Directed Evolution and Immobilization of Lactobacillus brevis Alcohol Dehydrogenase for Chemo-Enzymatic Synthesis of Rivastigmine.

Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues, such as the involvement of toxic metals and harsh reaction conditions. Herein, we report a chemo-enzymatic synthesis of Rivastigmine. The key chiral intermediate was synthesized by an engineered alcohol dehydrogenase from Lactobacillus brevis (LbADH). A semi-rational approach was employed to improve its catalytic activity and thermal stability. Several LbADH variants were obtained with a remarkable increase in activity and melting temperature. Exploration of the substrate scope of these variants demonstrated improved activities toward various ketones, especially acetophenone analogs. To further recycle and reuse the biocatalyst, one LbADH variant and glucose dehydrogenase were co-immobilized on nanoparticles. By integrating enzymatic and chemical steps, Rivastigmine was successfully synthesized with an overall yield of 66 %. This study offers an efficient chemo-enzymatic route for Rivastigmine and provides several efficient LbADH variants with a broad range of potential applications.

Efficient Production of 3-Amino-2-Hydroxy Acetophenone by Multi-Enzyme Biosynthesis.

We developed a synthetic route for producing 3-amino-2-hydroxy acetophenone (3AHAP) from m-nitroacetophenone (3NAP) using an in vitro approach. Various reaction systems were evaluated, and a direct reaction method with crude enzyme and supersaturated substrates for optimal catalytic efficiency was chosen. The reaction system included three enzymes and was enhanced by adjusting enzyme molar ratios and optimizing ribosomal binding sites. We performed substrate docking and alanine scanning to identify key sites in the enzymes nitrobenzene nitroreductase (nbzA) and hydroxylaminobenzene mutase (habA). The optimal mutant was obtained through site-directed mutagenesis, and incorporated into the reaction system, resulting in increased product yield. After optimization, the yield of 3AHAP increased from 75 mg/L to 580 mg/L within 5 hours, the highest reported yield using biosynthesis. This work provides a promising strategy for the efficient and sustainable production of 3AHAP, which has critical applications in the chemical and pharmaceutical industries.

Paeonol inhibits inflammatory response and protects chondrocytes by upregulating sirtuin 1.

Paeonol is the bioactive component in Paeonia lactiflora Pall., Cynanchum paniculatum and Paeonia × suffruticosa Andr. Paeonol has been previously demonstrated to inhibit the release of tumor necrosis factor α (TNF-α) and interluekin 6 (IL-6) in chondrocytes. Sirtuin 1 (SIRT1) is downregulated in degraded cartilage and paeonol could induce nuclear accumulation of SIRT1. Therefore, the present study aims to investigate the possible role of paeonol in chondrocyte inflammation and cartilage protection in osteoarthritis (OA) as well as its regulation of SIRT1. Primary chondrocytes from rat knee joints were transfected with short hairpin (sh) - SIRT1 and (or) paeonol prior to IL-1ß exposure, and then inflammatory response, apoptosis, and extracellular matrix (ECM) degradation in the cells were evaluated concurrent with the activation of the nuclear factor κß (NF-κß) signaling pathway. Increased levels of TNF-α, IL-17, IL-6, matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-13 along with decreased tissue inhibitor of metalloproteinases 1 and type II collagen levels were found in IL-1ß-stimulated chondrocytes. Chondrocyte apoptosis was elevated and the NF-κß signaling pathway was activated in response to IL-1ß treatment. Paeonol enhanced SIRT1 expression to inactivate the NF-κß signaling pathway, thereby ameliorating inflammatory cytokine secretion, ECM degradation, and chondrocyte apoptosis. In conclusion, the results of the present study confirm the potential of paeonol as a candidate OA drug.

An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants.

A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. '84K'). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and ß-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.

A novel organic cation transporter involved in paeonol transport across the inner blood-retinal barrier and changes in uptake in high glucose conditions.

Paeonol exerts various pharmacological effects owing to its antiangiogenic, antioxidant, and antidiabetic activities. We aimed to investigate the transport mechanism of paeonol across the inner blood-retinal barrier both in vitro and in vivo. The carotid artery single injection method was used to investigate the retina uptake index of paeonol. The retina uptake index (RUI) value of [³H]paeonol was dependent on both concentration and pH. This value decreased significantly in the presence of imperatorin, tramadol, and pyrilamine when compared to the control. However, para-aminohippuric acid, choline, and taurine had no effect on the RUI value. Conditionally immortalized rat retina capillary endothelial cells (TR-iBRB cell lines) were used as an in vitro model of the inner blood-retinal barrier (iBRB). The uptake of [³H]paeonol by the TR-iBRB cell lines was found to be time-, concentration-, and pH-dependent. However, the uptake was unaffected by the absence of sodium or by membrane potential disruption. Moreover, in vitro structural analog studies revealed that [³H]paeonol uptake was inhibited in the presence of organic cationic compounds including imperatorin, clonidine and tramadol. This is consistent with the results obtained in vivo. In addition, transfections with OCTN1, 2 or plasma membrane monoamine transporter (PMAT) small interfering RNA did not affect paeonol uptake in TR-iBRB cell lines. Upon pre-incubation of these cell lines with high glucose (HG) media, [3H]paeonol uptake decreased and mRNA expression levels of angiogenetic factors, such as hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) increased. However, after the pretreatment of unlabeled paeonol in HG conditions, the mRNA levels of VEGF and HIF-1 were comparatively reduced, and the [3H]paeonol uptake rate was restored. After being exposed to inflammatory conditions induced by glutamate, TNF-α, and LPS, paeonol and propranolol pretreatment significantly increased the uptake of both [3H]paeonol and [3H]propranolol in TR-iBRB cell lines compared to their respective controls. Our results demonstrate that the transport of paeonol to the retina across the iBRB may involve the proton-coupled organic cation antiporter system, and the uptake of paeonol is changed by HG conditions.

Analysis of carotenoid profile changes and carotenogenic genes transcript levels in Rhodosporidium toruloides mutants from an optimized Agrobacterium tumefaciens-mediated transformation method.

Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium-mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T-DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 105 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and ß-carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1-13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ-carotene and ß-carotene, and the proportion of γ-carotene and ß-carotene were all over 92%. TAIL-PCR was carried out to found T-DNA insertion in these mutants, and insertion hotspot was found. RT-qPCR results showed that CTA1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.

Chromoselective Photocatalysis Enables Stereocomplementary Biocatalytic Pathways*.

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93 % ee).

Exploring efficacy of natural-derived acetylphenol scaffold inhibitors for α-glucosidase: Synthesis, in vitro and in vivo biochemical studies.

The discovery of novel α-glucosidase inhibitors and anti-diabetic candidates from natural or natural-derived products represents an attractive therapeutic option. Here, a collection of acetylphenol analogues derived from paeonol and acetophenone were synthesized and evaluated for their α-glucosidase inhibitory activity. Most of derivatives, such as 9a-9e, 9i, 9m-9n and 11d-1e, (IC50 = 0.57 ± 0.01 µM to 8.45 ± 0.57 µM), exhibited higher inhibitory activity than the parent natural products and were by far more potent than the antidiabetic drug acarbose (IC50 = 57.01 ± 0.03 µM). Among these, 9e and 11d showed the most potent activity in a non-competitive manner. The binding processes between the two most potent compounds and α-glucosidase were spontaneous. Hydrophobic interactions were the main forces for the formation and stabilization of the enzyme - acetylphenol scaffold inhibitor complex, and induced the topography image changes and aggregation of α-glucosidase. In addition, everted intestinal sleeves in vitro and the maltose loading test in vivo further demonstrated the α-glucosidase inhibition of the two compounds, and our findings proved that they have significant postprandial hypoglycemic effects.

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants.

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP+ ) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.

Expression and function of the ectopic olfactory receptor OR10G7 in patients with atopic dermatitis.

BACKGROUND: Ectopic olfactory receptors (ORs) are found in the skin, but their expression and biological function in normal skin and skin form patients with atopic dermatitis (AD) are unknown. OBJECTIVES: We sought to characterize the expression of ORs in the skin and assess OR-mediated biological responses of primary human keratinocytes in the presence of odorant ligands. METHODS: OR expression was examined by using whole-transcriptome sequencing of skin tape strips collected from patients with AD and healthy control (HC) subjects. OR10G7 and filaggrin 1 (FLG-1) expression was analyzed by using RT-PCR and immunostaining in skin biopsy specimens and primary human keratinocytes from patients with AD and HC subjects. ATP and cyclic AMP production by control and OR10G7 small interfering RNA-transfected keratinocytes in response to odorant stimulation with acetophenone and eugenol was assessed. RESULTS: A total of 381 OR gene transcripts were detected in the skin samples, with the greatest OR expression detected in the skin tape strips corresponding to the upper granular layer of the skin. OR10G7 expression was significantly increased in skin biopsy specimens from patients with AD compared with those from HC subjects (P = .01) and inversely correlated with FLG-1 expression (P = .009). OR10G7 expression was greatest in undifferentiated keratinocytes from patients with AD and was downregulated with progressive differentiation. Primary human keratinocytes produced ATP, an essential neurotransmitter in sensory pathways, in response to acetophenone and eugenol, odorants previously identified as potential ligands for this receptor. This response was abolished in OR10G7 small interfering RNA-transfected keratinocytes. CONCLUSIONS: OR10G7 is expressed at significantly greater levels in undifferentiated keratinocytes from patients with AD compared with HC subjects. OR10G7 is likely involved in transmission of skin-induced chemosensory responses to odorant stimulation, which might modulate differential nociceptive responses in AD skin.

Taguchi analysis and asymmetric keto-reduction of acetophenone and its derivatives by soil filamentous fungal isolate: Penicillium rubens VIT SS1.

Microbial asymmetric reduction of ketone is an efficient tool for the synthesis of chiral alcohols. This research focuses on exploring the soil fungal isolates for their ability toward the keto reduction of acetophenone and its derivatives to their corresponding chiral alcohols using growing cells. Bioreduction of acetophenone, 4-fluoro acetophenone, 4-methyl acetophenone, and 3-hydroxy acetophenone was carried out using different fungal cultures isolated from soil. Among the fungal isolates, Penicillium sp. and Aspergillus sp. showed significant bioconversion with varying enantio-selectivity. However, the Penicillium sp. has shown the maximum ability of bioreduction. The potential isolate was characterized using the internal transcribed spacer (ITS) region and found to be Penicillium rubens VIT SS1 (Genbank accession number: MK063869.1), which showed higher conversion and selectivity > 90%. The biocatalyst production and the reaction conditions were optimized using Taguchi analysis. The process conditions such as pH, temperature, media components, cosolvent, and substrate dosing were evaluated for the bioreduction of 3-hydroxy acetophenone, which is a key chiral intermediate of Phenylephrine and Rivastigmine using P. rubens VIT SS1. This study concludes about the potential of fungal cultures for sustainable synthesis of key chiral intermediates of Phenylephrine and Rivastigmine, similarly many aromatic chiral alcohols in simpler, novel, and cost-effective manner.

Pharmacokinetics and Metabolism of Liposome-Encapsulated 2,4,6-Trihydroxygeranylacetophenone in Rats Using High-Resolution Orbitrap Liquid Chromatography Mass Spectrometry.

2,4,6-trihydroxy-3-geranylacetophenone (tHGA) is a bioactive compound that shows excellent anti-inflammatory properties. However, its pharmacokinetics and metabolism have yet to be evaluated. In this study, a sensitive LC-HRMS method was developed and validated to quantify tHGA in rat plasma. The method showed good linearity (0.5-80 ng/mL). The accuracy and precision were within 10%. Pharmacokinetic investigations were performed on three groups of six rats. The first two groups were given oral administrations of unformulated and liposome-encapsulated tHGA, respectively, while the third group received intraperitoneal administration of liposome-encapsulated tHGA. The maximum concentration (Cmax), the time required to reach Cmax (tmax), elimination half-life (t1/2) and area under curve (AUC0-24) values for intraperitoneal administration were 54.6 ng/mL, 1.5 h, 6.7 h, and 193.9 ng/mL·h, respectively. For the oral administration of unformulated and formulated tHGA, Cmax values were 5.4 and 14.5 ng/mL, tmax values were 0.25 h for both, t1/2 values were 6.9 and 6.6 h, and AUC0-24 values were 17.6 and 40.7 ng/mL·h, respectively. The liposomal formulation improved the relative oral bioavailability of tHGA from 9.1% to 21.0% which was a 2.3-fold increment. Further, a total of 12 metabolites were detected and structurally characterized. The metabolites were mainly products of oxidation and glucuronide conjugation.

Comparative metabolome-based classification of Senna drugs: a prospect for phyto-equivalency of its different commercial products.

INTRODUCTION: The demand to develop efficient and reliable analytical methods for the quality control of nutraceuticals is on the rise, together with an increase in the legal requirements for safe and consistent levels of its active principles. OBJECTIVE: To establish a reliable model for the quality control of widely used Senna preparations used as laxatives and assess its phyto-equivalency. METHODS: A comparative metabolomics approach via NMR and MS analyses was employed for the comprehensive measurement of metabolites and analyzed using chemometrics. RESULTS: Under optimized conditions, 30 metabolites were simultaneously identified and quantified including anthraquinones, bianthrones, acetophenones, flavonoid conjugates, naphthalenes, phenolics, and fatty acids. Principal component analysis (PCA) was used to define relative metabolite differences among Senna preparations. Furthermore, quantitative 1H NMR (qHNMR) was employed to assess absolute metabolites levels in preparations. Results revealed that 6-hydroxy musizin or tinnevellin were correlated with active metabolites levels, suggesting the use of either of these naphthalene glycosides as markers for official Senna drugs authentication. CONCLUSION: This study provides the first comparative metabolomics approach utilizing NMR and UPLC-MS to reveal for secondary metabolite compositional differences in Senna preparations that could readily be applied as a reliable quality control model for its analysis.

Structural and dynamic basis of substrate permissiveness in hydroxycinnamoyltransferase (HCT).

Substrate permissiveness has long been regarded as the raw materials for the evolution of new enzymatic functions. In land plants, hydroxycinnamoyltransferase (HCT) is an essential enzyme of the phenylpropanoid metabolism. Although essential enzymes are normally associated with high substrate specificity, HCT can utilize a variety of non-native substrates. To examine the structural and dynamic basis of substrate permissiveness in this enzyme, we report the crystal structure of HCT from Selaginella moellendorffii and molecular dynamics (MD) simulations performed on five orthologous HCTs from several major lineages of land plants. Through altogether 17-µs MD simulations, we demonstrate the prevalent swing motion of an arginine handle on a submicrosecond timescale across all five HCTs, which plays a key role in native substrate recognition by these intrinsically promiscuous enzymes. Our simulations further reveal how a non-native substrate of HCT engages a binding site different from that of the native substrate and diffuses to reach the catalytic center and its co-substrate. By numerically solving the Smoluchowski equation, we show that the presence of such an alternative binding site, even when it is distant from the catalytic center, always increases the reaction rate of a given substrate. However, this increase is only significant for enzyme-substrate reactions heavily influenced by diffusion. In these cases, binding non-native substrates 'off-center' provides an effective rationale to develop substrate permissiveness while maintaining the native functions of promiscuous enzymes.

The use of apocynin inhibits osteoclastogenesis.

Reactive oxygen species (ROS) are produced by NADPH oxidase (NOX), an enzyme that reduces oxygen by using NADPH as a substrate. Apocynin (APO) is a catechol that is used as a NOX inhibitor, and N-acetyl-cysteine ​​(NAC) can reduce intracellular ROS levels. In this work, the effect of APO and NAC on osteoclast formation were evaluated. APO and NAC significantly decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive cells and the osteoclast area. We analyzed bone-marrow derived monocyte-macrophages (BMMs) that differentiated into osteoclasts after RANKL stimulation. Stimulation was associated with either APO or NAC treatment and osteoclastogenesis marker expression, including NFATc1, MMP-9, and DC-STAMP, was evaluated. APO decreased the intracellular calcium concentration by calcium channels other than ITPR1 and TPC2. On the other hand, APO reduced Tnfrsf11a (RANK) expression and did not alter Fam102a (EEIG1) expression. Therefore, our results demonstrate that APO inhibits osteoclastogenesis by the RANK-RANKL-related signaling pathways, decreases osteoclast markers, and reduces intracellular calcium concentration.

Enantioselective synthesis of enantiopure chiral alcohols using carbonyl reductases screened from Yarrowia lipolytica.

AIMS: We aimed to explore Yarrowia lipolytica carbonyl reductases as effective biocatalysts and to develop efficient asymmetric reduction systems for chiral alcohol synthesis. METHODS AND RESULTS: Yarrowia lipolytica carbonyl reductase genes were obtained via homologous sequence amplification strategy. Two carbonyl reductases, YaCRI and YaCRII, were identified and characterized, and used to catalyse the conversion of 2-hydroxyacetophenone (2-HAP) to optically pure (S)-1-phenyl-1,2-ethanediol. Enzymatic assays revealed that YaCRI and YaCRII exhibited specific activities of 6·96 U mg-1 (99·8% e.e.) and 7·85 U mg-1 (99·9% e.e.), respectively, and showed moderate heat resistance at 40-50°C and acid tolerance at pH 5·0-6·0. An efficient whole-cell two-phase system was established using reductase-expressing recombinant Escherichia coli. The conversion of 2-HAP (20·0 g l-1 ) conversion with the solvent of dibutyl phthalate was approximately 70-fold higher than in water. Furthermore, the two recombinant E. coli displayed biocatalyst activity and enantioselectivity towards several different carbonyl compounds, and E. coli BL21 (DE3)/pET-28a-yacrII showed a broad substrate spectrum. CONCLUSIONS: A new whole-cell recombinant E. coli-based bioreduction system for enantiopure alcohol synthesis with high enantioselectivity at high substrate concentrations was developed. SIGNIFICANCE AND IMPACT OF THE STUDY: We proposed a promising approach for the efficient preparation of enantiopure chiral alcohols.

Reductive capabilities of different cyanobacterial strains towards acetophenone as a model substrate - Prospect of applications for chiral building blocks synthesis.

Bioreductive capabilities of four morphologically different strains of cyanobacteria have been assessed in this work. Arthrospira maxima, Leptolyngbya foveolarum, Nodularia sphaerocarpa and Synechococcus bigranulatus were applied as catalysts for the reduction of acetophenone to the corresponding chiral phenylethyl alcohol. The process was modified regarding substrate concentration, duration of pre-cultivation period, duration of biotransformation, light regime and glucose addition to the culture media. Obtained results clearly showed that cyanobacteria were active towards acetophenone what resulted in the substrate reduction to (S)-1-phenylethanol with high enantiomeric excess. The reaction efficiency increased with the biotransformation time, but the higher concentration of substrate limited the process yield. Also, all tested strains performed reaction with the highest efficacy under continuous light regime. The most active strains - N. sphaerocarpa and S. bigranulatus carried out the conversion of 1 mM acetophenone with high efficiency of respectively 97.6% and 96.2% after 13 days of biotransformation. A. maxima reached 45.8% of conversion after 13 days of biotransformation whereas L. foveolarum did not exceed 20%. The enantiomeric excesses were respectively 98.8%- A. maxima, 91.7%- L. foveolarum, 72.6%- S. bigranulatus and N. sphaerocarpa 16.2%.

Key sites insight on the stereoselectivity of four mined aldo-keto reductases toward α-keto esters and halogen-substituted acetophenones.

Biocatalytic reduction catalyzed by aldo-keto reductases (AKRs) is a valuable approach for asymmetric synthesis of chiral alcohols. In this study, four novel aldo-keto reductases with significant activity and stereoselectivity toward a variety of α-keto esters and halogen-substituted acetophenones were identified by genome mining. Through analysis of the crystal structure and multiple-sequence alignment of the starting AKR YvgN from Bacillus subtilis, residues F25 and W113 were proposed as the key positions that might control the stereoselectivity of YvgN. F25S and F25S/W113F variants of YvgN were able to improve its activity and stereoselectivity toward some α-keto ester compounds and halogen-substituted acetophenone derivatives. In addition, similar enhancement of catalytic activity and stereoselectivity was also found in the other three AKRs with corresponding mutations of starting YvgN.

Reversible control of enantioselectivity by the length of ketone substituent in biocatalytic reduction.

Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from (S) to (R) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best (R)-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding (R)-alcohol with 97% ee. Our docking simulation suggested that when enantioselectivity inverted to (R), only pro-R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro-R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the (S) form.

Haloferax volcanii as immobilised whole cell biocatalyst: new applications for halophilic systems.

Enzyme-mediated synthesis of pharmaceutical compounds is a 'green' alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell-mediated biocatalysis by the halophilic archaeon H. volcanii.