Characterization of four diol dehydrogenases for enantioselective synthesis of chiral vicinal diols

Jiandong Zhang,Rui Dong ,Xiaoxiao Yang ,Lili Gao ,Chaofeng Zhang ,Fan Ren ,Jing Li,Honghong Chang

1 Department of Biological and Pharmaceutical Engineering,College of Biomedical Engineering,Taiyuan University of Technology,Taiyuan 030024,China

2 Department of Environmental Engineering,Taiyuan University of Technology,Taiyuan 030024,China

3 Shanxi Xuanran Pharmaceutical Technology Co,Ltd,Jinzhong 030600,China

Keywords:Diol dehydrogenases Kinetic resolution Enantioselective Chiral vicinal diols α-Hydroxy ketone

ABSTRACT Enantiopure vicinal diols are important building blocks used in the synthesis of fine chemicals and pharmaceutical compounds.Diol dehydrogenase(DDH)mediated stereoselective oxidation of racemic vicinal is an efficient way to prepare enantiopure vicinal diols.In this study,four new bacterial DDHs (AnDDH from Anoxybacillus sp. P3H1B,HcDDH from Hazenella coriacea, GzDDH from Geobacillus zalihae and LwDDH from Leptotrichia wadei) were mined from the GenBank database and expressed in E.coli T7.The four DDHs were purified and biochemically characterized for oxidation activity toward (R)-1-phenyl-1,2-ethanediol,with the optimal reaction condition of pH9.0 (AnDDH),10.0 (HcDDH) and 11.0(GzDDH and LwDDH) and the temperatures at 40 °C (AnDDH),50 °C (HcDDH) and 60 °C (GzDDH and LwDDH),respectively.The four enzymes were stable at the pH from 7.0 to 9.0 and below 40 °C.Kinetic parameters of four DDHs showed that the HcDDH from Hazenella coriacea had high activity toward a broad range of vicinal diols.A series of racemic vicinal diols were successfully resolved by recombinant E.coli (HcDDH-NOX) resting cells co-expression of an NADH oxidase (NOX),affording (S)-diols and (1S,2S)-trans-diols in ≥99% ee.The synthetic potential of HcDDH was proved by E.coli(HcDDH-NOX) via kinetic resolution of racemic trans-1,2-indandiol on a 100 ml scale reaction,(S, S)-trans-1,2-indandiol was prepared in 46.7% yield and ＞99% ee.In addition,asymmetric reduction of four α-hydroxy ketones (10-300 mmol·L-1) by E.coli (HcDDH-GDH) resting cells resulted in ＞99% ee and 69-98%yields of(R)-vicinal diols.The current research expands the toolbox of DDHs to synthesize chiral vicinal diols and demonstrated that the mined HcDDH is a potential enzyme in the synthesis of a broad range of chiral vicinal diols.

1.Introduction

Chiral vicinal 1,2-diols are very important chemicals can be utilized as building blocks in the synthesis of numerous industrial products and pharmaceuticals [1].For example,(R)-1,2-butanediol as a building block for the synthesis of antiepileptic drug levetiracetam[2].(R)-1-Phenyl-1,2-ethanediol as an intermediate for the synthesis of drugs used to treat psychiatric disorders[3,4].(S)-1-Phenyl-1,2-ethanediol as an intermediate for the preparation of chiral biphosphines,liquid crystals [5] and chiral phosphoramidite ligand [6].For cyclic vicinal diols,(S,S)-1,2-cyclohexanediol as a chiral auxiliary for the asymmetric synthesis of α,α-disubstituted α-amino acids [7] and as effective ligand for titanium alkoxide catalyzed asymmetric phosphonylation [8].Moreover,(S,S)-trans-1,2-dihydroxy-1,2,3,4-terahydronaphtha lene and(S,S)-trans-1,2-dihydroxyindan are precursors to the corresponding amino alcohols which are chiral ligands in transition metal-catalyzed reactions [9-11].

To access chiral vicinal diols,different chemical approaches were reported.Such as the Sharpless dihydroxylation of alkenes[12-13],enantioselective hydrolysis of epoxides [14-15] and asymmetric transfer hydrogenation of α-hydroxy ketones [16-19]were the classical methods.In addition,asymmetric aldol addition[20-21] and asymmetric hydrosilylation [22] were also reported for the synthesis of chiral vicinal diols.Compared to conventional chemical methods that often rely on the use of hazardous reagents and reaction conditions,biocatalysis as a green and sustainable method with the advantages of excellent selectivity and mild reaction conditions has been widely used in the synthesis of chiral vicinal diols.Such as naphthalene dioxygenase catalyzed stereospecific dihydroxylation of styrene [23],lipase-mediated kinetic resolution of racemic diols[24-25],epoxide hydrolases catalyzed hydrolysis of styrene oxide (SO) [26-29] and carbonyl reductases (or alcohol dehydrogenases) catalyzed asymmetric reduction of α-hydroxy ketone[30-34]or stereoselective oxidation of racemic vicinal diols [35-36].In addition,some cascade biocatalysis strategies have also been documented for the synthesis of chiral vicinal diols recently[37-39].Compared with other enzymatic methods for the production of chiral diols,diol dehydrogenase (DDH) mediated stereoselective oxidation of racemic diols is an efficient way to prepare the enantiopure vicinal diols.In 2013,Zhanget al.demonstrated the bacterialtrans-diol dehydrogenases as biocatalysts for the stereoselective oxidation of cyclic vicinaltrans-diols to chiral diols and α-hydroxy ketones [40].In 2015,Boydet al.reported twocis-diol dehydrogenases (benzenecisdiol dehydrogenase and naphthalene cis-diol dehydrogenase) as biocatalysts to catalyze regio-and stereoselective oxidation of cycloalkane-and cycloalkenecis-diols to yield chiral diols and αketols [41].Recently,two 2,3-butanediol dehydrogenases from two differentBacillushave been cloned and characterized,a selection of vicinal diols,vicinal diketones and α-hydroxy ketones were accepted as substrate by these enzymes [42-43].However,these biocatalytic processes are still exist some limitations,such as the limited range of substrate,large amount of cofactor,low reaction rates and low substrate concentration.Thus,there is an increasing demand for exploration of new DDHs with excellent catalytic properties.

In this study,four new bacterial diol dehydrogenases (DDHs)were identified by data mining based on the reported amino acid sequence of 2,3-butanediol dehydrogenase (BDHA) fromBacillus subtilisBGSC1A1.The four DDHs were successfully expressed inEscherichia coliT7,purified and biochemically characterized.Kinetic resolution of several racemic vicinal diols.(Fig.1) and asymmetric reduction of four α-hydroxy ketones (Fig.2) were demonstrated byE.coliresting cells co-expression of HcDDH with an enzyme for cofactor regeneration.The synthetic potentiality of HcDDH was also demonstrated byE.coli(HcDDH-NOX)viakinetic resolution of racemictrans-1,2-indandiol on a 100 ml scale reaction.

2.Materials and Methods

2.1.Chemicals

Fig.2.Diol dehydrogenase catalyzed asymmetric reduction of α-hydroxy ketones.

(±)-1,2-Butanediol(1a),(±)-1-phenyl-1,2-ethanediol(1b),(±)-1-(4-fluorophenyl)-1,2-ethanediol (1c),(±)-1-(4-chlorophenyl)-1,2-e thanediol (1d),trans-1,2-cyclohexanediol (1e),1-hydroxybutan-2-one (2a),2-hydroxyacetophenone (2b),4-fluoro-2-hydroxyaceto phenone(2c)and 4-chloro-2-hydroxyacetophenone(2d)were from Energy Chemical (Shanghai,China).(±)-Trans-1,2-indandiol (1f)and (±)-trans-1,2,3,4-tetrahydro-1,2-naphthalenediol (1g) were purchased from Saen Chemical Technology (Shanghai,China).The main components of culture medium were purchased from Sangon Biotech (Shanghai,China).All other chemicals were commercially available.

2.2.Bacterial strains and molecular biology tools

T7 express competentEscherichia coli(E.coliT7)was from New England BioLabs (NEB) (Beijing,China).Plasmid pET28a (+) was from Novagen (Shanghai,China).E.coli(pETduet-NOX) for the expression of NADH oxidase (NOX) andE.coli(pETduet-GDH) for the expression of glucose dehydrogenase (GDH) and the recombinant plasmids pETduet-NOX[44]and pETduet-GDH[34]were preserved in our lab.Restriction enzymes and T4 DNA ligase were from New England BioLabs,inc.(NEB,Beijing,China).Pfu DNA polymerase,isopropyl β-D-1-thiogalactopyranoside (IPTG),PCR product purification kit and plasmid isolation kit were from Sangon Biotech (Shanghai,China).Ampicillin and kanamycin were from Sigma Aldrich.

2.3.Construction of the recombinant E.coli strains

Four diol dehydrogenases (GenBankTM:LwDDH:BBM48176.1,AnDDH:KXG09708.1,HcDDH:TCS92404.1,GzDDH:WP_060787977.1) were mined from GenBank (BLASTP program).The four DDH genes were synthesized by TSINGKE Biological Technology (Anhui,China).The LwDDH gene fromLeptotrichia wadeiand GzDDH gene fromGeobacillus zalihaeNBRC 101842 were cloned into expression vector pET28a (+) using the enzymesNdeI andXhoI.AnDDH gene fromAnoxybacillus sp.P3H1B and HcDDH gene fromHazenella coriaceawere cloned into expression vector pET28a(+)using the enzymesNheI andXhoI.The resulting recombinant plasmids(pET28a-LwDDH,pET28a-GzDDH,pET28a-AnDDH and pET28a-HcDDH) were transformed into theE.coliT7 competent cells to forming the recombinant strainE.coli(LwDDH),E.coli(GzDDH),E.coli(AnDDH) andE.coli(HcDDH),respectively.

Fig.1.Diol dehydrogenase catalyzed enantioselective oxidation of racemic vicinal diols.

To construct the recombinantE.coli(HcDDH-NOX) cells,the recombinant plasmids pETduet-NOX and pET28a-HcDDH were simultaneous transformed intoE.coliT7 competent cells.The transformed cultures were plated on LB agar plate containing 100 μg·ml-1of ampicillin and 50 μg·ml-1of kanamycin.The coexpression of HcDDH and NOX was confirmed by SDS-PAGE and the activities of HcDDH and NOX were tested.

To construct the recombinantE.coli(HcDDH-GDH) cells,the recombinant plasmids pETduet-GDH and pET28a-HcDDH were simultaneous transformed intoE.coliT7 competent cells.The transformed cultures were plated on LB agar plate containing 100 μg·ml-1of ampicillin and 50 μg·ml-1of kanamycin.The coexpression of HcDDH and GDH was confirmed by SDS-PAGE and the activities of HcDDH and GDH were tested.

2.4.Production of recombinant DDHs in E.coli and purification

The recombinant cells were pre-cultured in 5 ml of LB medium with 50 μg·ml-1kanamycin at 37°C for 12 h.One milliliter of precultures was inoculated into 50 ml of terrific broth (TB) medium with 50 μg·ml-1kanamycin.The cells were incubated at 37 °C and 200 r·min-1 until an OD600of 0.6-0.8 was reached.Then 0.5 mmol·L-1isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce the gene expression,the cells were grown for another 12 h at 25 °C and 200 r·min-1.The cells were harvested by centrifugation,washed twice with 50 mmol·L-1sodium phosphate buffer(pH 8.0),and resuspended in the chilled sodium phosphate buffer(50 mmol·L-1,pH 8.0)containing 300 mmol·L-1NaCl.After disruption by sonication,the suspension was centrifuged at 12,000gand 4°C for 30 min,the supernatant of cell lysate was collected and the soluble His6-tagged protein was purified using a Ni-NTA column according to the manufacturer’s instructions.The fractions containing the DDH activity were pooled and concentrated by an ultrafiltration tube (30000).The purified DDHs were used for enzyme characterization.The protein concentration was determined by the Bradford method [45].

2.5.DDH activity assay

The DDH activity assays were conducted by photometrical measurement of NADH production at 340 nm using a UV-vis spectrophotometer (MINI-1240,Shimadzu).The standard assay mixture containing 100 mmol·L-1sodium phosphate buffer (pH 8.0),0.5 mmol·L-1NAD+,10 mmol·L-1(R)-1-phenyl-1,2-ethanediol and an appropriate amount of enzyme in a total volume of 1 ml.Reactions were started by addition of the enzyme solution,and the initial reaction velocity was determined from the linear increase of the absorption at 340 nm.The assay without substrate or enzyme was used as a negative control.The amount of enzyme that reduced one μmol of NAD+per minute at 30 °C under the given conditions was defined as one unit.

2.6.Effects of pH and temperature on DDHs activity

The purified DDHs activity assays were performed at pH 7.0-12.0 under standard assay condition to assess the effect of pH on enzyme activity,three buffer systems including sodium phosphate buffer (100 mmol·L-1,pH7.0-8.0),Tris-HCl buffer (100 mmol·L-1,pH8.0-9.0) and glycine-NaOH buffer (100 mmol·L-1,pH 9.0-12.0) were used,respectively.The assays were conducted at 30°C.For determination of the temperature optimum,the purified DDHs activities were estimated at various temperatures ranging from 20 to 70°C in sodium phosphate buffer(100 mmol·L-1)under the optimized pH.The pH stability of the purified DDHs was estimated by incubation of the DDH in sodium phosphate buffer(100 mmol·L-1,pH 7.0-8.0),Tris-HCl buffer (100 mmol·L-1,pH 8.0-9.0) and glycine-NaOH buffer (100 mmol·L-1,pH 9.0-12.0) at 4 °C,respectively.Thermostability of DDHs was determined by incubating of the enzyme in 1 ml sodium phosphate buffer(100 mmol·L-1,pH 8.0)at the temperature from 4 to 60°C.At certain time intervals,the residual DDHs activities were measured under standard assay conditions.

2.7.Determination of kinetic parameters

The kinetic parameters of purified DDHs were determined under the optimum reaction conditions with the substrate concentration in the range of 0.1-6 mmol·L-1.The Michaelis-Menten parameters,like Michaelis constant (KM) values and maximum specific activities (Vmax) were obtained by using nonlinear regression fitting of the Michaelis-Menten equation.Each assay was repeated at least three times.

2.8.Biooxidation of racemic vicinal diols 1a-g by E.coli(HcDDH-NOX)

RecombinantE.coli(HcDDH-NOX)was inoculated into 50 ml of terrific broth (TB) medium with 50 μg·ml-1kanamycin and 100 μg·ml-1ampicillin.The cells were incubated at 37 °C and 200 r·min-1until an OD600of 0.6-0.8 was reached.Then 0.5 mmol·L-1isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce the gene expression,the cells were grown for another 18 h at 25 °C and 200 r·min-1.The cells were harvested by centrifugation,washed twice with 50 mmol·L-1sodium phosphate buffer (pH 8.0),and lyophilized.The biooxidation reactions were conducted in 5 ml reaction mixture containing 100 mmol·L-1glycine-NaOH buffer (pH 10.0),20-100 mmol·L-1racemic vicinal diols 1a-g and 10 g cdw·L-1E.coli(HcDDH-NOX) resting cells at 30 °C and 250 r·min-1.For quantitative assessment of diols 1a-e concentration and enantiomeric excess (ee),0.5 ml aliquots were taken out after the reactions were run for 3-12 h,saturated with NaCl,extracted with equal volume of ethyl acetate containing 20 mmol·L-1ofn-dodecane as an internal standard.The ethyl acetate phases were dried with anhydrous sodium sulfate,the concentrations and the ee values of diols 1a-e were measured by gas chromatography(GC).For the diols 1f and 1g,at regular time intervals,a 100 μl sample was taken,mixed with 900 μl acetonitrile(ACN) containing 10 mM benzylacetone as an internal standard,after centrifugation,the supernatant was subjected to HPLC analysis.For chiral HPLC analysis of the 1f and 1g,the samples were prepared by taking 300 μl aliquots,removing the cells via centrifugation,and extracting with 300 μl chloroform,after centrifugation (12,000 r·min-1) for 10 min,chloroform portion was transferred into a clean tube and dried by evaporation,300 μl of isopropyl alcohol was added to dissolve the residues in the tube,after centrifugation,the solvents were subjected to chiral HPLC analysis.All experiments were performed in duplicate.

2.9.Preparation of (S,S)-trans-1f by E.coli (HcDDH-NOX) via enantioselective oxidation of (±)-trans-1f

The recombinantE coli(HcDDH-NOX) resting cells (10 g·cdw L-1) were suspended in 100 ml glycine-NaOH buffer(100 mmol·L-1,pH 10.0).After the substrate (±)-trans-1f (0.3 g,2 mmol) was added,the reaction was continued for 9 h at 30 °C and 200 r·min-1.After the reaction was finished,the reaction mixture was centrifugated at 12,000gfor 10 min.The supernatant was saturated with NaCl and extracted with ethyl acetate (3 × 50 ml).The combined organic phase was dried overnight with anhydrous sodium sulfate,filtered and concentrated under reduced pressure by rotary evaporation,the crude product was purified by flash chromatography on a silica gel column and the product was dried overnight under vacuum.

2.10.Asymmetric reduction of α-hydroxy ketones by E.coli (HcDDHGDH)

The recombinantE.coli(HcDDH-GDH) was inoculated into 50 ml of terrific broth (TB) medium with 50 μg·ml-1kanamycin and 100 μg·ml-1ampicillin.The cells were grown at 37 °C and 200 r·min-1until the OD600reached up to 0.6-0.8.Then 0.5 mmol·L-1isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce the gene expression,the cells were grown for another 18 h at 25 °C and 200 r·min-1.The cells were harvested by centrifugation,washed twice with 100 mmol·L-1sodium phosphate buffer (pH 6.5) and lyophilized.The asymmetric reduction reactions were performed in 5 ml sodium phosphate buffer(100 mmol·L-1,pH 6.5) containing 20-300 mmol·L-1α-hydroxy ketones 2a-d,5%-20% DMSO,20-300 mmol·L-1glucose and 10 g cdw·L-1E.coli(HcDDH-GDH).The reactions were continued at 30 °C and 250 r·min-1for 6-12 h,0.5 ml aliquots were taken out,saturated with NaCl,and extracted with equal volume of ethyl acetate containing 20 mmol·L-1of n-dodecane as an internal standard.The ethyl acetate phase was dried over anhydrous sodium sulfate and subjected to GC analysis.

2.11.Analysis method

The concentrations and ee values of diols 1a-e were analyzed by GC (GC-14C,Shimadzu,Japan) with a flame ionization detector coupled with a chiral column (CP-Chirasil-Dex CB,25 m × 0.32 m m×0.25 μm;Agilent Technologies,lnc.),as described by Cuiet al.[34].The detailed GC analysis conditions and retention times of vicinal diols were described in Supplementary Material.

Concentrations of dioltrans-1f-g were analyzed by HPLC with an EC-C18 column (2.1-150 mm,5 μm).The sample was detected at 210 nm at a flow rate of 0.4 ml·min-1(ACN:H2O=70:30);Retention time:6.9 min for internal standard (benzylacetone);4.6 min for 1f;5.3 min for 2f.4.6 min for 1g;5.3 min for 2g.The enantiomeric excess of vicinal diol 1f was analyzed by HPLC with a chiral column AS-H (Chiral Technologies,Chiralpak AS-H,250-4.6 mm,5 μm).The sample was measured at 210 nm at a flow rate of 0.5 ml min-1(n-hexane:isopropanol=9:1).Retention time:20.24 min for (S,S)-1f,25.39 min for (R,R)-1f,38.3 min for (R)-2f.The enantiomeric excess of vicinal diol 1g was analyzed by HPLC with a chiral column OJ (Chiral Technologies,Chiralcel OJH,250-4.6 mm,5 μm).The sample was measured at 210 nm at a flow rate of 0.5 ml min-1(n-hexane:isopropanol=9:1).Retention time:16.42 min for (R,R)-1g and 20.14 min for (S,S)-1g,22.69 min for (R)-2g.

3.Results and Discussion

3.1.Data mining of diol dehydrogenases

In order to mine new DDHs with potential diol oxidation activity,one representative and well-documented 2,3-butanediol dehydrogenase,BDHA (accession no.BSU_06240) fromBacillus subtilis[40] was used for homologous sequence searching in the BLASTP database.The amino acid sequence similarity was controlled between 40%and 70%.The sequences containing key features characteristic of alcohol dehydrogenase (Prosite signature) and the butanediol dehydrogenase (PRATT patch) [42] were selected for further screening.Four of them (AnDDH,LwDDH,GzDDH and HcDDH)were selected and evaluated for expression and suitability for biotransformation.The genomic annotation indicated that AnDDH was a putative sorbitol dehydrogenase fromAnoxybacillussp.P3H1B,LwDDH was a putative L-iditol 2-dehydrogenase fromLeptotrichia wadei,HcDDH and GzDDH were putative NADHdependent butanediol dehydrogenase fromHazenella coriaceaandGeobacillus zalihaeNBRC 101842,respectively.The amino acid sequence identities with BDHA were 54.1% (AnDDH),56.9%(GzDDH),60.6% (HcDDH) and 65.3% (LwDDH) (Fig.S1),respectively.The protein sequences analysis revealed that four enzymes harbor the zinc containing alcohol dehydrogenases Prosite signature,as well as the (R,R)-butanediol dehydrogenase PRATT patch.Amino acids for the ligation of the catalytic Zn2+-ion and putative NAD(P) binding sites were also identified.The four DDHs have not been investigated or characterized for the enantioselective oxidation of vicinal diols.

3.2.Protein expression and purification

The four diol dehydrogenases (AnDDH,HcDDH,LwDDH,and GzDDH)genes were synthesized and cloned into the vector pET28a(+),the constructed recombinant plasmids pET28a-AnDDH,pET28a-HcDDH,pET28a-LwDDH and pET28a-GzDDH were then transformed intoE.coliT7.The protein production inE.coliT7 cells was analyzed by SDS-PAGE.As shown in Fig.3,the four DDHs were successfully expressed in the soluble form with a His*6 tag.The protein bands with a size of about 40,000 (including 2000 of His*6 tag),which are in line with the expected molecular mass as predicted from the amino acid sequences.After purification with a Ni-NTA column(Fig.3),the four DDHs were subjected to enzyme activity analysis toward 1-phenyl-1,2-ethanediol 1b,the results showed that the four DDHs have high activity toward (R)-1-phenyl-1,2-ethanediol (5.42 U·mg-1for AnDDH,4.96 U·mg-1for HcDDH,0.46 U·mg-1for LwDDH and 3.29 U·mg-1for GzDDH),and no activity was detected toward (S)-1-phenyl-1,2-ethanediol.Throughout purification,the four DDHs activities were measured in 100 mmol·L-1sodium phosphate buffer (pH8.0) with (R)-1-phenyl-1,2-ethanediol (10 mmol·L-1) and NAD+(0.5 mmol·L-1)as substrates.No activities were observed with NADP+,revealed that the four DDHs were NADH-dependent dehydrogenases.

Fig.3.SDS-PAGE of mined diol dehydrogenases.A:lane 1:marker,lanes 2:cell-free extract of E.coli (AnDDH),lanes 3-6:column flow-through fraction,lane 7:purified AnDDH.B:lane 1 marker,lanes 2 cell-free extract of E.coli (HcDDH),lanes 3-7:column flow-through fraction,lane 8 purified HcDDH.C:lane 1:marker,lanes 2:cell-free extract of E.coli(LwDDH),lanes 3-7:column flow-through fraction,lane 8:purified LwDDH.D:lane 1:marker,lanes 2:cell-free extract of E.coli(DDH),lanes 3-7:column flow-through fraction,lane 8:purified GzDDH.

3.3.Effects of pH and temperature on the activity of DDHs

As shown in Fig.4A,most of the DDHs showed very low diol oxidation activity at the pH below 7.0.The diol oxidation activities of four DDHs could be detected in a wide alkaline region.The optimum pH for the diol oxidation activities were at pH 9.0 (AnDDH),10.0 (HcDDH),11.0 (LwDDH and GzDDH),respectively.These are in agreement with the DDHs fromRhodococcus erythropolis[46,47] andSaccharomyces cerevisiae[48],which the maxima oxidation activities were reported between pH 9.0 and pH 11.0.The effect of temperature on DDHs activities were examined from 20 °C to 70 °C under the optimum pH conditions,as shown in Fig.4B,the oxidation activity of four enzymes increases first and then decreases with the increase of temperature.The optimum temperatures of the purified four enzymes were at 40°C(LwDDH),50 °C (AnDDH and HcDDH) and 60 °C (GzDDH),respectively.

Fig.4.Effect of pH (A) and temperature (B) on the activity of mined DDHs.

3.4.Effect of pH and temperature on the stability of DDHs.

For the pH stability,the purified enzymes were placed in buffers with different pH values and stored at 4 °C.The reactions were sampled regularly and the residual enzyme activities were measured.As shown in Fig.5,the four purified enzymes revealed good stability in the pH range of 7.0-9.0,and ＞40% residual activities were observed after 24 h.Among them,HcDDH had an excellent stability in a pH range from 7.0 to 10.0,showed residual activities of more than 50% over a period of 24 h.When the pH was above 10.0,the activities of four DDHs were decreased significantly.For the thermostability,the four enzymes were incubated in a water bath at 4-60 °C for 24 h,and samples were taken at regular time to detect residual enzyme activity,as shown in Fig.6,the four enzymes had good stability at the temperatures from 4 to 40 °C,and the enzyme activity dropped sharply if the temperature exceeds 50 °C.Interestingly,AnDDH and HcBDHA revealed good thermal stability at 50 °C,and more than 20% residual enzyme activity could be detected after 12 h incubation.As we know,AnDDH and HcBDHA are from the strainAnoxybacillussp.P3H1B andHazenella coriaceawhich are belong to thermophilic bacteria and thermoactinomycetaceae,respectively.

Fig.5.The pH stability of ADHs.A:AnDDH.B:HcDDH.C:LwDDH.D:GzDDH.Standard condition:1 ml reaction system,10 mmol·L-1 (R)-1-phenyl-1,2-ethanediol,0.5 mmol·L-1 NAD+,sodium phosphate buffer (pH 8.0,100 mmol·L-1) and 4 mg·ml-1 enzyme.

Fig.6.The thermostability of ADHs.A:AnDDH.B:HcDDH.C:LwDDH.D:GzDDH.Standard condition:1 ml reaction system,10 mmol·L-1 (R)-1-phenyl-1,2-ethanediol,0.5 mmol·L-1 NAD+,sodium phosphate buffer (100 mmol·L-1,pH 8.0) and 4 mg·ml-1 enzyme.

3.5.Kinetic parameters of four DDHs

The kinetic parameters of four DDHs toward different vicinal diols were obtained under optimum assay conditions by varying the substrate concentration from 0.1 to 6.0 mmol·L-1.As shown in Table 1,theKMof AnDDH toward the substrates 1a-g were 0.2 7-0.93 mmol·L-1,with theVmax0.79-9.51 μmol·mg-1protein,respectively (Table 1).TheKMof HcDDH toward the substrates 1a-g were 0.17-2.47 mmol·L-1,with theVmax0.24-6.86 μmol·mg-1protein,respectively.Although theVmaxof LwDDH toward 1a(9.67 μmol·mg-1protein) was the highest among the four DDHs,the activities of LwDDH toward other vicinal diols (0.18-4.06 μm ol·mg-1protein) were relative lower than other DDHs.The activities of GzDDH toward 1a,1b and 1e (3.06 μmol·mg-1protein,4.46 μmol·mg-1protein and 4.29 μmol·mg-1protein) were relative higher than other tested substrates,with theKMfrom 0.16-1.82 mmol·L-1,respectively.Interestingly,among the four DDHs,HcDDH showed the highest activity toward the cyclic vicinal diols 1f and 1g (0.24 μmol·mg-1protein and 1.88 μmol·mg-1protein),demonstrated the broad substrate spectrum of HcDDH.Although AnDDH showed the highest activity toward cyclic dioltrans-1e(9.51 μmol·mg-1protein),the activities toward other two cyclic diol trans-1f and 1g were very low.Then,the HcDDH was selected as the best candidate for subsequent oxidation and reduction reactions.

3.6.Kinetic resolution of racemic vicinal diols by E.coli(HcDDH-NOX)

After the HcDDH was selected,a group of racemic vicinal diols were tested as substrates of the HcDDH.Before these,an NADH oxidase (NOX) gene fromLactobacillus pentosus[44] was coexpressed with HcDDH inE.coliT7 for regeneration of NAD+cofactor.As shown in Fig.S2B,HcDDH and NOX were successfully coexpressed inE.coliT7,the specific activities of HcDDH and NOX in crude cell free extract were 6.0 U·mg-1protein and 3.0 U·mg-1protein,respectively.Then,kinetic resolution of racemic vicinal diols 1a-g was conducted with the recombinantE.coli(HcDDHNOX) cells.As shown in Table 2,all of the tested racemic vicinal diols were resolved byE.coli(HcDDH-NOX)cells,with the conversion rates of 50-68%,and leave the unreacted substrate in ≥99%ee.For substrates 1a and1b,100 mmol·L-1substrate could be completely resolved within 6 h,leave the (S)-1a and 1b in ＞99% ee.For substrates 1c and 1d,it will take 9 h to resolve 20 mmol·L-1racemic substrates.For cyclic vicinal diolstrans-1e-g,100 mmol·L-1oftrans-1e could be completely resolved within 20 h,leave the (S,S)-trans-1e in 99% ee.While 20 mmol·L-1trans-1f-g could be resolved within 9 h,leave the (S,S)-trans-1f-g in 99% ee.Thus far,few biocatalysts were reported for the oxidation of racemic cyclic vicinal diols to yield chiral diols.2,3-Butanediol dehydrogenase(BDHA) fromBacillus subtiliscatalyzed oxidation of racemic cyclictrans-diols 1e (20 mmol·L-1) and 1g (10-20 mmol·L-1),affording(S,S)-trans-1e in ＞99%ee,while(S,S)-trans-1g was obtained in only93.4%-96% ee [40].Twocis-diol dehydrogenases (benzenecis-diol dehydrogenase and naphthalene cis-diol dehydrogenase) have been investigated for regio-and stereoselective oxidation ofcisdiols 1e-f to yield chiral diols and α-ketols,however,the unexpected racemization of α-ketols and formation of diol bioproduct made the catalytic process very complicated [41].In comparison,HcDDH catalyzed oxidation process is much more enantioselective and thus gives higher yield of chiral diols.

Table 1 Kinetic parameters of four DDHs

Table 2 Kinetic resolution of racemic vicinal diols by E.coli (HcDDH-NOX)①

3.7.Preparation of (1S,2S)-trans-1,2-dihydroxyindan 1f via enantioselective biooxidation of racemic trans-1,2-dihydroxyindan 1f by E.coli (HcDDH-NOX)

The synthetic potential of HcDDH was demonstrated byE.coli(HcDDH-NOX)on 100 ml-scale biotransformationviakinetic resolution of racemictrans-1,2-dihydroxyindan 1f.As shown in Fig.7,theE.coli(HcDDH-NOX) resting cells (10 g·cdw L-1) were used to kinetic resolution of racemictrans-1,2-dihydroxyindan 1f(300 mg),the reaction mixture was kept at 30 °C and 200 r·min-1for 9 h,the conversion of 1f could reach to 50%,and leave the unreacted (1S,2S)-trans-1f in ＞99% ee.After purification by flash chromatography on a silica gel column,(1S,2S)-trans-1f was obtained in ＞99% purity and 46.7% isolated yield (140.0 mg).(1S,2S)-Trans-1,2-dihydroxyindan is an important precursor to thecis-1-amino-2-indanol,itself the essential component of a wide variety of chiral ligands and chiral auxiliaries [49,50].

Fig.7.The time course for the biooxidation of racemic trans-1,2-dihydroxyindan 1f with the resting cells of E.coli (HcDDH-GDH).Reaction condition:100 ml glycine-NaOH buffer (pH 10.0,100 mmol·L-1),including 20 mmol·L-1 racemic trans-1,2-dihydroxyindan 1f,5% (V/V) DMSO,10 g cdw·L-1 E.coli (HcDDH-GDH),30 °C,200 r·min-1.

3.8.Asymmetric reduction of α-hydroxy ketones by E.coli (HcDDHGDH)

DDH catalyzed asymmetric reduction of α-hydroxy ketones is another strategy for the preparation of chiral vicinal diols.In this study,the potentially of HcDDH for the synthesis of chiral vicinal diolsviaasymmetric reduction of α-hydroxy ketone was investigated by recombinantE.coli(HcDDH-GDH),which co-expression of HcDDH with a glucose dehydrogenase(GDH)gene fromBacillus subtilis[35] for the regeneration of NADH cofactor (Fig.S2A).The functional expression of both HcDDH and GDH genes were demonstrated by testing their activities (3.5 U·mg-1protein for HcDDH and 1.2 U·mg-1protein for GDH) in cell-free extract.The reaction conditions were first optimized with the 2-hydroxyacetophenone(2-HAP) 2b as a model substrate.The highest yield of (R)-1b(98%) could be obtained at pH 6.5,when pH below 6.5 or higher than 7.0,the yields of (R)-1b were evidently decreased (Fig.8A).The effects of temperature on the product yields were then investigated.The highest yield of (R)-1b (98%) could be obtained at 35 °C,the higher temperature or lower temperature will result in decreased yields of (R)-1b (Fig.8B).The effect of different ratio of glucose and 2-HAP (glucose/2-HAP,molar ratio) on the product yield was tested with 10 g·cdw L-1ofE.coli(HcDDH-GDH) and 100 mmol·L-12-HAP,as shown in Fig.8C,the highest yield of(R)-1b (98%) could be obtained with the ratio of glucose and 2-HAP at 1:1.Asymmetric reduction of 50-300 mmol·L-1of 2-HAP 2b was also investigated with 10 g cdw·L-1ofE.coli(HcDDHGDH) (Fig.8D).For 50-200 mmol·L-1of 2b,＞90% yields of (R)-1b could be obtained within 3 h.Increasing the 2b to 300 mmol·L-1,the product yield was decreased to 70%.

Fig.8.Effects of pH,temperature,glucose/2b (molar ratio) and substrate concentration on the yield of product.A:pH,B:temperature,C:glucose/2b (molar ratio),D:substrate concentration.Reaction condition:5 ml reaction system,A:100 mmol·L-1 2-HAP,E.coli(HcDDH-GDH)(10 g cdw·L-1),100 mmol·L-1 glucose,10%DMSO,sodium citrate buffer(100 mmol·L-1,pH 6.0-6.5),sodium phosphate buffer(100 mmol·L-1,pH 7-8)and Tris-HCl buffer(100 mmol·L-1,pH 8-9),30°C,200 r·min-1 for 3 h;B:sodium citrate buffer(100 mmol·L-1,pH 6.5),100 mmol·L-1 2-HAP,10%DMSO,E.coli(HcDDH-GDH)(10 g cdw·L-1),100 mmol·L-1 glucose,20-40°C,200 r·min-1 for 3 h;C:sodium citrate buffer(100 mmol·L-1,pH 6.5),100 mmol·L-1 2-HAP,10%DMSO,E.coli(HcDDH-GDH)(10 g cdw·L-1),25-200 mmol·L-1 glucose,35°C,200 r·min-1 for 3 h;D:sodium citrate buffer (100 mmol·L-1,pH 6.5),50-300 mmol·L-1 2-HAP,10% DMSO, E.coli (HcDDH-GDH) (10 g cdw·L-1),50-300 mmol·L-1 glucose,35 °C,200 r·min-1 for 3 h.

Fig.9.Time courses for the asymmetric reductive of 2-HAP with the resting cells of E.coli (HcDDH-GDH).Reaction condition:5 ml sodium phosphate buffer (pH 6.5,100 mmol·L-1),including 50-200 mmol·L-1 2-HAP,50-200 mmol·L-1 glucose,10%(V/V) DMSO,10 g cdw·L-1 E.coli (HcDDH-GDH),35 °C,200 r·min-1.

Under the optimized reaction conditions (pH 6.5,glucose(mmol·L-1)/substrate (mmol·L-1)=1/1,35 °C),the time course for the asymmetric reduction of 50-200 mmol·L-12-HAP were conducted with theE.coli(HcDDH-GDH) resting cells (10 g cdw·L-1) without addition of the external of NAD+.As shown in Fig.9,for reduction of 50-100 mmol·L-12-HAP,the yields of(R)-1b could reach 98% by 10 g cdw·L-1ofE.coli(HcDDH-GDH) within 5.0 h.For 200 mmol·L-12-HAP,98% yield of (R)-1b could be obtained after prolonging the reaction time to 7 h.The optical purity of the product was ＞99%ee in all cases.Further increase the 2-HAP concentration to 400 mmol·L-1,the substrate was presented in suspended solid form in the reaction mixture,and the product yield was sharply decreased to 20% (data not show).Then the cosolvent (DMSO) concentration was increased up to 20% so that the 2-HAP could be dissolved completely,however,the product yield was not greatly improved (＜25%).The substrate inhibition and high concentration of DMSO maybe the main reasons for such a low yield.Cui et al.demonstrated that the high concentration of DMSO (＞15%) in the reaction system had a detrimental effect on activity of the diol dehydrogenase [40].

Moreover,another three α-hydroxy ketones 2a,2c and 2d were tested as a substrate of HcDDH.As shown in the Table 3,the activities of purified HcDDH toward 2a-d were from 1.4 to 3.0 U·mg-1.Asymmetric reduction of aliphatic α-hydroxy ketone 2a(50 mmol·L-1) by 10 g·cdw·L-1E.coli(HcDDH-GDH) at 35 °C,resulted in 94% yield and ＞99% ee of 1a.For the substrate 2c and 2d (30 mmol·L-1) with para substituent groups on an aromatic ring,69% and 73% yields could be obtained after 3 h reaction,and the product (R)-1c-d were obtained in ＞99% ee.Overall,space-time yields of 24.2-99.4 g·L-1·d-1could be achieved by the whole cells ofE.coli(HcDDH-GDH).Compared with other reported bioreduction systems in Table 4,theE.coli(HcDDHGDH)could catalyze the asymmetric reduction of 2-HAP at a maximal load of 27.2 g·L-1without additional cofactor,making it very competitive and promising tools for practical application in the preparation of chiral vicinal diols.

Table 3 Asymmetric reduction of α-hydroxy ketones by E.coli (HcDDH-GDH)①

Table 4 Comparison on asymmetric reduction of 2-HAP

4.Conclusions

In summary,we have identified four new DDHsviagenome mining.The four enzymes were successfully over-expressed inE.coli,purified and characterized.HcDDH with the highest activity and enantioselectivity toward a broad range of vicinal diols was selected and co-expressed with an NOX inE.coli,the resting cells ofE.coli(HcDDH-NOX) was employed for the kinetic resolution of a set of racemic diols,all substrates examined were completely resolved,leave the un-reacted vicinal diols in ≥99% ee.Moreover,the synthetic potential of HcDDH was proved byE.coli(HcDDHNOX)viakinetic resolution of racemictrans-1,2-dihydroxyindan 1f on a 100 ml scale reaction,(S,S)-trans-1,2-dihydroxyindan 1f was obtained with 46.7% isolated yield and ＞99% ee.Asymmetric reduction of four α-hydroxy ketones was also implemented by the resting cells ofE.coli(HcDDH-GDH),(R)-vicinal diols were obtained in ＞99% ee and 73%-98% yields.The current research expands the toolbox of DDH in the synthesis of chiral vicinal diols and proved that the mined HcDDH is a promising enzyme for biotechnology application in the production of chiral vicinal diols.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant No.21772141),the Shanxi Province Science Foundation for Youths (grant No.201701D221042),the Key Research and Development (R &D) Project of Shanxi Province(201803D31050).

Supplementary Material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2021.05.019.