synthesis/hydrolysis ratio for the synthesis of cephalexin. CONCLUSIONS: Presented results provide useful data regarding fermentation strategy, intracellular biosynthetic potential, and consequences of the heterologous expression of PGAEc in P. pastoris X-33. Aberrant processing of the precursor of PGAEc in the cytosol yielded the mature enzyme with modified traits.

Cloning of an epoxide hydrolase-encoding gene from Aspergillus niger M200, overexpression in E. coli, and modification of activity and enantioselectivity of the enzyme by protein engineering.

The gene encoding an epoxide hydrolase from Aspergillus niger M200 has been cloned and its sequence determined. The gene is interrupted by seven introns, one exon being only nine nucleotides long. The non-coding 5'- and 3'-regions of the mRNA are composed of 47 and 76 nucleotides, respectively. Overexpression of the fungal epoxide hydrolase in E. coli TOP10 has led to a 15-fold increase in specific activity (compared to the wild-type strain). Saturation mutagenesis at codon 217 resulted in the discovery of nine enzyme variants showing in several cases profound differences in activity and enantioselectivity towards various epoxides when compared to the data of the wild-type enzyme. The site 217 is located at the entrance of the tunnel that provides the substrate with access to the active site. The exchange of Ala at this position for Cys has led to a doubled enantioselectivity (E-value of 5.0) towards benzyl glycidyl ether. The same substitution resulted in a threefold-enhanced activity of the enzyme towards allyl glycidyl ether and styrene oxide without affecting enantioselectivity. The variant A217L showed an enhanced enantioselectivity towards tert-butyl glycidyl ether reaching an E-value of 100 (from 60 for the wild-type enzyme). Replacement of A217 by Val has led to higher activity towards allyl glycidyl ether by a factor of six. The substitutions Ala-->Glu and Ala-->Gln increased the enantioselectivity towards allyl glycidyl ether and styrene oxide by over 50% to E-values of 10 and 16, respectively. The study underlines that single amino acid exchanges in the substrate tunnel region can lead to significant improvements in enantioselectivity and activity of the epoxide hydrolase from A. niger M200.