CHEN Bingmei (陳秉梅), XU Xiaoping (許小平),**, HOU Zhiguo (侯志國(guó)), LI Zhongqin(

1李忠琴)2 and RUAN Wenbing (阮文兵)12 College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350108, China College of Fisheries, Jimei University, Xiamen 361021, China

1 INTRODUCTION

(R)-α-hydroxyphenylacetic acid [(R)-HPA] and its derivatives are valuable chiral synthons and resolving agent in fine chemical and pharmaceutical industries [1]. (R)-HPA is often employed as a precursor for the manufacture of semi-synthetic penicillin and cephalosporin [2, 3], and it is also used as a chiral block to synthesize anti-tumor and anti-obesity agents[4]. Many methods, including chemical process and biotransformation, have been reported for the preparation of enantiomerically pure (R)-HPA [5-10]. Biotransformation, owing to its high enantioselectivity,mild reaction conditions and environmental compatibility, has been preferential in the asymmetric production of many chiral drugs and their intermediates [11],and can be catalyzed either by isolated enzymes or by whole cells. For instance, Choiet al. [12] reported one pot synthesis of (R)-mandelic acid ester from racemic phenylglyoxylic acid (PGA) in an aqueous/organic two-phase system with two-enzyme (lipase and recombinant mandelate racemase) process performed in hollow-fiber membrane bioreactor. However, as these reactions often involve nicotinamide cofactors [usually NADPH (nicotinamide adenine dinucleotide phosphate)], few applications on a large scale have been reported in spite of many reports regarding the improvement of cofactor regeneration [13, 14]. As a result, the use of whole cells rather than isolated enzymes is preferred to avoid the enzyme purification and cofactor regeneration [15].

Recently, we have succeeded in isolating a new bacterium strain with an ability of synthesizing high optical pure (R)-HPA. With 16S rDNA (ribosomal DNA) sequencing and phylogenic analysis it is identified asBacillus. sp. B26, and to the best of our knowledge, the biosynthesis of (R)-HPA employingBacillushas not been reported yet. In this paper, we describe the isolation and identification of microorganism for the asymmetric synthesis of (R)-HPA. In addition, the mutation of strains is also employed to increase the biotransformation efficiency.

2 MATERIALS AND METHODS

2.1 Materials

(R)-HPA and (S)-HPA were purchased from Sigma(St. Louis, MO USA). Phenylglyoxylic acid (PGA,＞98%) was supplied by Pharmaceutical & Chemical Co., Ltd of Taizhou, China. Methyl alcohol of HPLC grade was purchased from Merck, Germany. Hydroxypropyl-β-cyclodextrin (HP-β-CD) was supplied by Fluka (Neu Ulm, Germany). Strains (including 53 bacterium strains and 47 fungus strains, originally isolated from soil samples of Fujian province) were preserved in the lab of Chemical Engineering Department, Fuzhou University. All other chemicals were obtained commercially and of analytical grade.

2.2 Medium and fermentation conditions

2.2.1Medium

Cell growth (CG) medium (g?L-1): maltose 10,casein peptone 10, beef extract 5, NaCl 2, pH7.2.

Fermentation (FM) medium (g·L-1): maltose 10.9,casein peptone 11.5, beef extract 6.7, NH4H2PO44,K2HPO41.2, KH2PO42.5, MgSO4·7H2O 0.15, MnSO40.012, ZnSO40.008, pH 7.2.

All the above culture mediums were sterilized at 121 °C for 20 min. For solid medium agar (2% w/v)was added.

2.2.2Fermentation conditions

Strains maintained in the tube containing agar slant culture-medium were inoculated to 20 ml CG medium in 50 ml silicone stopper plugged flasks and incubated at 32 °C with an agitation speed of 180 r·min-1in a rotating shaker. When the cells reached logarithmic phase (about 20 h), cultures were used as inoculums (1%, volume fraction) to be transferred to the 50 ml flasks containing 20 ml of FM medium under the same fermentation condition. Substrate PGA, with the final concentration 10 mmol·L-1, was added directly into the fermentation broth under aseptic conditions to start the biotransformation reaction. Samples were withdrawn after 24 h, centrifuged and assayed by HPLC (High Performance Liquid Chromatography)and HPCE (High Performance Capillary Electrophoresis) for the yield and enantiomeric excess of HPA(eep) in the FM medium, respectively.

2.3 Isolation of organism

Strains were subjected to fermentation conditions mentioned in Section 2.2.2 to evaluate their potential HPA dehydrogenase activity by measuring the yield andeepof generated HPA. The one yielding the highest output and enantiomeric purity of HPA was chosen for further investigation.

2.4 Analytical methods

Biomass concentrations were estimated by measuring the optical density of the bacterial suspension at 600 nm with a UV-Vis spectrophotometer (SP-752,Shanghai Spectrum Instruments Co., China).

The concentrations of PGA and HPA in the fermentation broth were determined by HPLC (Varian ProStar) equipped with Agilent HC-C18 column(Φ4.6 mm×250 mm, 5 μm) and monitored with a UV detector at 220 nm. Mobile phase is phosphate buffer(25 mmol·L-1) with 6 mmol·L-1tetrabutyl ammonium bromide (TBAB) as ionpair reagent and methanol(19∶81, volume fraction) at a flow rate of 1.0 ml·min-1, column temperature 30 °C. The retention time for racemic HPA and PGA was 15.0 min and 37.5 min, respectively. The yield of HPA is defined as

wherecHPAandcPGArepresent the concentration of yielding HPA and initial concentration of PGA.

Optical purity of HPA isomers in the fermentation medium was determined by HPCE (P/ACETMMDQ, Beckman Coulter Co., USA) containing a 60 cm×75 μm (I.D.) uncoated fused silica capillary. The capillary electrophoresis conditions were Tris-H3PO4solution (100 mmol·L-1, pH 7.6) containing HP-β-CD(150g?L-1) as mobile phase, at detection of 20 kV and 214 nm. The retention time for (R)- and (S)- HPA was 21.6 min and 22.1 min, respectively.

The enantiomeric excess of (R)- HPA (eep) is calculated by

where [(R)-HPA] and [(S)-HPA] are the concentration of (R)-HPA and (S)-HPA, respectively.

All experiments were performed in triplicates,and analyses were carried out in duplicates. The data given here are the means of the measurements.

Relative molecular mass of HPA was determined by high performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS) method.The separation was performed on RP-HPLC (Reversed Phase-High Performance Liquid Chromatography)system. The MS analysis was carried out on a LCQ(Liquid Chromatography Quadrupole) series ion trap mass spectrometer with an electrospray ionization source in the positive ion mode. Electrospray ionization was performed using a spray needle voltage of 4.5 kV and a capillary interface temperature of 275°C.Other parameters are: cone voltage, 15 V; noble gas(N2), 25 ml?min-1.

2.5 Identification of the strain and phylogenetic analysis

Genomic DNA of the isolate was extracted according to Sambrooket al. [16] with some changes.The differences were as follows: after incubation with Proteinase K, the mixture was extracted once with equal volume of phenol/chloroform/isopentanol (25∶24∶1)to remove protein and then twice with chloroform/isopentanol (24∶1) to remove traces of phenol. The quantity of DNA was measured by electrophoresis on a 1.0% (w/v) agarose TAE (tris-acetic acid-Na2EDTA·2H2O)-gels using ethidium bromide(EB) staining and all images were realized with a GelDoc-ItTS Imaging System (UVP). The 16S rDNA was selectively amplified using polymerase chain reaction (PCR) with Taq DNA polymerase (MBI-Fermentas) and the universal primer pair of 27F(5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R(5′-TACGGYTACCTTGTTACGACTT-3′). PCR product was analyzed by electrophoresis on 1.0% agarose TAE-gels and sequenced in both directions by Sangon Biotech (Shanghai, China).

The nucleotide sequence was searched for homology using the Nucleotide BLAST program at NCBI website and submitted to Genbank by BankIt submission tool. The 16S rDNA sequence of isolated strain was aligned with homologous sequences obtained from the NCBI database by MEGA5 [17] and CLUSTAL W multiple sequence alignment program. Phylogenetic and molecular evolutionary analyses were performed with the default setting of MEGA5 using the neighbor-joining method [18]. The bootstrap consensus tree was inferred from 1000 replicates [19].

2.6 UV mutagenesis and UV-LiCl cooperative mutagenesis

2.6.1UV irradiation

For UV mutagenesis, 108exponentially growing cells after washing were spread onto a sterilized petri dish (9 cm in diameter) with a magnetic rotor exposed to UV light (15 W) at a distance of 35 cm with exposure time ranging from 0 to 160 s at an interval of 20 s.Three replicates were carried out for each interval.After exposure, the plates were immediately transferred to ice water with a view to deactivating the enzymes concerning restoration. 5 ml each of the UV-irradiated cell suspensions after serially dilution under red light (in view of photoreversal) was transferred to individual petri plate containing FM agar medium with PGA final concentration of 5 mmol·L-1and incubated in dark to avoid photo reactivation.Colonies appeared within 48 h of incubation at 32 °C were computed and the viability cell count was carried out by spread plate technique and the percent of survival was calculated with [20]

whereSis the the percent of survival,Niis the initial viable cell count andNdis the viable cell count after mutation. The lethality rate is defined as 1-S.

2.6.2UV-LiCl cooperative mutagenesis

For UV-LiCl cooperative mutagenesis, the UV-irradiated for 30 and 40 s cell suspensions after serially dilution were spread on individual plates containing FM agar medium with PGA and different doses of LiCl, and the plates were incubated in dark at 32 °C for 48 h. The variation curve of lethality rateversusLiCl doses of 0, 0.3, 0.5, 0.7 and 0.9 g·L-1was plotted.

2.7 Screening and isolation of potential mutant strains

A two stage screening method was employed for isolating the potential mutant strains. In addition, the screening criteria were based on colony morphology and size.

Firstly, from different petri dishes, about 120 colonies (60 colonies for each treatment) having at lest 90% death rate were selected for further study. Secondly, the isolates were analyzed for their activities of HPA dehydrogenase by monitoring the yield andeepof HPA produced in FM medium. Only those colonies exhibiting the maximum yield andeepwere selected as potential strains for biotransformation of PGA to HPA for further exploration.

2.8 Comparison of biotransformation employing the wild strain and mutants

The efficiency of production of HPA by the wild strain and two mutants under study was performed following the procedure described in section 2.2.2,where PGA concentration was 5, 10, 15, 20 and 25 mmol·L-1. The time course of HPA production was also studied with respect to conversion of PGA and yield of HPA under the same conditions as section 2.2.2, where PGA concentration was 15 mmol·L-1instead of 10 mmol·L-1. Samples were withdrawn in 5 h intervals after adding PGA, centrifuged and assayed by HPLC and HPCE.

3 RESULTS AND DISCUSSION

3.1 Isolation of microorganisms for biotransformation of PGA to HPA

Among all the microorganisms screened, 10 bacterium strains were able to asymmetrically biosynthesis ofRconfiguration witheepmore than 90% except B17, while none of the fungi displayed any desired activity and some of them could not utilize PGA at all(data not shown). The yield of HPA andeepwere not invariant, depending on the microorganisms used in the reaction. As can be seen in Table 1, strain B26 is most active in stereoselective biosynthesis of (R)-HPA with an optical purity of 99.1% and the yield of 47.5%,indicating that HPA dehydrogenase in B26 possesses excellent stereospecificity. The relative molecular mass of product is agreed with HPA standard (Fig. 1),which further confirms that the product is HPA.Therefore, B26 (Fig. 2) was chosen as original strain for further study.

Table 1 Screening of microorganisms for biotransformation of PGA to HPA

Figure 1 Mass spectrum of standard of HPA and product

3.2 16S rDNA sequencing and phylogenetic analysis of the isolate strain B26

The partial 16S rDNA sequence of strain B26(comprising 1448 nucleotides) was successfully amplified, determined and submitted to the GenBank database where accession number HM852450 was allotted for the submission sequence. Performing BLAST analyses, the sequence was most closely related to variousBacilluswith maximal identity as high as 99% and a phylogenetic tree was constructed based on 16S rDNA sequence (Fig. 3). Presently the acceptable positional standard is that those strain sequences with the similarity higher than 97%-98% are regarded as belonging to the same genus [21, 22]. Therefore, the isolate was identified asBacillussp. B26. Notably,B.sp. B26 (the third one from the bottom) was grouped with the pair ofBacillussp. enrichment culture clone SYW5 (FJ601635.1) andBacilluscereusstrain FM-4(EU794727.1) (both of them are of 99% maximum identity and 99% query coverage withBacillussp. B26).

Figure 2 B26 under biological microscopes

Figure 3 Phylogenetic tree analysis of the isolated 16S rDNA with different Bacillus strains from the NCBI database by MEGA5 (The tree was obtained using the neighbor-joining method and computed by the p-distance model and pairwise deletion option. The bootstrap values were based on 1000 replicates.)

3.3 The effect of UV mutagenesis and UV-LiCl cooperative mutagenesis

It is well known that UV radiation will cause mutations in cells, due to mistakes in repair of the resultant thymine dimers. Characterized by its simple operation and high efficiency, UV induced mutation is extensively used for improvement of strains with higher productivity. LiCl is a metal salt mutagen, and it is always used with other mutagen since it has no effect when used alone. It can be found from Fig. 4 (a)that lethality rate of the organism increases drastically with an increase in the duration of UV irradiation.This may be due to the lethal DNA damage caused by UV irradiation. The penetration of UV irradiation through the cell membrane and destruction of DNA may alter protein structure. Accordingly, Fig. 4 (b)shows that lethality rate of the organism increases sharply with the increase of the duration of UV irradiation [in accordance with Fig. 4 (a)] and the doses of LiCl. LiCl may affect DNA reparation after UV irradiation or cause new damage to DNA frame, changing the mutants. Finally, the colonies exhibiting the maximal size in diameter indicating a certain tolerance towards PGA were selected from different plates with lethality rate no less than 90%. To obtain the best producer strain of (R)-HPA, those selected stains were subjected to fermentation for further evaluation in terms of the yield andeepof HPA.

Figure 4 The lethality rate of UV mutagenesis and UV-LiCl cooperative mutagenesis on Bacillus sp. B26 UV dose/s: ▲ 40; ■ 30(Reaction condition: temperature, 32 °C; pH, 7.2; shake speed, 180 r·min-1; PGA concentration, 10 mmol·L-1; reaction time, 24 h)

Among the total 120 mutants selected after mutagenesis, some increased the yield of HPA, while others had no observable improvement or even decreased the production of HPA (data not shown). Two of the positive mutants labeledBacillussp. UV-38 andBacillussp. ULi-11, which were mutated by UV light and UV-LiCl cooperative mutagens respectively, were selected for further study, since both of them significantly increased the yield of HPA, as high as 60.6%(eep, 99.3%) and 61.0% (eep, 99.4%), respectively.Furthermore,B. sp. UV-38 andB. sp. ULi-11 were stable after subcultured for 6 ages (one week for each age) with no visible lessening in HPA production (data no shown). The illustration results of the mutants through colonial morphology inspection and microscopic examination are coincident with the wild,which further confirmed that the mutants genetically belong to the wild.

3.4 Comparison for biotransformation of the wild strain and mutants

The wild strainB. sp. B26 and mutantsB. sp.UV-38 andB. sp. ULi-11 were subjected to PGA biotransformation. A comparative plot of biotransformation efficiency of the mutated and control strains is shown in Fig. 5. It is noticeable that in the case of non-mutated and UV-LiCl cooperative mutated, a maximum HPA yield at PGA concentration of 15 mmol·L-1is achieved, while UV-mutated strain reaches its maximum HPA yield at PGA concentration of 10 mmol·L-1, and the maximum value is highly enhanced by the mutants. It might result from the changes taking place in the cell membrane caused by UV light, which vary the mass transfer efficiency of PGA from external to internal. As can be seen from Fig. 5, the yield drops dramatically when PGA concentration is more than 15 mmol·L-1in the case ofB.sp. B26. That is due to the inactivation or inhibition of enzymes at high substrate concentrations. For mutated strains, the yield of PGA decreases more slowly than that ofB. sp. B26 when PGA concentration is 15-20 mmol·L-1and then follows monotonical decrease to zero when the concentration is more than 20 mmol·L-1. It seems that the mutants harbor mutation(s) in specific genes related to tolerance PGA. On the other hand, slightly lessening ineepvalue is observed when PGA concentrations range from 5 to 15 mmol·L-1. Higher than 15 mmol·L-1, theeepvalue declines gradually, which is in line with those from Xiaoet al. [23]. This indicates that the transformed configuration of responsible enzyme caused by substrate inhibition reduces the enantioselectivity.

Figure 5 Effect of PGA concentrations on HPA yield and eep by B. sp. B26, B. sp. UV-38 and B. sp. ULi-11□■ B. sp. B26; ○● B. sp. UV-38; △▲ B. sp. ULi-11(Reaction condition: temperature, 32 °C; pH, 7.2; shake speed,180 r·min-1; reaction time, 24 h; PGA concentration, 5-25 mmol·L-1)

The time courses of HPA production byB. sp.B26,B. sp. UV-38 andB. sp. ULi-11 were studied and are compared in Fig. 6. The HPA production by mutated strains in comparison with control strain takes more time for adaptation, especiallyB. sp. ULi-11,during the initial phase. The mutated strains, however,are more efficient after 15 h of fermentation,B. sp.ULi-11 in particular. Further incubation (after 25 h)does not increase HPA production, but PGA consumption continues, resulting in the extra growth of cells,as seen from Fig. 6 (a). This reveals that PGA and HPA are consumed for essential metabolism after depletion of essential nutrients such as maltose and casein peptone. The HPA production is enhanced by 39.1% byB. sp. ULi-11 (yield, 65.4%) compared toB.sp. B26 (yield, 47.0%) when cultured for 25 h.

Figure 6 Time courses of biotransformation of PGA by B. sp. B26, B. sp. UV-38 and B. sp. ULi-11(Reaction condition: temperature, 32 °C; pH, 7.2; shake speed,180 r·min-1; PGA concentration, 15 mmol·L-1; reaction time,5-30 min)

4 CONCLUSIONS

Bacterium B26 was isolated, which has a high ability to convert PGA into (R)-HPA, and identified asBacillussp. B26 by 16S rDNA sequencing and phylogenic analysis. In order to improve the production of HPA and toleration to PGA, UV irradiation and UV-LiCl cooperative mutagenesis techniques were employed and two ideal mutantsB. sp. UV-38 andB.sp. ULi-11 were obtained, respectively. The results indicated thatB. sp. UV-38 andB. sp. ULi-11 could produce HPA with higher yield without decreasing optical purity and tolerate higher concentrations of PGA compared to the wild one and that mutantB. sp.ULi-11 was better thanB. sp. UV-38. These characteristics are interesting regarding its use in industry.Thus,Bacillussp. ULi-11 is an excellent strain for use in the efficient production of (R)-HPA from PGA.

NOMENCLATURE

cconcentration, mmol·L-1

eeenantiomeric excess

Mrelative molecular mass

Nthe number of the viable cell

OD600optical density at 600 nm

Sthe percent of survival

ttime, s

zelectric charge, C

Subscripts

ddispose

iinitial

pproduct

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