Optically pure methyl (R)-o-chloromandelate and (R)-acetyl-o-mandelic acid are key intermediates for the synthesis of (S)-clopidogrel, which could be prepared with 100 % theoretical yield by sequential hydrolysis and racemization. At the moment, efficient sequential hydrolysis and racemization are hindered by the low catalytic activity of mandelate racemase (MR) toward (S)-o-chloromandelic acid ((S)-2-CMA). In the present work, we proposed to improve the catalytic performance of MR toward (S)-2-CMA by directed evolution and developed an enantioselective oxidation system for high-throughput screening (HTS) of MR libraries. Based on this HTS method, a triple mutant V22I/V29I/Y54F (MRDE1) with 3.5-fold greater relative activity as compared to the native MR was obtained. Kinetic analysis indicated that the enhanced catalytic efficiency mainly arose from the elevated kcat. Further insight into the source of improved catalytic activity was gained by molecular simulations, finding that substrate binding and product release were possibly made easier by decreased steric bulk and increased hydrophobicity of substrate binding sites. In addition, the substrate (S)-2-CMA in the enzyme-substrate complex of MRDE1 seemed to have a lower binding free energy comparing with the complex of wild-type MR. The HTS method developed in this work and the successful directed evolution of MR based on this method provide an example for racemase engineering and may inspire directed evolution of other racemases toward enhanced catalytic performance on non-natural substrates.

D-Fructose-6-phosphate aldolase A (FSAA) from Escherichia coli was engineered for enhanced catalytic efficiency towards cinnamaldehyde. The result implies that the site 59 residue possibly affects the activity of FSAA towards cinnamaldehyde through a residue interaction network rather than binding to the catalytic water as generally regarded.

•Enhanced precursor supply for CoQ10 by disabling or repressing carotenogenesis.•Knockdown of the carotenogenesis pathway by ppsR overexpression.•Improved CoQ10 production by co-overexpressing of crtE and ppsR under tac promoter.In Rhodobacter sphaeroides, synthesis of coenzyme Q10 (CoQ10) shares the same precursor geranylgeranyl diphosphate (GGPP) with carotenoids. Therefore, suppression of carotenoids synthesis is supposed to pose positive effects on accumulation of CoQ10. In this paper, the carotenogenesis pathway was repressed using two different strategies, deletion of carotenogenic genes and overexpression of ppsR, a transcriptional regulator for photosynthesis genes, respectively. Knockout of carotenoids synthesis (crt) genes resulted in undetectable carotenoids as expected. However, the production of CoQ10 and biomass were both decreased to half as compared to the wild-type strain. In contrast, upon overexpression of ppsR, the production of carotenoids was decreased from 15.7 mg/L to 2.2 mg/L, and the CoQ10 production and content were enhanced by 28% and 34.2%, respectively. To further enhance the production of CoQ10, crtE was constitutively co-overexpressed with ppsR to improve the supply of GGPP as a key precursor for the isoprenoid side chain of CoQ10. The CoQ10 production and content of the resulting strain RspPE were increased to 73.2 mg/L and 5.67 mg/g, respectively, representing 47% and 55% improvement as compared to the wild type. This result demonstrated that appropriate reduction of carotenoids rather than complete blocking could enhance the CoQ10 production in Rhodobacter. sphaeroides.

•Strengthening isoprene-forming pathway by improving expression and activity of isoprene synthase.•Adoption of GAL4 overexpression for metabolic regulation of isoprene biosynthesis.•Directed evolution of isoprene synthase based on DMAPP toxicity.•Highest isoprene production ever reported in engineered eukaryotes.Current studies on microbial isoprene biosynthesis have mostly focused on regulation of the upstream mevalonic acid (MVA) or methyl-erythritol-4-phosphate (MEP) pathway. However, the downstream bottleneck restricting isoprene biosynthesis capacity caused by the weak expression and low activity of plant isoprene synthase (ISPS) under microbial fermentation conditions remains to be alleviated. Here, based on a previously constructed Saccharomyces cerevisiae strain with enhanced precursor supply, we strengthened the downstream pathway through increasing both the expression and activity of ISPS to further improve isoprene production. Firstly, a two-level expression enhancement system was developed for the PGAL1-controlled ISPS by overexpression of GAL 4. Meanwhile, the native GAL1/7/10 promoters were deleted to avoid competition for the transcriptional activator Gal4p, and GAL80 was disrupted to eliminate the dependency of gene expression on galactose induction. The IspS expression was obviously elevated upon enhanced Gal4p supply, and the isoprene production was improved from 6.0 mg/L to 23.6 mg/L in sealed-vial cultures with sucrose as carbon source. Subsequently, a novel high-throughput screening method was developed based on precursor toxicity and used for ISPS directed evolution towards enhanced catalytic activity. Combinatorial mutagenesis of the resulting ISPS mutants generated the best mutant ISPSM4, introduction of which into the GAL4-overexpressing strain YXM29 achieved 50.2 mg/L of isoprene in sealed vials, and the isoprene production reached 640 mg/L and 3.7 g/L in aerobic batch and fed-batch fermentations, respectively. These results demonstrated the effectiveness of the proposed combinatorial engineering strategy in isoprene biosynthesis, which might also be feasible and instructive for biotechnological production of other valuable chemicals.

The structure-guided rational design of an NADH-dependent short-chain dehydrogenase/reductase (SDR) reversed the stereoselectivity towards halogenated acetophenones from Prelog to anti-Prelog. The enzyme–substrate interactions involving an aromatic ring and a halogen atom were proven to play critical roles in determining the stereoselectivity of these ketone reductions besides the steric repulsion.

•Enzymatic activity toward halogenated compounds may be impeded by the substitution.•Halogen bond is specific in the complex of proteins and halogenated compounds.•Halogen bond introduction is an efficient strategy in protein engineering.Chlorine substitution in mandelate turned its derivatives into less active substrates for d-mandelate dehydrogenase (DMdh). To improve the catalysis of chloro-mandelate, a halogen bond was introduced into the protein–substrate complex by site mutation. The catalytic activity of the resulting mutant A89H toward o-chloromandelate was improved by 5 times. The effect of halogen bond was also observed in the dehydrogenation of m-chloromandelate and p-chloromandelate. Based on these results, a new insight was proposed for the role of halogen bond in enzyme activity, and the introduction of halogen bond was shown as an efficient strategy to optimize the catalytic activity toward halogenated substrates.

Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce phosphate monoesters and diacylglycerol. It has many applications in the enzymatic degumming of plant oils. PLCBc, a bacterial PLC from Bacillus cereus, is an optimal choice for this activity in terms of its wide substrate spectrum, high activity, and approved safety. Unfortunately, its large-scale production and reliable high-throughput screening of PLCBc remain challenging. Herein, we summarize the research progress regarding PLCBc with emphasis on the screening methods, expression systems, catalytic mechanisms and inhibitor of PLCBc. This review hopefully will inspire new achievements in related areas, to promote the sustainable development of PLCBc and its application.

Further improvement of the enantioselectivity of hydrolases with moderate enantioselectivity is of important significance to fulfill the requirement in industrial application. Herein, a strategy based on sequential hydrolysis and racemization was adopted, using esterase BioH from Escherichia coli as an example. After coupling with a mandelate racemase, the E value of esterase BioH toward methyl (S)-o-chloromandelate was enhanced from 73 to 162, demonstrating the effectiveness of this strategy.

Astaxanthin is a highly valued carotenoid with strong antioxidant activity and has wide applications in aquaculture, food, cosmetic, and pharmaceutical industries. The market demand for natural astaxanthin promotes research in metabolic engineering of heterologous hosts for astaxanthin production. In this study, an astaxanthin-producing Saccharomyces cerevisiae strain was created by successively introducing the Haematococcus pluvialis β-carotenoid hydroxylase (crtZ) and ketolase (bkt) genes into a previously constructed β-carotene hyperproducer. Further integration of strategies including codon optimization, gene copy number adjustment, and iron cofactor supplementation led to significant increase in the astaxanthin production, reaching up to 4.7 mg/g DCW in the shake-flask cultures which is the highest astaxanthin content in S. cerevisiae reported to date. Besides, the substrate specificity of H. pluvialis CrtZ and BKT and the probable formation route of astaxanthin from β-carotene in S. cerevisiae were figured out by expressing the genes separately and in combination. The yeast strains engineered in this work provide a basis for further improving biotechnological production of astaxanthin and might offer a useful general approach to the construction of heterologous biosynthetic pathways for other natural products.

Methyl (R)-o-chloromandelate (R-CMM) is an intermediate for the platelet aggregation inhibitor clopidogrel. Its preparation through enzymatic resolution of the corresponding ester has been hindered by the lack of an enzyme with satisfying enantioselectivity and activity. In the present work, we aimed to improve the enzymatic enantioselectivity towards methyl (S)-o-chloromandelate (S-CMM) by rational design, using esterase BioH as a model enzyme. Based on the differences in the binding mode of S- and R-enantiomers at the active cavity of the enzyme, the steric and electronic interactions between the key amino acids of BioH and the enantiomers were finely tuned. The enantioselectivity of esterase BioH towards S-CMM was improved from 3.3 (the wild type) to 73.4 (L123V/L181A/L207F). Synergistic interaction was observed between point mutations, and insight into the source of enzymatic enantioselectivity was gained by molecular dynamics simulations. The results can provide a reference for the enzyme design of other enzymes towards S-CMM for the enhancement of enantioselectivity.

Many studies have demonstrated that the properties of enzymes expressed in eukaryotes can be affected by the position and extent of glycosylation on enzyme. In this study, two potential glycosylation sites (the 8th and the 58th asparagine) were identified and the effect of propeptide glycosylation on Rhizomucor miehei lipase (RML) expressed in Pichia pastoris was investigated. To better understand the effect of glycosylation on the activity of RML, three mutants (M1, generated by N8A; M2, generated by N58A; and M3, generated by N8A and N58A) were designed to generate deglycosylated enzymes. The results showed that deglycosylated RML exhibited a twofold higher activity compared to the wild type. However, it was also found that glycosylation on the propeptide was important for the removal of the propeptide by Kex2 protease and secretion of the enzyme. Thus, our study provided a further understanding into the role of glycosylation on enzyme function.

“Amano” lipase AS(lipase from Aspergillus niger), which naturally hydrolyzes triglycerides, was found promiscuously to catalyze multi-component reactions of aromatic aldehydes with malononitrile and β-naphthol to prepare naphthopyran derivatives in anhydrous organic solvents in moderate to good yields.

Ten lipases and esterases have been examined to catalyse the reaction between p-nitrobenzaldehyde and methyl vinyl ketone, the Baylis–Hillman reaction, to form 3-[hydroxyl-(4-nitrophenyl)-methyl]-but-3-en-2-one. Among these enzymes, Escherichia coli BioH esterase had the best activity. Optimal conditions for this reaction were: 0.1 mmol aldehyde, 0.1 mmol activated alkene, 30 mg E. coli BioH, 1 ml acetonitrile at 30 °C for 96 h. In addition to the named substrates, four other aldehydes and three activated alkenes were also investigated to determine the substrate range of the enzyme. The structures of nine products were confirmed by NMR and yields of the corresponding products ranged from 21 to 46 %.

Esterase BioH, which is obligatory for biotin synthesis in Escherichia coli, was found to exhibit a promiscuous ability to catalyse Aldol and Knoevenagel reactions with moderate to good yields. The reaction conditions including organic solvent, molar ratio of ketone to aldehyde, enzyme amount, and reaction time were investigated to evaluate the effect of different reaction conditions on yield. Target compounds were afforded in the best yield of 91.2% for Aldol reaction and 54.7% for Knoevenagel reaction. In addition, because the enzyme could be prepared with a low cost, this protocol could provide an economic route to conduct Aldol and Knoevenagel reactions, which expand the field of enzymatic promiscuity.

A novel high-throughput screening method for directed co-evolution of endoglucanase and β-glucosidase was constructed based on the synergy of the two enzymes using a bicistronic operon. To our knowledge, this is the first report of directed co-evolution of two cellulosic enzymes using an insoluble screening substrate via their synergistic reactions.

Despite directed evolution being a practical and efficient method of improving the properties of enzymes, a trade-off between the targeted property and other essential properties often exists which hinders the efficiency of directed evolution. In our previous work, mutant CVH of an esterase from Rhodobacter sphaeroides with high enantioselectivity was obtained by directed evolution, unfortunately its activity cannot catch another mutant YH. To compensate the trade-off of mutant CVH, site-directed saturation mutagenesis was conducted on four residues, three (Asn62, Met121, and Leu145) were hot spots determined from directed evolution, and one (Tyr27) was introduced to make up the large distance between a mutation (Asn62) and the substrate. A new mutant (HMVY) with high enantioselectivity and comparable activity to YH was obtained. According to the kinetic analysis and molecular dynamics simulations, it was understood that the high enantioselectivity and poor activity of mutant CVH was caused by different decrement of efficiency constants to two isomers, (R)-, (S)-methyl mandelate, and the high enantioselectivity and activity of mutant HWVY was caused by improved activity towards the preferred substrate ((S)-methyl mandelate), which provided the interpretation of the trade-off compensation. This work could provide a way to compensate the trade-off of enantioselectivity and activity in the process of enzyme evolution.

We constructed a biosynthetic pathway of isoprene production in Escherichia coli by introducing isoprene synthase (ispS) from Populus alba. 1-deoxy-d-xylulose 5-phosphate synthase (dxs), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (dxr) and isopentenyl diphosphate (IPP) isomerase (idi) were overexpressed to enhance the isoprene production. The isoprene production was improved 0.65, 0.16, and 1.22 fold over the recombinant BL21 (pET-30a-ispS), respectively, and idi was found to be a key regulating point for isoprene production. In order to optimize the production of isoprene in E. coli, we attempted to construct polycistronic operons based on pET-30a with genes dxs, dxr, and idi in various orders. The highest isoprene production yield of 2.727 mg g−1 h−1 (per dry weight) was achieved by E. coli transformed with pET-30a-dxs/dxr/idi. Interestingly, the gene order was found to be consistent with that of the metabolic pathway. This indicates that order of genes is a significant concern in metabolic engineering and a sequential expression pattern can be optimized according to the biosynthetic pathway for efficient product synthesis.

The present work created an esterase variant from Rhodobacter sphaeroides (RspE) with enhanced selectivity in hydrolytic kinetic resolutions by directed evolution. A “model” substrate, methyl mandelate, was introduced in the high-throughput screening procedure. E values of a variant CH (Asn62Cys/Leu145His) for six different esters were 10–83, which were a relative improvement compared to 2–20 for the wild type. Our subsequent crystal structure interpretation and molecular dynamics simulations helped shed light on the source of enantioselectivity modified by directed evolution. Though mutations displayed no “direct” interaction with the substrate, they were hypothesized to strengthen the intramolecular interaction in the catalytic cavity of variant. Conformation analysis revealed that the enhanced enantioselectivity of variant CH for the seven substrates applied in this study was derived from the decrease in size of the substrate binding pocket.

Propeptides are short sequences that facilitate the folding of their associated proteins. The present study found that the propeptide of Rhizomucor miehei lipase (RML) was not proteolytically removed in Escherichia coli. Moreover, RML was not expressed if the propeptide was removed artificially during the cloning process in E. coli. This behavior in E. coli permitted the application of directed evolution to full-length RML, which included both propeptide and catalytic domain, to explore the role played by the propeptide in governing enzyme activity. The catalytic rate constant, kcat, of the most active mutant RML protein (Q5) was increased from 10.63 ± 0.80 to 71.44 ± 3.20 min−1 after four rounds of screening. Sequence analysis of the mutant displayed three mutations in the propeptide (L57V, S65A, and V67A) and two mutations in the functional region (I111T and S168P). This result showed that improved activity was obtained with essential involvement by mutations in the propeptide, meaning that the majority of mutants with enhanced activity had simultaneous mutations in propeptide and catalytic domains. This observation leads to the hypothesis that directed evolution has simultaneous and synergistic effects on both functional and propeptide domains that arise from the role played by the propeptide in the folding and maturation of the enzyme. We suggest that directed evolution of full-length proteins including their propeptides is a strategy with general validity for extending the range of conformations available to proteins, leading to the enhancement of the catalytic rates of the enzymes.

Thirty-one ester hydrolases were cloned from Escherichia coli K-12 and an efficient screening strategy was applied to screen and characterize them, emphasizing on their enantioselectivity. We are the first to investigate the enantioselectivity of these enzymes, although their activity had been reported by other researchers. The enzyme XL3 from gene b0349, XL10 from gene b0494, XL15 from gene b3412, XL27 from gene b2154 and XL31 from gene b3825 exhibited high activity towards p-nitrophenyl esters with short chain. The enzyme XL15 from gene b3412 was demonstrated for the first time to show high enantioselectivity to (R)-1-phenylethyl acetate both in hydrolysis and esterification with enantioselectivity value (E) > 100 at the conversion of 31.2 and 36.8%, respectively.

A green and fully enzymatic procedure for the resolution of chiral alcohols through lipase/esterase-catalyzed acylation and subsequent lipase-catalyzed aminolysis using anhydrous ammonia was demonstrated. Both enantiomers can be obtained in high ee values (up to >99%) under ambient reaction conditions. The solvent and acyl donors can be recycled, and the enzyme can be reused for up to five times.

Escherichia coli BioH, which is obligatory for biotin synthesis, was found to be an organic solvent tolerant esterase with high enantioselectivity for the kinetic resolution of sec-alcohols using free enzyme powder. With this esterase, a variety of racemic sec-alcohols were efficiently resolved with ee values of up to 99%.An organic solvent tolerant esterase was found exhibiting high enantioselectivity for kinetic resolution of sec-alcohols and a variety of sec-alcohols were efficiently resolved with ee values of up to 99%.

An efficient enzymatic method for the synthesis of (S)-3-substituted glutaric acid monoesters which was aided by molecular docking has been described. The reaction was proceeded under mild conditions, and the desired products were afforded with up to 98% ee in the yield of 93%. The results demonstrate that molecular docking is efficient to facilitate selection of substrates in enzymatic reaction.An efficient enzymatic method for the synthesis of (S)-3-substituted glutaric acid monoesters which was aided by molecular docking has been described. The reaction was proceeded under mild conditions, and the desired products were afforded with up to 98% ee in the yield of 93%. The results demonstrate that molecular docking is efficient to facilitate selection of substrates in enzymatic reaction.

The purpose of this theoretical discussion is to help researchers understand how some parameters and reaction type (first-order and Michaelis−Menten kinetic) could impact effective enantioselectivity of enzyme in a fixed-bed reactor, including adsorption equilibrium constant (adsorption effect), Biot number, Peclet number, and bed length parameter. The theoretical analysis clearly derives that reaction rate is able to impact the effective enantioselectivity of enzyme through controlling the loading of enzyme on the solid support. In addition, adsorption phenomenon and reaction type have no effect on the effective enantioselectivity in a fixed-bed reactor. Among these parameters, Bi (<10) shows significant effect on the effective enantioselectivity, which indicates that external mass transfer limitation must be controlled. The interesting analysis suggests that cross-link enzyme aggregates without carrier could be an optional way to overcome mass transfer limitation for immobilized enzyme, and if solid support is applied, adsorption method, which is able to facilitate substrates to contact with enzyme compared to other immobilization methods, could be the best choice to immobilize enzyme when the kinetic resolution of racemate is conducted in a fixed-bed reactor.

Adsorption, a typical physical phenomenon, always neglected in the kinetic resolution of the racemate catalyzed by enzymes immobilized on the porous solid support, was theoretically analyzed. A concept that the adsorption effect plays an important role to affect the superficial performance of the enzyme which is often applied to evaluate the performance of the enzyme was described. Adsorption intensity, or so-called mass equilibrium constant (K), is the most important factor to affect the performance of the enzyme compared with adsorption rate coefficient (k) and adsorption equilibrium (adsorption isotherm) if the solid support with chiral selectivity is applied. The conclusion can be drawn that the solid support with a weak adsorption effect on the racemate may favorite the enzyme to exhibit a good performance in the kinetic resolution of the racemate. The adsorption phenomenon should be considered as an explanation to the performance changing of the enzyme after immobilized.

A new and efficient method was developed for the first time using the targeted substrate, triacylglycerol, for the rapid screening of a large library of mutants for lipase directed evolution. The high-throughput method applies calcium chloride to convert the fatty acid produced from hydrolysis reaction to hydrochloric acid which is easy to release H+ detected by pH indicator. This new method exhibits significant advantages compared with the conventional method of applying a substituent of the actual targeted substrate, p-nitrophenyl palmitate as a high-throughput screening substrate. The results determined by the new method correlate well with those obtained from alkali titration and HPLC analysis. By applying the new method, a significantly enhanced mutant enzyme was obtained with lipase activity increased more than five-fold compared to wild type after only two cycles of directed evolution. In addition, the esterification activity of the mutant was also improved and the regioselectivity of the lipase was not changed.Graphical abstractDownload full-size imageHighlights► The new high-throughput method applies calcium chloride to convert the fatty acid produced from hydrolysis of triacylglycerol reaction to hydrochloric acid which is easily detected by pH indicator. ► The new high-throughput screening method was successfully implemented in the directed evolution of RML and a significantly enhanced mutant enzyme was obtained. ► This new method exhibits significant advantages compared with the conventional method of applying a substituent of the actual targeted substrate, p-nitrophenyl palmitate as a high-throughput screening substrate.

Prediction of enzyme enantioselectivity in silico could be of major utility for avoiding the expensive and time-consuming experiments. Herein, we aimed to develop a new approach to construct a quantitative enantioselectivity prediction model with high accuracy for Candida antarctica lipase B (CALB). In the work, Autodock was used to generate substrate conformations for improving the calculation efficiency, followed by the quantitative structure–activity relationship (QSAR) analysis. The effects of acyl donors and 5 molecular interaction fields (steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor fields) on model construction were investigated. The results indicated that the application of actual acyl donors was indispensible for model construction. Inclusion of the relevant molecular interaction fields could significantly improve the predictive accuracy which suggested that enantioselectivity was a consequence of multiple molecular interactions. The final model was derived based on four molecular interaction fields (steric, electrostatic, hydrophobic, hydrogen bond acceptor fields) with actual acyl donors owning higher predictive accuracy (Rpred2=0.92) than previous report (Rpred2=0.79). Furthermore, the contour map produced by the model facilitated us to better elucidate the molecular basis of enzyme enantioselectivtiy, and was potential for the application of rational design of the enzyme.Graphical abstractDownload full-size imageHighlights• Quantitative enantioselectivity prediction model combining QSAR with Autodock. • Explore the effects of acyl donors and relevant force fields on model construction. • The model based on actual acyl donors and core force fields predicts more accurate. • The model helps us to better elucidate the origin of enzyme enantioselectivtiy. • The contour map produced was potential for the rational design of enzyme.

•Enhanced CoQ10 production in R. sphaeroides by a novel synergic regulation method.•Overexpression of gapA-1 effectively improved the CoQ10 content in R. sphaeroides.•The order of gapA-1 and vgb in polycistrons influenced both cell growth and activity.•Highest CoQ10 production among R. sphaeroides constructed by metabolic engineering.The physiological role of Coenzyme Q10 (CoQ10) as an electron carrier suggests its association with redox potential. Overexpression of glyceraldehyde-3-phosphate dehydrogenase type I (gapA-1) in Rhodobacter sphaeroides elevated the NADH/NAD+ ratio and meanwhile enhanced the CoQ10 content by 58%, but at the sacrifice of biomass. On the other hand, Vitreoscilla hemoglobin was heterologously expressed to enhance the oxygen uptake ability of the cells, leading to 127% improvement of biomass. Subsequent coexpression of gapA-1 and vgb resulted in a CoQ10 titer of 83.24 mg/L, representing 71% improvement as compared to the control strain RspMCS. When gapA-1 and vgb genes were co-expressed in a previously created strain RspMQd [1], 163.5 mg/L of CoQ10 was produced. Finally, 600 mg/L of CoQ10 production was achieved in fed-batch fermentation. These results demonstrated the synergic effect of redox potential regulation and oxygen uptake improvement on enhancing CoQ10 production in R. sphaeroides.

•A novel whole-cell biocatalyst with anti-Prelog stereoselectivity was reported.•Activity and selectivity of the biocatalyst were notablely enhanced by 10% ethanol.•The whole-cell biocatalyst showed strong tolerance against 10% ethanol.•The biocatalyst exhibited excellent performance over a broad pH range of 6.0–9.5.•The biocatalyst showed a broad substrate spectrum and excellent selectivity (>99% ee).Robust biocatalysts are in high demand for the reduction of prochiral ketones to anti-Prelog chiral alcohols. A recently isolated bacterial strain ZJUY-1401 exhibited high reduction activity and excellent anti-Prelog stereospecificity toward prochiral ketones. Based on the colony and microscopic morphology, physiological tests, and 16S rDNA sequence, the isolate was identified as Empedobacter brevis. Upon addition of either NADH or NADPH, the whole cells of E. brevis ZJUY-1401 showed enhanced catalytic activity. The activity and stereoselectivity of the biocatalyst toward acetophenone were significantly increased in the presence of 10% (v/v) ethanol as cosubstrate. Important properties concerning the application of E. brevis ZJUY-1401 include the excellent catalytic performance over a broad pH range from 6.0 to 9.5, temperature optimum of 35 °C, and noticeable tolerance against ethanol. Under the optimal conditions, E. brevis ZJUY-1401 was highly active for the reduction of acetophenone derivatives, giving corresponding alcohols in excellent enantiomeric purity (>99% ee). These results imply that E. brevis ZJUY-1401 is a promising biocatalyst for the preparation of anti-Prelog chiral alcohols.Empedobacter brevis ZJUY-1401, a novel whole-cell biocatalyst with excellent anti-Prelog stereoselectivity, was for the first time successfully applied in the asymmetric reduction of prochiral ketones.Download full-size image

⿢A novel biocatalyst EbSDR8 with anti-Prelog stereoselectivity was reported.⿢NADH can be efficiently regenerated via addition of isopropanol as cosubstrate.⿢The recombinant whole-cell could catalyze the reaction without NADH addition.⿢The biocatalyst exhibited excellent performance over a broad pH range of 7.0⿿10.5.⿢EbSDR8 showed a broad substrate spectrum and excellent selectivity (>99% ee).Empedobacter brevis ZJUY-1401 is capable of producing anti-Prelog alcohols with excellent stereoselectivity. The gene encoding a short-chain dehydrogenase/reductase from E. brevis ZJUY-1401 (EbSDR8) was cloned and heterologously expressed in Escherichia coli, and the purified recombinant protein was characterized. The subunit of EbSDR8 is composed of 250 amino acids with a calculated molecular mass of 26.4 kDa. Important properties regarding the application of EbSDR8 include utilization of cheaper coenzyme, the excellent catalytic performance over a broad pH range from 7.0 to 10.5, and temperature optimum of 35 °C. The enzyme showed moderate thermostability, with half-lives of 4.4 h at 35 °C and 3.1 h at 45 °C, respectively. In the presence of isopropanol as a cosubstrate, the whole-cell of recombinant E. coli expressing EbSDR8 could efficiently catalyze the asymmetric reduction without addition of any NADH into the reaction system. EbSDR8 displayed good activity and excellent stereoselectivity toward a spectrum of acetophenone derivatives, providing anti-Prelog alcohols with >99% ee for the majority of the substrates. These results suggest that EbSDR8 is a powerful chiral tool for the production of anti-Prelog alcohols.Download high-res image (206KB)Download full-size image

•A virtual screening method based on the binding energy was developed.•The method could efficiently predict the mutational effect on catalysis.•Mutants exhibiting enhanced activities to non-natural substrates were obtained.•Enhanced interaction between the active site and substrate led to higher activity.Mandelate racemase (MR) is a promising candidate for the dynamic kinetic resolution of racemates. However, the poor activity of MR towards most of its non-natural substrates limits its widespread application. In this work, a virtual screening method based on the binding energy in the transition state was established to assist in the screening of MR mutants with enhanced catalytic efficiency. Using R-3-chloromandelic acid as a model substrate, a total of 53 mutants were constructed based on rational design in the two rounds of screening. The number of mutants for experimental validation was brought down to 17 by the virtual screening method, among which 14 variants turned out to possess improved catalytic efficiency. The variant V26I/Y54V showed 5.2-fold higher catalytic efficiency (kcat/Km) towards R-3-chloromandelic acid than that observed for the wild-type enzyme. Using this strategy, mutants were successfully obtained for two other substrates, R-mandelamide and R-2-naphthylglycolate (V26I and V29L, respectively), both with a 2-fold improvement in catalytic efficiency. These results demonstrated that this method could effectively predict the trend of mutational effects on catalysis. Analysis from the energetic and structural assays indicated that the enhanced interactions between the active sites and the substrate in the transition state led to improved catalytic efficiency. It was concluded that this virtual screening method based on the binding energy in the transition state was beneficial in enzyme rational redesign and helped to better understand the catalytic properties of the enzyme.Download full-size image

•An alkaline phytase from Bacillus subtilis 168 was engineered using directed evolution.•Catalytic activity in acidic and neutral environments was successfully improved.•Underlying mechanism for the enhanced activity was revealed by molecular docking.As a feed additive, the desirable features of phytase include thermostability, strict substrate specificity, and high stability in a wide pH range. Alkaline phytases from Bacillus subtilis are promising candidates because of their high inherent thermostability and substrate specificity, but the application is restricted due to their poor specific activity in acidic and neutral conditions. Directed evolution was utilized in this paper to improve the activity of phytase from B. subtilis 168 in neutral and acidic environments. After two rounds of evolution, a number of positive mutants were picked out from the mutant library. Compared with the wild type, variants D24G, S51A, D24G/K265N, D24G/K70R/K111E/N121S showed 29.7%, 13.5%, 42.7%, 42.8% improvement in specific activity under the optimal reaction conditions (pH 7.0, 60 °C) and 76.6%, 79.5%, 84.2%, 121.1% improvement at pH 4.5 and 37 °C, respectively. The catalytic efficiency (kcat/Km) of D24G, D24G/K265N, D24G/K70R/K111E/N121S at pH 7.0 was increased by 132%, 110%, 131% as compared with that of the wild type, and the improvement at pH 4.5 was 98.0%, 114.5%, 163.3%, respectively. Molecular docking results revealed that D24, S51 and K70 had a relatively large influence on the catalytic activity probably because they are adjacent to the active site cleft.

•An esterase was cloned from a marine bacterium and overexpressed in Escherichia coli.•The enzyme was alkalitolerance and halotolerance.•The enzyme preferred short chain p-nitrophenyl esters, and was not a metalloenzyme.•The enzyme was useful in the synthesis of methyl (R)-3-(4-fluorophenyl)glutarate.•(R)-3-MFG was obtained in 71.6% ee and 73.2% yield after 36 h reaction.An esterase, designated as PE8 (219 aa, 23.19 kDa), was cloned from a marine bacterium Pelagibacterium halotolerans B2T and overexpressed in Escherichia coli Rosetta, resulting an active, soluble protein which constituted 23.1% of the total cell protein content. Phylogenetic analysis of the protein showed it was a new member of family VI lipolytic enzymes. Biochemical characterization analysis showed that PE8 preferred short chain p-nitrophenyl esters (C2–C6), exhibited maximum activity toward p-nitrophenyl acetate, and was not a metalloenzyme. PE8 was an alkaline esterase with an optimal pH of 9.5 and an optimal temperature of 45 °C toward p-nitrophenyl acetate. Furthermore, it was found that PE8 exhibited activity and enantioselectivity in the synthesis of methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG) from the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG). (R)-3-MFG was obtained in 71.6% ee and 73.2% yield after 36 h reaction under optimized conditions (0.6 M phosphate buffer (pH 8.0) containing 17.5% 1,4-dioxane under 30 °C). In addition, PE8 was tolerant to extremely strong basic and high ionic strength solutions as it exhibited high activity even at pH 11.0 in 1 M phosphate buffer. Given its highly soluble expression, alkalitolerance, halotolerance and enantioselectivity, PE8 could be a promising candidate for the production of (R)-3-MFG in industry. The results also demonstrate the potential of the marine environment as a source of useful biocatalysts.Download full-size image

D-Fructose-6-phosphate aldolase A (FSAA) from Escherichia coli was engineered for enhanced catalytic efficiency towards cinnamaldehyde. The result implies that the site 59 residue possibly affects the activity of FSAA towards cinnamaldehyde through a residue interaction network rather than binding to the catalytic water as generally regarded.

A novel high-throughput screening method for directed co-evolution of endoglucanase and β-glucosidase was constructed based on the synergy of the two enzymes using a bicistronic operon. To our knowledge, this is the first report of directed co-evolution of two cellulosic enzymes using an insoluble screening substrate via their synergistic reactions.

The structure-guided rational design of an NADH-dependent short-chain dehydrogenase/reductase (SDR) reversed the stereoselectivity towards halogenated acetophenones from Prelog to anti-Prelog. The enzyme–substrate interactions involving an aromatic ring and a halogen atom were proven to play critical roles in determining the stereoselectivity of these ketone reductions besides the steric repulsion.