Arthrobacter Citreus Research Articles

Structural and functional analysis of a bile salt hydrolase from the bison microbiome

The bile salt hydrolases (BSHs) are significant constituents of animal microbiomes. An evolving appreciation of their roles in health and disease has established them as targets of pharmacological inhibition. These bacterial enzymes belong to the N-terminal nucleophile superfamily and are best known to catalyze the deconjugation of glycine or taurine from bile salts to release bile acid substrates for transformation and or metabolism in the gastrointestinal tract. Here, we identify and describe the BSH from a common member of the Plains bison microbiome, Arthrobacter citreus (BSHAc). Steady-state kinetic analyses demonstrated that BSHAc is a broad-spectrum hydrolase with a preference for glycine-conjugates and deoxycholic acid (DCA). Second-order rate constants (kcat/KM) for BSHAc-catalyzed reactions of relevant bile salts-glyco- and tauro-conjugates of cholic acid and DCA- varied by ∼30-fold and measured between 1.4× 105 and 4.3× 106M-1s-1. Interestingly, a pan-BSH inhibitor named AAA-10 acted as a slow irreversible inhibitor of BSHAc with a rate of inactivation (kinact)of ∼2h-1 and a second order rate constant (kinact/KI) of ∼24M-1s-1 for the process. Structural characterization of BSHAc reacted with AAA-10 showed covalent modification of the N-terminal cysteine nucleophile, providing molecular details for an enzyme-stabilized product formed from this mechanism-based inhibitor's α-fluoromethyl ketone warhead. Structural comparison of the BSHs and BSH:inhibitor complexes highlighted the plasticity of the steroid-binding site, including a flexible loop that is variable across well-studied BSHs.

Arthrobacter zhaoxinii sp. nov. and Arthrobacter jinronghuae sp. nov., isolated from Marmota himalayana.

Four yellow-coloured strains (zg-Y815T/zg-Y108 and zg-Y859T/zg-Y826) were isolated from the intestinal contents of Marmota himalayana and assigned to the 'Arthrobacter citreus group'. The four strains grew optimally on brain heart infusion agar with 5 % defibrinated sheep blood plate at 30 °C, pH 7.0 and with 0.5 % NaCl (w/v). Comparative analysis of their 16S rRNA genes indicated that the two strain pairs belong to the genus Arthrobacter, showing the highest similarity to Arthrobacter yangruifuii 785T (99.52 %), which was further confirmed by the 16S rRNA gene and genome-based phylogenetic analysis. The comparative genomic analysis [digital DNA-DNA hybridization, (dDDH) and average nucleotide identity (ANI)] proved that the four strains are two different species (zg-Y815T/zg-Y108, 71.7 %/96.8 %; zg-Y859T/zg-Y826, 87.3 %/98.5 %) and differ from other known species within the genus Arthrobacter (zg-Y815T, 19.6-32.3 %/77.2-88.0 %; zg-Y859T, 19.5-29.3 %/77.4-86.3 %). Strain pairs zg-Y815T/zg-Y108 and zg-Y859T/zg-Y826 had the same major cellular fatty acids (iso-C16 : 0 and anteiso-C15 : 0), with MK-8(H2) as their dominant respiratory quinone (70.6 and 61.7 %, respectively). The leading polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylinositol. The detected amino acids and cell-wall sugars of the two new species were identical (amino acids: alanine, glutamic acid, and lysine; sugars: rhamnose, galactose, mannose, glucose, and ribose). According to the phylogenetic, phenotypic, and chemotaxonomic analyses, we concluded that the four new strains represented two different novel species in the genus Arthrobacter, for which the names Arthrobacter zhaoxinii sp. nov. (zg-Y815T= GDMCC 1.3494T = JCM 35821T) and Arthrobacter jinronghuae sp. nov. (zg-Y859T = GDMCC 1.3493T = JCM 35822T) are proposed.

Biotransamination of Furan-Based Aldehydes with Isopropylamine: Enzyme Screening and pH Influence.

Furan-based amines are highly valuable compounds which can be directly obtained via reductive amination from easily accessible furfural, 5-(hydroxymethyl)furfural (HMF) and 2,5-diformylfuran (DFF). Herein the biocatalytic amination of these carbonyl derivatives is disclosed using amine transaminases (ATAs) and isopropylamine (IPA) as amine donors. Among the different biocatalysts tested, the ones from Chromobacterium violaceum (Cv-TA), Arthrobacter citreus (ArS-TA), and variants from Arthrobacter sp. (ArRmut11-TA) and Vibrio fluvialis (Vf-mut-TA), afforded high levels of product formation (>80 %) at 100-200 mM aldehyde concentration. The transformations were studied in terms of enzyme and IPA loading. The pH influence was found as a key factor and attributed to the imine/aldehyde equilibrium that can arise from the high reactivity of the carbonyl substrates with a nucleophilic amine such as IPA.

Potential of some microbial isolates on diesel hydrocarbons removal, bio surfactant production and biofilm formation.

Potential of Arthrobacter citreus B27Pet, Bacillus thuringiensis B48Pet and Candida catnulata to produce biosurfactant using four different carbon sources (naphthalene, hexadecane, diesel and petroleum crude oil) was investigated. Removal of petroleum crude oil from aqueous culture and degradation of diesel were also determined using single and mixed culture of strains. The biofilm existence in single and mixed culture of strains was considered using naphthalene, hexadecane and diesel in culture medium. Cell surface hydrophobicity of A. citreus was higher than other isolates which also showed maximum surface tension reduction and emulsification index. As a whole, remarkable biosurfactant production occurred using petroleum crude oil as a carbon source in medium. A. citreus was found to be more robust than other tested strains in removal efficiency of crude oil due to its biosurfactant production capability. Statistically significant positive correlation was observed between biofilm existence and surface tension using diesel and hexadecane as carbon source. Overall diesel biodegradation efficiency by the mix culture of three applied strains was about 75% within a short period of time (10 days) which was accompanied with high biofilm production.

Transcriptional response of the xerotolerant Arthrobacter sp. Helios strain to PEG-induced drought stress.

A new bacterial strain has been isolated from the microbiome of solar panels and classified as Arthrobacter sp. Helios according to its 16S rDNA, positioning it in the “Arthrobacter citreus group.” The isolated strain is highly tolerant to desiccation, UV radiation and to the presence of metals and metalloids, while it is motile and capable of growing in a variety of carbon sources. These characteristics, together with observation that Arthrobacter sp. Helios seems to be permanently prepared to handle the desiccation stress, make it very versatile and give it a great potential to use it as a biotechnological chassis. The new strain genome has been sequenced and its analysis revealed that it is extremely well poised to respond to environmental stresses. We have analyzed the transcriptional response of this strain to PEG6000-mediated arid stress to investigate the desiccation resistance mechanism. Most of the induced genes participate in cellular homeostasis such as ion and osmolyte transport and iron scavenging. Moreover, the greatest induction has been found in a gene cluster responsible for biogenic amine catabolism, suggesting their involvement in the desiccation resistance mechanism in this bacterium.

A Novel Antimicrobial Metabolite Produced by Paenibacillus apiarius Isolated from Brackish Water of Lake Balkhash in Kazakhstan.

Four aerobic bacteria with bacteriolytic capabilities were isolated from the brackish water site Strait Uzynaral of Lake Balkhash in Kazakhstan. The morphology and physiology of the bacterial isolates have subsequently been analyzed. Using matrix assisted laser desorption ionization-time of flight mass spectrum and partial 16S rRNA gene sequence analyses, three of the isolates have been identified as Pseudomonas veronii and one as Paenibacillus apiarius. We determined the capability of both species to lyse pre-grown cells of the Gram-negative strains Pseudomonas putida SBUG 24 and Escherichia coli SBUG 13 as well as the Gram-positive strains Micrococcus luteus SBUG 16 and Arthrobacter citreus SBUG 321 on solid media. The bacteriolysis process was analyzed by creating growth curves and electron micrographs of co-cultures with the bacteriolytic isolates and the lysis sensitive strain Arthrobacter citreus SBUG 321 in nutrient-poor liquid media. One metabolite of Paenibacillus apiarius was isolated and structurally characterized by various chemical structure determination methods. It is a novel antibiotic substance.

Antibiotic resistance in mucosal bacteria from high Arctic migratory salmonids.

Two related salmonids, Arctic char (Salvelinus alpinus) and lake whitefish (Coregonus clupeaformis) sampled from the high Arctic region of Nunavut, Canada are anadromous fish, migrating annually from the same ice‐covered freshwater waterbodies to spend summers in the marine waters of the Arctic Ocean. Microbiota associated with the skin‐associated mucus undergo community change coincident with migration, and irrespective of this turnover, antibiotic resistance was detected in mixed bacterial cultures initiated with mucus samples. Although as expected most bacteria were unculturable, however, 5/7 isolates showed susceptibility to a panel of five common antibiotics. The fish were sampled under severe conditions and at remote locations far from human habitation. Regardless, two isolates, ‘Carnobacterium maltaromaticum sm‐2’ and ‘Arthrobacter citreus sm’, showed multi‐resistance to two or more antibiotics including ampicillin and streptomycin indicating multiple resistance genes. It is unknown if these fish bacteria have ‘natural’ resistance phenotypes or if resistance has been acquired. As result of these observations, we urge long‐term monitoring of drug‐resistant bacteria in the region and caution the assumption of a lack of drug‐resistant organisms even in such extreme environments.

A highly efficient method with low energy and water consumption in biodegradation of total petroleum hydrocarbons of oily sludge

The hydrocarbons in petroleum sludge are environmental pollutants. It is crucial to eliminate this type of pollution. In this study, a comprehensive and operational study has been conducted on the total petroleum hydrocarbons (TPH) biodegradation in oily sludge. The experiments were performed in a semi-solid phase by two degrader bacterium, Arthrobacter citreus and Rhodococcus jostii, and they were compared with the slurry phase. Solid samples were prepared in three mixing modes of oily sludge with clay. Experiments were conducted by semi-solid bioreactors and other methods on samples contaminated with petroleum hydrocarbons. The performance of the semi-solid bioreactor for the removal of clay-free oily sludge samples showed the best results, in which biodegradation of TPH was 90.33%. GC analyses were conducted on samples before and after biodegradation. It was observed that the contaminants were decomposed uniformly by the microorganisms, except for a combination with a large peak in 12 min. For the best case, the GC-MS test was performed before and after biodegradation. Compounds with a high concentration in the sludge were significantly reduced. Only one heavy aromatic compound was detected in 51.628 min, which decomposed quite slowly and produced a large peak. It was found that 78.2% of the compounds were removed completely.

Biodegradation of total petroleum hydrocarbons in oily sludge: a comparative study of biostimulation, bioaugmentation, and combination of methods

AbstractBACKGROUNDOne of the major parts of global environmental pollution is petroleum‐derived products. The purpose of this study was the biodegradation of total petroleum hydrocarbons (TPH) in oily sludge using biostimulation and bioaugmentation methods. The oily sludge was dredged from the bottoms of kerosene and oil gas storage tanks, and it was characterized according to American Society for Testing and Materials D1796 and the CHNS (equipment for determination Carbon, Hydrogen, Nitrogen and Sulfour) elements analysis. The TPH biodegradation potential of the indigenous bacteria in the oily sludge was investigated via biostimulation applying mineral salt medium(N). Besides it was compared with bioaugmentation with two oil degrader bacterium, namely Arthrobacter citreus (strain E) and Rhodococcus jostii (strain D), being separated from the oil‐contaminated soil. The gravimetric and GC (Gas Chromatography) methods were used for TPH removal assays.RESULTSThe results indicated that the biodegradation efficiency was dependent on treatment time and microbial species. Increasing pH and TPH biodegradation % for samples were observed over time. The highest removal degradations of hydrocarbons (75.1%) was reported using the inoculum mixture of strains D & E (pH = 7.02) at the 5th week.CONCLUSIONThis method could be used for bioremediation purposes at petroleum hydrocarbon‐contaminated sites as a biological technique. © 2020 Society of Chemical Industry

An Arthrobacter citreus strain suitable for degrading \u03b5-caprolactam in polyamide waste and accumulation of glutamic acid

ε-Caprolactam-a toxic xenobiotic compound present in industrial polyamide waste was found to be degraded by caprolactam-degrading bacteria. Arthrobacter citreus was able to utilize up to 20 g ε-caprolactam/l as the sole source of carbon more efficiently as compared to the other Gram positive caprolactam-degrading bacteria Rhodococcus rhodochrous and Bacillus sphaericus. The cells of A. citreus remained viable in medium up to 40 g caprolactam/l. The degradation of 10 g caprolactam/l by A. citreus, when supplied as the sole source of carbon and nitrogen lead to the formation of 6-aminocaproic acid which was detected in broth and there was also an increase in the ammonium content. One of the other metabolites found to consistently accumulate in extracellular medium during the utilization of caprolactam by A. citreus was glutamic acid, though not reported in case of other caprolactam-degrading bacteria. A. citreus could metabolise caprolactam to form non toxic products such as 6-aminocaproic acid and glutamic acid which are amino acids of physiological and commercial importance. In the presence of 6-aminocaproic acid, the rate of caprolactam utilization by A. citreus was decreased but not inhibited and the viable count of cells was found to increase using both the substrates simultaneously. A. citreus was also suitable for degradation of caprolactam in presence of low phosphate as prevalent in soil, and in sterile soil without the supplementation of any other carbon or nitrogen, as well as in native non sterile soil where other microorganisms are present.

Unexpected formation of N'-phenyl-thiophosphorohydrazidic acid O,S-dimethyl ester from acephate: chemical, biotechnical and computational study.

By the nucleophilic attack of phenylhydrazine on acephate in aqueous medium, a stable product N′-phenyl-thiophosphorohydrazidic acid O,S-dimethyl ester (1) was obtained and characterized by elemental, spectral and thermal analysis. Thermodynamic parameters, Ea, ΔH°, ΔS° and ΔG°, have found to be 62.15, −67.95, −0.068 and −20.05 kJ mol−1 according to the Coats–Redfern equation. Analysis of interaction of 1 with BSA protein was done by using the UV–Vis and FTIR spectroscopic methods. The observed binding constants was 1.12 (±0.09) × 104 M−1. The biological effect of 1 was checked on different plant growth-promoting (PGPR) strains such as Rhizobium leguminosarum, Pseudomonas fluorescens, Arthrobacter citreus, Bacillus brevis and Salmonella typhimurium and compared with parent molecule acephate where 1 has shown less toxicity against PGPRs as compared to acephate. The experimental results for geometric parameters and values of peak position in IR spectra have found to match excellently with the computational studies performed by GAMESS software package. Theoretically, twelve new analogs of 1 were prepared and their comparative reactivity (HOMO–LUMO energy) and biodecomposition (on the basis of polarizability) is discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-015-0313-6) contains supplementary material, which is available to authorized users.

An improved process for biocatalytic asymmetric amine synthesis by in situ product removal using a supported liquid membrane

Chiral amines are important building blocks in the pharmaceutical industry, and the biocatalytic synthesis of these compounds using ω-transaminases has been increasingly studied in recent years. In principal, asymmetric synthesis of chiral amines from a prochiral ketone is the preferable route, but it is often hampered by an unfavourable equilibrium position and product inhibition. An effective method for product removal is therefore necessary to drive the reaction towards product formation. In a recent study (Rehn et al., 2014) [29] we reported on the successful use of a supported liquid membrane (SLM) for the in situ product removal (ISPR) of (S)-α-methylbenzylamine (MBA) produced by Arthrobacter citreus ω-transaminase present in immobilized Escherichia coli cells.In the present work, we thoroughly discuss the factors influencing the performance of the SLM system and considerations for its successful use. Moreover, the system is further improved by implementing continuous control of the reactor pH using the amine donor substrate, and regeneration of the SLM unit at regular intervals to maintain the extraction performance, allowing the accumulation of 1.0M (121g/l) product in the stripping phase during operation for 91h.

Antibacterial metabolites and bacteriolytic enzymes produced by Bacillus pumilus during bacteriolysis of Arthrobacter citreus.

The marine isolate Bacillus pumilus SBUG 1800 is able to lyse living cells of Arthrobacter citreus on solid media as well as pasteurized A. citreus cells in liquid mineral salt medium. The cultivation of B. pumilus in the presence of pasteurized A. citreus is accompanied by an enhanced production of 2,5-diketopiperazines (DKPs). DKPs inhibit bacterial growth, but do not seem to cause bacteriolysis. This study shows that B. pumilus also lyses living cells of A. citreus in co-culture experiments as an intraguild predator, even if the inoculum of B. pumilus is low. In order to characterize the bacteriolytic process, more precisely changes in the extracellular metabolome and proteome have been analyzed under different culture conditions. Besides the known DKPs, a number of different pumilacidins and bacteriolytic enzymes are produced. Two lipopeptides with [M + H](+) = 1008 and [M + H](+) = 1022 were detected and are proposed to be pumilacidin H and I. While the lipopeptides lyse living bacterial cells in lysis test assays, a set of extracellular enzymes degrades the dead cell material. Two of the cell wall hydrolases involved have been identified as N-acetylmuramoyl-L-alanine amidase and beta-N-acetylglucosaminidase. These findings together with electron microscopic and cell growth monitoring during co-culture experiments give a detailed view on the bacteriolytic process.

Chemical composition, antimicrobial and antifungal activities of essential oils of the leaves of Aegle marmelos (L.) Correa growing in Egypt

The essential oil obtained by hydrodistillation of Aegle marmelos (L.)Correa leaves were analyzed by GC/MS yield (0.9% v/w). Twenty seven components were identified representing 97.76 % of the total oil composition. The major components were α-phellenderene (20.97%), α-pinene (17.76%) and δ-carene (16.37%) and other abundant components as γ-cadinene (8.01%), trans-2-hydroxycinnmic acid (6.85%) and β-myrcene (4.32%). The essential oil exhibited significant antibacterial activity against Gram-positive bacteria as Streptococcus faecalis with inhibition zone (30 mm) and Gram-negative bacteria as Pseudomonas aeruginosa (28 mm). Moreover, moderate activity was observed against Bacillus subtilis (23 mm), Staphylococcus aureus (23 mm), Sarcina lutea (20 mm), Arthrobacter citreus (20 mm) and Escherichia coli (25 mm) in comparison with antibiotics. The antifungal activity against Aspergillus niger (30 mm) and Candida albicans (30 mm) was higher than the antifungal antibiotics. Moreover, the oil inhibited the germination of Aspergillus niger and Fusarium oxysporum spores at different concentrations.

Supported liquid membrane as a novel tool for driving the equilibrium of ω-transaminase catalyzed asymmetric synthesis

An attractive option to produce chiral amines of industrial importance is through asymmetric synthesis using ω-transaminase. However, reaching high yields often requires a strategy for shifting the equilibrium position. This paper describes a novel strategy for handling this problem. It involves the use of a supported liquid membrane (SLM) together with a packed bed reactor. The reactor contains Escherichia coli cells with ω-transaminase from Arthrobacter citreus, immobilized by flocculation with chitosan. The SLM consists of a hollow fibre membrane contactor in which the pores contain undecane. The system enables continuous extraction of the amine product and was used to successfully shift the equilibrium in asymmetric synthesis of (S)-α-methylbenzylamine (MBA). A conversion of 98% was reached, compared to 50% without product extraction. Moreover, a selective extraction of the produced MBA was realized. A high product concentration of 55g/l was reached after 80h, and the system showed promising potential for continuous operation.

2,5-Diketopiperazines Produced by Bacillus pumilus During Bacteriolysis of Arthrobacter citreus

We report the detection by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry analyses of the secreted 2,5-diketopiperazines (DKPs) cyclo(-Ala-Pro), cyclo(-Gly-Pro), cyclo(-Val-Pro), cyclo(-Ile-Pro), cyclo(-Leu-Pro), cyclo(-Pro-Pro), cyclo(-HyP-Pro), cyclo(-Met-Pro), and cyclo(-Phe-Pro) produced by Bacillus pumilus. The study focuses on a marine isolate and a laboratory test strain of B. pumilus with capabilities to lyse pregrown living cell lawns of different bacterial species, among them Arthrobacter citreus. Chromatographic methods were used to analyze induced bioactive compounds. At least 13 different DKPs are produced by B. pumilus. Both strains respond with an increased production of the DKPs cyclo(-Gly-Pro), cyclo(-Ala-Pro), and cyclo(-Val-Pro) to the presence of pasteurized A. citreus cells after 4 h in a nutrient-poor liquid medium. In agar diffusion assays, these DKPs did not cause lysis zones in living cell lawns, but they did inhibit further growth of several pregrown test bacteria in microplates even at concentrations as low as 1 μg ml(-1). Antibiotic substances produced by B. pumilus after 20 h of cultivation in a special lysis medium showed lytic activity in cell-free extracts of B. pumilus culture supernatants.

Biodegradation of Phenolic Pollutants

Phenolic compounds are important constituents of petrochemical waste waters arising from transformation processes. Phenol together with substituted halophenols, nitrophenols and alkyl phenols are the main constituents. An Arthrobacter citreus spp. strain MSA - 2005 initially selected for growth at the expense of phenol as the sole source of carbon and energy, was enriched with phenolic compounds such as 4-nitrophenol, 4- chlorophenol and 4-methyl phenol. Metabolization of these compounds upto their maximum levels in the mineral salt medium was studied. The complete metabolization of 4-nitrophenol upto 15mM, 4- chlorophenol up to 12mM and 4-methyl phenol upto 6mM against phenol upto 22mM was achieved in 24 hours. The metabolism of these compounds by the isolated strain occurred through the involvement of catechol derivatives as key intermediates which are further hydrolysed to hydroxy quinol which enters the β -keto adipate pathway. Results indicate that the cells contain mono and di- oxygenase enzymes necessary for these conversions. The degradation rates of these phenolic compounds, by agar immobilized cells has also been reported.

Bacteriolytic Bacillus species isolated from brackish waters of the Southern Baltic Sea

The present study examines antagonistic relationships between different microorganisms inhabiting brackish water and includes a systematic screening for bacteriolytic prokaryotes in the brackish waters of the Southern Baltic Sea (Nordruegenscher Bodden) sampled in July/August 2009. Ten of the 35 marine bacteriolytic isolates belong to the genus Bacillus. Five isolates (B. pumilus, B. subtilis, B. megaterium, B. licheniformis) lysed living microbial cells such as the bacteria Arthrobacter citreus, Arthrobacter protophormiae, Micrococcus luteus, Pseudomonas putida and the yeast Trichosporon mucoides. These and other bacteria (Aeromonas sp., Bacillus subtilis, Chromobacterium violaceum, Citrobacter freundii, Enterobacter aerogenes, Proteus mirabilis, Pseudomonas aeruginosa and Serratia marcescens) were also lysed as autoclaved and pasteurized cells on agar plates. One isolate of Bacillus pumilus showed a distinct bacteriolysis activity against pasteurized cells of A. citreus in liquid culture. Our results suggest that Bacillus species may play a role as opportunistic predators in the marine microbial food web.

Activity and stability of different immobilized preparations of recombinant E. coli cells containing ω-transaminase

Production of chiral amines using ω-transaminases has been thoroughly studied in recent years. Immobilized ω-transaminases, however, have been used on relatively few occasions despite potential benefits such as reuse of enzyme and ease of product purification. In this study principally different methods including surface immobilization, entrapment and sweep flocculation using titanium oxide, Ca-alginate and chitosan respectively were evaluated for the immobilization of recombinant Escherichia coli cells. The enzyme expressed was a modified Arthrobacter citreus ω-transaminase with improved thermostability. The preparations were compared in terms of cell loading capacity, operational stability in repeated batches and storage stability using the conversion of methylbenzylamine to acetophenone.The use of chitosan for cell immobilization proved to be the method of choice since it was both very simple and effective. At a very high cell loading of 3.2g cells/g chitosan >60% activity was observed. The preparation was reused in eight successive 1-h batches with >90% remaining activity. To further demonstrate its usability the preparation was used for asymmetric synthesis of (S)-4′-cyano-(α)-methylbenzylamine in three repeated bathes (cycle time >20h), using isopropylamine as the amine donor. Storage stability was comparable with that of non-immobilized cells. It was concluded that the chitosan method due to its properties and simplicity would be advantageous for use also on a larger scale.

Reversed Enantiopreference of an ω‐Transaminase by a Single‐Point Mutation

AbstractAltering the characteristics of an active‐site loop in an (S)‐selective ω‐transaminase from Arthrobacter citreus (variant CNB05‐01) influences the enantioselectivity. This active‐site loop belongs to the second subunit of the dimeric enzyme structure that participates in the coordination of pyridoxal‐5′‐phosphate (PLP) in the so called “phosphate group binding cup”. Three amino acid residues (E326, V328, and Y331) in this loop are selected by homology modeling for site‐directed mutagenesis aiming to increase the enzyme enantioselectivity for 4‐fluorophenylacetone. By combining these mutations, five enzyme variants are created. The performance of these variants is explored using a model system consisting of isopropylamine and 4‐fluorophenylacetone or 4‐nitroacetophenone in asymmetric synthesis using a whole‐cell system approach. Three of the five variants show increased enantioselectivity for 4‐fluorophenylacetone compared to CNB05‐01. Variant CNB05‐01/Y331C increases the enantioselectivity from 98 % ee to over 99.5 % ee. A single‐point mutation, V328A, turn the (S)‐selective ω‐transaminase into an (R)‐selective enzyme. This switch in enantioselectivity is substrate dependent, exhibiting (R) selectivity for 4‐fluorophenylacetone and retaining (S) selectivity for 4‐nitroacetophenone. The shift in enantiopreference is further confirmed by molecular docking simulations. Homology modeling is shown to be a powerful tool to target important amino acid residues in this enzyme in order to improve enantioselectivity by rational design.