The interaction of antitumor drug, cisplatin (cis-[PtCl2(NH3)2], CDDP) with insulin from porcine pancreas has been investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high resolution hybrid ion trap/time-of-flight mass spectrometry (MALIDI-TOF/TOF–MS and ESI-IT/TOF MS). The MALDI-TOF/TOF–MS results demonstrated that the presence of cisplatin complex resulted in the reduction of the disulfide bond in porcine pancreas after the incubations of the two substances were performed in vitro. It indicated that the presence of cisplatin would destroy the native configuration of insulin, which may lead to the inactivation of insulin. High resolution mass values and the characteristic isotopic pattern of the platinated insulin ions allowed the analysis of platinated mono-, di- and triadducts of cisplatin and insulin in the incubations under different conditions. The laser-induced dissociation of the monoadduct obtained in MALDI source was carried out and one platinum was found to bind to insulin B chain was determined. The platinum binding sites were further identified to be the N terminus (B chain), cysteine 7 (B chain) and cysteine 19 (B chain) residues by electrospray ionization tandem mass spectrometry. The identification of the interaction between insulin and cisplatin broadens the horizon of the knowledge in the interaction of the proteins and metallodrugs.

A concise strategy to improve the p-NPP (p-nitrophenyl palmitate) catalytic activity and enantioselectivity towards secondary alcohols of Pseudomonas cepacia lipase (PcL) has been described. The PcL was modified by I3−, N-acetyl imidazole (NAI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and ethylenediamine (EDA) in the absence or presence of n-hexane, respectively. After being modified by the four modification reagents, the enantioselectivity (E value) of the PcL towards secondary alcohols was enhanced by 2- to 4-fold. The catalytic activity of EDA-PcL was increased by about 6-fold. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of modified PcL showed that Tyr4, Tyr29, Tyr45, Tyr95, Asp36 and Asp55 were the modified sites. When Tyr29 was modified, the E value of PcL towards secondary alcohols was largely improved. MALDI-TOF-MS characterization and molecular dynamics simulation of the lipase indicated that Tyr29 located inside the catalytic cavity had a significant impact on the E value. The strong steric hindrance of acetyl and iodine ion to the groups on the chiral center of the substrates is responsible for the improvement. In addition, the enhancement of hydrophobicity on the surface of the lipase due to the sidechain replacement of Asp with uncharged hydrophobic groups also improved the E value.

•This work is the first time reported in research of structure and bioactivity of an alkali-soluble single-component polysaccharide (named CPTC-2) isolated and purified from Taxus chinensis var. mairei by ion-exchange and gel-permeation chromatography in series.•The authors used a variety of methods to analyze the structure properties of polysaccharide and get a fully understanding of the structure of CPTC-2. Moreover, the combination of MTS assay and flow cytometry method was adopted to study the antitumor function of CPTC-2.•Finally, the partial relationship between structure of the polysaccharide and its antitumor activity was obtained.An alkali-soluble single-component polysaccharide, named CPTC-2, was isolated and purified from the leaves of Taxus chinensis var. mairei. by ion-exchange and gel-permeation chromatography in series. The weight-average molecular mass (Mw) of CPTC-2 was about 73.53 kDa determined by gel permeation chromatography (GPC). The structural characteristics of CPTC-2 were analyzed by gas chromatography (GC), Infrared (IR) spectrum, nuclear magnetic resonance (NMR) spectroscopy, periodate oxidation and Smith degradation studies, as well as methylation analysis. The results showed that CPTC-2 consisted of glucose, mannose, xylose, arabinose, rhamnose, and galactose with a molar ratio of 1.00:0.32:0.27:3.34:1.22:1.84. CPTC-2 was mainly composed of one type of sugar, α-glycosidic linkage. It had a backbone composed of α-(1 → 3) Araf, α-(1 → 5) Araf and α-(1 → 4) Galp with branches composed of α-(1 → 3,5) Araf and β-(1 → 3,6) Manp. In vitro anti-tumor experiments with SGC-7901 cells were performed and assessed by MTS (MPEG-2 Transport Stream) method and flow cytometry. The results showed CPTC-2 could inhibit the growth of SGC-7901 cells in a concentration-dependent manner via increased apoptosis. The relationship between the structure of CPTC-2 and its anti-tumor activity indicated that the α-configuration glycosidic bond residues may be essential for the anti-tumor activity of this polysaccharide.

tert-Butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) is a valuable chiral synthon, which is used for the synthesis of the cholesterol-lowering drugs atorvastatin and rosuvastatin. To date, only the alcohol dehydrogenases from Lactobacillus brevis (LbADH) and Lactobacillus kefir (LkADH) have demonstrated catalytic activity toward the asymmetric reduction of tert-butyl 6-chloro-3,5-dioxohexanoate (CDOH) to (S)-CHOH. Herein, a tetrad mutant of LkADH (LkTADH), A94T/F147L/L199H/A202L, was screened to be more efficient in this bioreduction process, exhibiting a 3.7- and 42-fold improvement in specific activity toward CDOH (1.27 U/mg) over LbADH (0.34 U/mg) and wild-type LkADH (0.03 U/mg), respectively. The molecular basis for the improved catalytic activity of LkTADH toward CDOH was investigated using homology modeling and docking analysis. Two major issues had a significant impact on the biocatalytic efficiency of this process, including (i) the poor aqueous stability of the substrate and (ii) partial substrate inhibition. A fed-batch strategy was successfully developed to address these issues and maintain a suitably low substrate concentration throughout the entire process. Several other parameters were also optimized, including the pH, temperature, NADP+ concentration and cell loading. A final CDOH concentration of 427 mM (100 g/L) gave (S)-CHOH in 94 % yield and 99.5 % e.e. after a reaction time of 38 h with whole cells expressing LkTADH. The space–time yield and turnover number of NADP+ in this process were 10.6 mmol/L/h and 16,060 mol/mol, respectively, which were the highest values ever reported. This new approach therefore represents a promising alternative for the efficient synthesis of (S)-CHOH.

The enantioselective biocatalytic hydrolysis of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (rac-cyanodiester, rac-CNDE) to obtain a key (S)-intermediate is crucial for the synthesis of Pregabalin. The gene estZF172 from Pseudomonas CGMCC No. 4184, which encoded a novel esterase EstZF172 with excellent stereoselective catalysis capacity of (R)-CNDE (E = 95), was obtained from genomic library construction through activity screening. EstZF172 was identified to be a new member of the bacterial esterase/lipase Family VIII by sequence alignment, phylogenetic tree analysis and homology model analysis, with preference toward shortchain p-nitrophenyl esters. The esterase was functionally expressed in E. coli BL21(DE3), exhibiting a 120-fold improvement in catalytic activity (4027.5 U/L) over the wild strain (33.43 U/L) toward CNDE. For the chiral hydrolysis of rac-CNDE catalyzed by recombinant cells, the optimum operating temperature and pH were determined to be 35°C and 8.5, respectively, based on the biochemical characterization of the purified EstZF172. Finally, the yield and ee of (S)-CNDE reached 47.7% and > 99.5% after reaction for 7 h with a substrate loading of 127.5 g/L (500 mM). The results suggested that EstZF172 is a potential biocatalyst for the synthesis of an important chiral intermediate of Pregabalin.

A novel and efficient racemization catalyst, Pd/layered double-hydroxide-dodecyl sulfate anion, was prepared and used in the dynamic kinetic resolution (DKR) of arylamines. The undesired enantiomer was completely racemized at 55 °C, allowing the catalyst to be compatible with biocatalysts. DKR proceeded smoothly and showed a broad substrate scope, with good conversion and high product enantiomeric excesses (eep). The system could be reused more than 30 times without loss of conversion and eep value.A novel and efficient racemization catalyst, Pd/layered double-hydroxide-dodecyl sulfate anion, was prepared and used in the dynamic kinetic resolution (DKR) of arylamines. The undesired enantiomer was completely racemized at 55 °C, allowing the catalyst to be compatible with biocatalysts. DKR proceeded smoothly and showed a broad substrate scope, with good conversion and high product enantiomeric excesses (eep). The system could be reused more than 30 times without loss of conversion and eep value.

The biocatalytic cascade conversion of ethyl 4-chloroacetoacetate (COBE) to ethyl (R)-4-cyano-3-hydroxybutyrate ((R)-HN) for the preparation of atorvastatin represents significant economic and environmental benefits, and is catalyzed by alcohol dehydrogenase and halohydrin dehalogenase (HHDH). However, as the activity of HHDH is inhibited by COBE, the cascade reaction is an inefficient one-pot reaction. In this study, substrate inhibition kinetics analysis was performed and the inhibition by COBE was found to be competitive reversible inhibition. Molecular simulation analysis was used to determine the inhibition mechanism by COBE. The results showed that COBE bound to the active center of HHDH via the formation of hydrogen bonds with the OH groups of S132 and Y145. Site saturation mutagenesis of residues around the active site and at the entrance of the access tunnel was performed, and two target mutant residues were identified, F136 and W249. Small focused mutagenesis on these two residues was performed and the F136V/W249F mutant was successfully found to relieve the activity inhibition of HHDH by COBE. The half inhibiting concentration of mutant F136V/W249F was found to be 20-fold higher than wild-type HHDH. The efficiency of the multi-enzymatic one-pot system for the synthesis of (R)-HN from COBE using mutant F136V/W249F was improved significantly.

The resolutions of racemic diastereomeric mixtures of menthyl propionate was performed by Pseudomonas alcaligenes lipase (PaL) to produce (2S, 5R) L-menthol. Because of the inherently low diastereopreference of PaL, covalent docking and molecular dynamic (MD) simulations were used to investigate possible avenues of improvement. Rational site-directed mutagenesis of PaL revealed residues V180 and A272 to be the hotspots for diastereopreference. The double V180L/A272F mutant exhibited the highest degree of diastereopreference, as the diastereomeric ratio of (2S, 5R) L-menthol increased towards both (2R, 5S) L-neomenthol (dr1) and (2R, 5R) D-isoneomenthol (dr2) (diastereomeric ratios dr1 and dr2 increased to 4.65 and 2.13 times that of wild-type PaL). MD simulation analysis indicated that these mutations decrease the flexibility of the surrounding protein regions. The combination of increased steric exclusion and decreased flexibility results in less favorable binding of the non-target substrates, (2R, 5S) L-neomenthyl propionate and (2R, 5R) D-isoneomenthyl propionate, to the V180L/A272F mutant. These results confirmed and further improved our previously proposed model of the diastereomer recognition mechanism based on the combined effect of steric exclusion and regional flexibility.

Lipase-catalyzed remote resolution of the tertiary alcohol, citalopram intermediate (diol acetate), has been achieved. The chiral discrimination was obtained by the Novozym435-catalyzed alcoholysis of the primary hydroxyl ester which was four bonds away from the center. The influence of acyl acceptor structure and the organic solvents on the reaction rate and enantioselectivity were investigated. Based on the thermodynamic analysis, the difference of activation free energy between the two enantiomers which dominated the enantioselectivity was significantly affected by the organic solvents, while the acyl acceptor showed less effect. In addition, the enantiomer discrimination was driven by both the difference of activation enthalpy and activation entropy. The thermodynamic analysis provides further insights into the prediction and optimization of enantioselectivity and reaction rate in remote resolution.

A new and efficient dynamic kinetic resolution (DKR) process of secondary aromatic alcohols was developed with acid resins as racemization catalysts. Acid resin CD8604 was shown to have excellent racemization activity and good biocompatibility. When employing CD8604 and complex acyl donors as racemization catalyst and acyl donor, respectively, enantiomerically pure aromatic acetate was obtained with excellent yield and ee values through the DKR process. It is noteworthy that the system could be reused more than 10 times with little loss of yield and ee value.

The enzyme-aggregate coating method was performed to immobilize Arthrobacter sp. lipase in order to achieve better catalytic properties comparable to the conventional covalent attachment and covalent attachment plus cross-linking. The glutaraldehyde-activated amino-silica gel which was synthesized by sol–gel technique was used as the support, and the catalytic characteristics of the lipase preparations were tested in the asymmetric acylation of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one (HMPC) in organic solvents. The results showed that the immobilized lipase by enzyme-aggregate coating possessed both higher activity and stability than those by other methods, e.g. it obtained an activity of 82.6 U/g and remained 42% and 93% of the original activity after incubation in vinyl acetate at 60 °C for 16 h and 9 times recycles, respectively, while the covalently attached lipase got an activity of 67.4 U/g and left 33% and 73% of the original under the same conditions, and the enzyme prepared by covalent attachment plus cross-linking exhibited the lowest activity yield. Moreover, excellent enantioselectivity (E ≥ 400) was achieved by all the three prepared lipases in our paper (E = 85 for the free enzyme).

A new method to separate citalopram intermediate (diol), one of chiral aromatic amino-alcohols, from its acetate after lipase-catalyzed kinetic resolution using liquid–liquid extraction was developed. Diol was firstly derivatized with anhydrides and then extracted with different solvent systems. The effects of pH and initial diol acetate concentration on the total distribution coefficients of diol acetate were investigated. It was found that succinic anhydride was the best derivative agent and water/toluene was the optimal extraction system. The extraction conditions with initial diol acetate concentration of 10 mmol/L, pH 8.0 were applied to the practical extraction process of the mixture of diol and diol acetate obtained by lipase-catalyzed reaction. After three extraction stages at 25 °C, the purity of diol was 99.5% and the yield achieved 86.2%.

A protein thin film-modified electrode sensor, that features both generalizability and simplicity in design toward reagentless detection of hydrogen peroxide with high sensitivity and reliability, is reported here. Within this electrode device, the film active material of the nanocomposite of myoglobin and zirconium (iv) ion-adenosine monophosphate dianion particles forming via monolayer adsorption of protein, is fabricated on a glassy carbon surface using self-assembly technique. The electrode modification helps in facilitating the direct electron transfer kinetics of protein at the formal potential (E°′) of 12.3 mV versus SHE (pH 7.0). As a result, the potential applied in H2O2 determination through reduction can be shifted to −3.7 mV, a useful characteristic for further applications. Furthermore, the electrode configuration provides sufficient operational stability for sensing. Detection limit of 0.06 μM and the linear calibration range up to 148.47 μM H2O2 are obtained for this sensor. The sensor assay can retain a value of 91.7% initial activity within 1 month.

An efficient and convenient strategy for synthesis of enantiomerically pure S-2-(1-hydroxy-3-butenyl)-5-methylfuran was for the first time described utilizing a lipase-mediated asymmetric acylation in organic solvents. Rhizopus arrhizus lipase was chosen as the biocatalyst, and different immobilization methods including sol–gel encapsulation and covalent attachment were adopted to improve its catalytic characteristics. Various influential factors of the reaction were also investigated. Finally, the results showed that the lipase covalently attached onto epoxy resin exhibited the highest enantioselectivity and operational stability. Under optimized reaction conditions, i.e., n-hexane as the solvent, 5/1 (mol/mol) of vinyl acetate to 2-(1-hydroxy-3-butenyl)-5-methylfuran and 30 °C, the ee value of S-1 reached up to above 98% at 52% conversion with an E value of 99.

A facile method for the preparation of tert-butyl (3R,5S)-6-hydroxy-3,5-O-isopropylidene-3,5-dihydroxyhexanoate is described by a chemoenzymatic approach. In this method, one hydroxyl stereocenter at C5 is obtained with a high ee value (up to 98.0%) via an enzymatic transesterification resolution of l-chloro-3-(4-methylbenzyloxy)-2-propanol. The other hydroxyl stereocenter at C3 was built with 98.0% de, by acid-hydrolysis of a 1,3-diol-acetonide syn/anti-10. It is noteworthy that the reduction of β-hydroxy ketone 8 with sodium borohydride can be carried out smoothly in aqueous isopropyl alcohol with a high diastereomeric ratio syn/anti (drs:a) of 4.0:1.(R)-l-Chloro-3-benzyloxy-2-propanolC10H13ClO2Ee = 97.3% [by chiral HPLC][α]D20=+1.5 (c 3.3, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2R)(R)-1-Chloro-3-(2,4-dichlorobenzyloxy)-2-propanolC10H11Cl3O2Ee = 96.8% [by chiral HPLC][α]D20=+2.2 (c 3.4, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2R)(R)-1-Chloro-3-(4-methyoxybenzyloxy)-2-propanolC10H15ClO3Ee = 98.2% [by chiral HPLC][α]D20=+1.9 (c 3.9, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2R)(R)-1-Chloro-3-(4-fluorobenzyloxy)-2-propanolC10H12ClFO2Ee = 98.1% [by chiral HPLC][α]D20=+2.4 (c 1.8, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2R)(R)-1-Chloro-3-(4-chlorobenzyloxy)-2-propanolC10H12Cl2O2Ee = 96.7% [by chiral HPLC][α]D20=+2.2 (c 3.2, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2R)(R)-l-Chloro-3-(4-methylbenzyloxy)-2-propanolC11H15ClO2Ee = 98.0% [by chiral HPLC][α]D20=+2.0 (c 3.5, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2R)(S)-2-Acetoxy-1-chloro-3-benzyloxy-2-propanolC12H15ClO3Ee = 88.5% [by chiral HPLC][α]D20=+7.3 (c 3.3, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2S)(S)-2-Acetoxy-1-chloro-3-(2,4-dichlorobenzyloxy)-2-propanolC12H13Cl3O2Ee = 84.5% [by chiral HPLC][α]D20=+6.8 (c 3.4, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2S)(S)-2-Acetoxy-1-chloro-3-(4-methyoxybenzyloxy)-2-propanolC13H17ClO4Ee = 90.1% [by chiral HPLC][α]D20=+8.8 (c 3.5, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2S)(S)-2-Acetoxy-1-chloro-3-(4-fluorobenzyloxy)-2-propanolC12H14ClFO5Ee = 91.5% [by chiral HPLC][α]D20=+8.4 (c 3.7, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2S)(S)-2-Acetoxy-1-chloro-3-(4-chlorobenzyloxy)-2-propanolC12H14Cl2O2Ee = 96.0% [by chiral HPLC][α]D20=+8.9 (c 3.1, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2S)(S)-2-Acetoxy-l-chloro-3-(4-methylbenzyloxy)-2-propanolC13H17ClO5Ee = 96.0%[by chiral HPLC][α]D20=+8.7 (c 2.2, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (2S)tert-Butyl (S)-6-(4-methybenzyloxy)-5-hydroxy-3-oxohexanoateC18H26O5Ee = 98.0% [by chiral HPLC][α]D20=-13.0 (c 2.5, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (5S)tert-Butyl (3R,5S)-6-(4-methybenzyloxy)-3,5-O-isopropylidene-3,5-dihydroxyhexanoateC21H32O5De = 98.0% [by GC][α]D20=-6.5 (c 2.0, CHCl3)Source of chirality: enzymatic resolution and chemical resolutionAbsolute configuration: (3R,5S)tert-Butyl (3R,5S)-6-hydroxy-3,5-O-isopropylidene-3,5-dihydroxyhexanoateC13H24O5De = 98.0% [by GC][α]D20=+9.9 (c 2.0, CHCl3)Source of chirality: enzymatic resolution and chemical resolutionAbsolute configuration: (3R,5S)

The kinetic resolution of 4-arylmethoxy-3-hydroxybutanenitriles was investigated by lipase-catalyzed transesterification in organic solvents. A high enantioselectivity was obtained via reaction with vinyl acetate in a mixed solvent (n-heptane/acetonitrile 1:1), which was catalyzed by the lipase from Artgribacter sp. A better selectivity was demonstrated when the number of substituents on the aryl ring increased. (S)-4-Arylmethoxy-3-hydroxybutanenitriles can be obtained with enantiomeric excesses of up to 98.0% by this method. Furthermore we have developed a novel route to synthesize tert-butyl (S)-6-benzyloxy-5-hydroxy-3-oxohexanoate, a key intermediate for the preparation of HMG-CoA reductase inhibitors (statins).(S)-4-Benzyloxy-3-hydroxybutanenitrileC11H13NO2Ee = 98.0% [by chiral HPLC][α]D20=-3.3 (c 3.3, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (3S)(S)-4-(4-Chlorobenzyloxy)-3-hydroxybutanenitrileC11H12ClNO2Ee = 96.2% [by chiral HPLC][α]D20=-2.6 (c 3.2, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (3S)(S)-4-(2,4-Dichlorobenzyloxy)-3-hydroxybutanenitrileC11H11Cl2NO2Ee = 96.1% [by chiral HPLC][α]D20=-0.6 (c 3.7, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (3S)(S)-4-(4-Methlybenzyloxy)-3-hydroxybutanenitrileC12H15NO2Ee = 97.0% [by chiral HPLC][α]D20=-2.6 (c 3.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: (3S)(S)-4-(4-Methoxybenzyloxy)-3-hydroxybutanenitrileEe = 96.3% [by chiral HPLC][α]D20=-1.8 (c 3.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 3S(S)-4-(2,4,6-Trimethylbenzyloxy)-3-hydroxybutanenitrileEe = 92.7% [by chiral HPLC][α]D20=+3.0 (c 3.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 3S(S)-4-(4-Fluorobenzyloxy)-3-hydroxybutanenitrileEe = 99.0% [by chiral HPLC][α]D20=-1.4 (c 3.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 3S(R)-3-Acetyloxy-4-benzyloxy-3-hydroxybutanenitrileEe = 70.0% [by chiral HPLC][α]D20=+3.4 (c 3.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 3R(R)-3-Acetyloxy-4-(4-chlorobenzyloxy)-3-hydroxybutanenitrileEe = 85.0% [by chiral HPLC][α]D20=+4.0 (c 5.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 3R(R)-3-Acetyloxy-4-(2,4-dichlorobenzyloxy)-3-hydroxybutanenitrileEe = 90.1% [by chiral HPLC][α]D20=+4.6 (c 4.5, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 3R(R)-3-Acetyloxy-4-(4-methylbenzyloxy)-3-hydroxybutanenitrileEe = 81.3% [by chiral HPLC][α]D20=+4.0 (c 3.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 3R(R)-3-Acetyloxy-4-(4-methoxybenzyloxy)-3-hydroxybutanenitrileEe = 82.3% [by chiral HPLC][α]D20=+4.2 (c 5.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 3R(R)-3-Acetyloxy-4-(2,4,6-trimethylbenzyloxy)-3-hydroxybutanenitrileEe = 99.0% [by chiral HPLC][α]D20=+8.0 (c 3.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 3R(R)-4-Acetyloxy-(4-fluorobenzyloxy)-3-hydroxybutanenitrileEe = 85.0% [by chiral HPLC][α]D20=+3.0 (c 3.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 3Rtert-Butyl (S)-6-(benzyloxy)-5-hydroxy-3-oxohexanoateEe = 98.0% [by chiral HPLC][α]D20=-13.6 (c 2.0, CHCl3)Source of chirality: enzymatic resolutionAbsolute configuration: 5S

•A new hydrogen-bond-based mechanism lipase's stereo-recognition was revealed.•The deviant characteristics of enantioselectivity toward chiral primary alcohol were explained.•Chemical modification of the lipase was adapted to alter its selectivity.•Restricted molecular dynamics simulation was employed to study the mechanism.The stereo-recognition of chiral primary alcohols by lipase from Pseudomonas cepacia was found to deviate from earlier observations. Enantioselectivity toward 14 pairs of chiral primary alcohol esters by this lipase was dependent on the existence of an Onon-α (oxygen at non-α-position of the acyloxy group) in the alcohol moiety, and decreased as the size of the acyl moiety increased. Chemical modification on the lipase and molecular dynamics simulations indicated that Tyr29 located within the catalytic cavity forms a hydrogen bond with the Onon-α of the preferred enantiomer of the primary alcohol ester. However, a larger acyl moiety suffered stronger hindrance from the catalytic cavity wall of the lipase, pushing the Onon-α away from Tyr29, and thus weakening the stereo-recognition.Download full-size image

Lipase's performance decreases severely in high-viscosity solvent-free system. In this article, common commercial lipases were screened for such a typical system: synthesis of 1,3-diolein by solvent-free esterification of glycerol and oleic acid. Among those enzymes, MjL (Mucor javanicus lipase) was of the best activity and reasonable selectivity. Catalytic performance of MjL was further improved by immobilization onto a novel kind of porous magnetic polymeric microsphere carrier. Glycerol-oleic-acid-esterification activity of the immobilized lipase increased by sevenfold compared with the enzyme in native form. Specific activity based on total protein content of the immobilized enzyme was 200-fold greater than that of the native enzyme. It was noteworthy that operational stability of the lipase was critically improved. The immobilized lipase showed no obvious activity loss after 30 batches of reaction, while the native enzyme kept only 25% after 5 batches. Further characterization by various techniques suggested that the carrier was superparamagnetic, and had core–shell microsphere structure, porous surface composed of hydrophobic polymer. These properties greatly benefited the immobilized lipase's activation, stabilization and recycling.Graphical abstractDownload full-size imageHighlights► It was the first time that surface modified magnetic carrier was employed for lipase immobilization in high-viscosity system. ► The carrier had advantages of both hydrophobic hyperactivation effect on lipase and magnetic material's convenience of separation. ► The immobilized Mucor javanicus lipase's activity increased by sevenfold. ► The immobilized lipase was able to be reused for 30 batches, while the native enzyme can only be used less than 5 batches.

Lipase from Arthrobacter sp. was immobilized onto low-cost diatomite materials using different protocols for the resolution of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one (HMPC) by asymmetric acylation. The support surface was grafted various functional groups including methacryloxypropyl, vinyl, octyl, dodecyl and γ-(aminopropyl)-glutaraldehyde. These modifications resulted in various mechanisms during the immobilization and thus introduced different characteristics to the prepared lipases. The interfacially adsorbed lipase onto dodecyl-modified support exhibited both higher activity and stability among these immobilized preparations. The modified enzyme-aggregate coating method was performed based on interfacial adsorption in our work, and the characteristics of this immobilized lipase were investigated and compared with those by cross-linking and interfacial adsorption methods. It was shown that the enzyme-aggregate coated lipase yielded the highest activity with a recovered activity of 8.5-fold of the free enzyme, and the highest operational stability with 85% of initial activity remained after 10 recycles. Excellent enantioselectivity (E ≥ 400, with e.e. = 99% of S-HMPC) was obtained for most lipase preparations in our paper (E = 85 for the free enzyme).

The rapid and incomplete oxidation of sugars, alcohols, and polyols by the gram-negative bacterium Gluconobacter oxydans facilitates a wide variety of biological applications. For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a promising precursor of the industrial substance L-(+)-tartaric acid, G. oxydans DSM2343 was genetically engineered to strain ZJU2, in which the GOX1231 and GOX1081 genes were knocked out in a markerless fashion. Then, a secondary alcohol dehydrogenase (GCD) from Xanthomonas campestris DSM3586 was heterologously expressed in G. oxydans ZJU2. The 5-KGA production and cell yield were increased by 10% and 24.5%, respectively. The specific activity of GCD towards gluconate was 1.75 ± 0.02 U/mg protein, which was 7-fold higher than that of the sldAB in G. oxydans. Based on the analysis of kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (qs) and specific 5-KGA production rate (qp), a dissolved oxygen (DO) control strategy was proposed. Finally, batch fermentation was carried out in a 15-L bioreactor using an initial agitation speed of 600 rpm to obtain a high μ for cell growth. Subsequently, DO was continuously maintained above 20% to achieve a high qp to ensure a high accumulation of 5-KGA. Under these conditions, the maximum concentration of 5-KGA reached 117.75 g/L with a productivity of 2.10 g/(L·h).

•The structure of a novel lipase was characterized.•Computational method identified the structure basis of enantioselectivity.•The structure basis of enantioselectivity was verified by site-directed mutation.In this >work, a lipase from Pseudomonas alcaligenes CGMCC4405 (PaL) was cloned and expressed. It was very attractive that the recombinant PaL exhibited excellent enantioselectivity (E > 200) in the resolution of racemic d,l-menthyl propionate to produce l-menthol. The structure basis of enantiopreference is a fundamental scientific problem which needs to be resolved. In our research, molecular dynamic simulation (MD) research was employed to research the different binding modes of d and l-menthyl propionate. The results showed that when bound with slow-reacting enantiomer (d-menthyl propionate), the steric requirements of the large substituent (isopropyl) of the d-menthyl propionate force a rotation of the imidazole ring of catalytic residue His271 and further pushed the active site His271 away from its proper orientation. Moreover, the average distance between alcohol oxygen (Oalc) and HNɛ of catalytic His271 increased to 3.7 Å, which was too far to form an essential hydrogen bond and further prevented efficient catalysis of slow enantiomer. This correlation of the distance between alcohol oxygen (Oalc) and HNɛ of catalytic His271 and the enantioselectivity was also confirmed by the result of site-directed mutagenesis.

A concise strategy to improve the p-NPP (p-nitrophenyl palmitate) catalytic activity and enantioselectivity towards secondary alcohols of Pseudomonas cepacia lipase (PcL) has been described. The PcL was modified by I3−, N-acetyl imidazole (NAI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and ethylenediamine (EDA) in the absence or presence of n-hexane, respectively. After being modified by the four modification reagents, the enantioselectivity (E value) of the PcL towards secondary alcohols was enhanced by 2- to 4-fold. The catalytic activity of EDA-PcL was increased by about 6-fold. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of modified PcL showed that Tyr4, Tyr29, Tyr45, Tyr95, Asp36 and Asp55 were the modified sites. When Tyr29 was modified, the E value of PcL towards secondary alcohols was largely improved. MALDI-TOF-MS characterization and molecular dynamics simulation of the lipase indicated that Tyr29 located inside the catalytic cavity had a significant impact on the E value. The strong steric hindrance of acetyl and iodine ion to the groups on the chiral center of the substrates is responsible for the improvement. In addition, the enhancement of hydrophobicity on the surface of the lipase due to the sidechain replacement of Asp with uncharged hydrophobic groups also improved the E value.