• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Characterization of a novel deep-sea microbial esterase EstC10 and its use in the generation of (R)-methyl 2-chloropropionate*

    2018-05-07 06:07:40GONGYanhui公顏慧MASanmei馬三梅WANGYongfei王永飛XUYongkai許永楷SUNAijun孫愛君ZHANGYun張?jiān)?/span>HUYunfeng胡云峰
    Journal of Oceanology and Limnology 2018年2期
    關(guān)鍵詞:張?jiān)?/a>

    GONG Yanhui (公顏慧) MA Sanmei (馬三梅) WANG Yongfei (王永飛) XU Yongkai (許永楷) SUN Aijun (孫愛君) ZHANG Yun (張?jiān)? HU Yunfeng (胡云峰) 5

    1 Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China

    2 Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences,Guangzhou 510301, China

    3 Department of Biotechnology, Jinan University, Guangzhou 510632, China

    4 Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, China

    5 South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou 510275, China

    1 INTRODUCTION

    Chiral 2-chloropanoic acids and their ester derivatives are important building blocks for the synthesis of a great variety of chiral herbicides, chiral pharmaceuticals and chiral final chemicals (Schulze and Wubbolts, 1999; Kurata et al., 2004; Shen, 2005).For example, enantiomerically pure (R)-2-chloropanoic acid is utilized as a starting material in the synthesis of nutrition agent alanyl-glutamine (Breuer et al., 2004).Enantiomerically pure (S)-2-chloropanoic acid and its ester derivatives are crucial starting materials for the synthesis of (R)-2-phenoxypropionic acid, one crucial herbicide widely used in agriculture (K?hler et al.,1994; Kurata et al., 2004). It has been identified that(S)-2-phenoxypropionic acid exhibits potentially high toxicities and does not exert biological activities similar to itsR-enantiomer (Colton et al., 1995). So in order to avoid unexpected enantiomers and reduce the toxicities brought by unexpected enantiomers, the synthesis of chiral 2-chloropanoic acids and their ester derivatives has been a hot spot in asymmetric synthesis.

    Chiral 2-chloropanoic acids and their ester derivatives contain one chiral carbon center connected with a chlorine atom and could be synthesized using traditional organic synthesis. However, traditional organic synthesis suff ers from some problems such as harsh working conditions and great pollution to the environment. Scientists are trying to generate chiral chemicals like chiral 2-chloropanoic acids and their ester derivatives using enzymes through green biocatalytic methods (Bui et al., 2000; Wang et al.,2014). Due to the lack of hydroxyl group, chiral 2-chloropanoic acids and their ester derivatives cannot be prepared from keto precursors through bioreduction using dehydrogenases (Moore et al., 2007).So the preparation of chiral 2-chloropanoic acids and their ester derivatives is mainly through kinetic resolutions catalyzed by esterases.

    Esterases (EC 3.1.1.1) are one important class of hydrolases and have been widely utilized in industry(Koeller and Wong, 2001). Chiral alcohols and acids could be enzymatically prepared by using esterases through kinetic resolutions such as transesterifications, esterifications and hydrolysis reactions(Cambou and Klibanov, 1984; Ballesteros et al.,1989; Otero et al., 1990; Carta et al., 1991). Before our study, there were only few reports about the preparation of chiral 2-chloropanoic acids and their ester derivatives using esterases through kinetic resolutions, possibly because the structural difference on both sides of the ester bond was too small to be recognized by esterases. We previously functionally characterized one novel microbial esterase EST12-7 and utilized EST12-7 as a biocatalyst in the asymmetric synthesis of enantiomerically pure (R)-methyl 2-chloropropionate (Cao et al., 2016).

    Because of the diverse environments of the oceans,many novel microorganisms and novel esterases should be able to be identified from the oceans. So we can isolate novel microorganisms and use novel microbial enzymes represented by esterases from the oceans as biocatalysts in diverse industries. Herein,we cloned and functionally characterized one novel esterase EstC10 fromBacillussp. CX01 isolated from the deep sea of the Western Pacific Ocean. Deep-sea microbial esterase EstC10 was further developed into another novel biocatalyst in the preparation of enantiomerically pure (R)-methyl 2-chloropropionate with high enantiomeric excess.

    2 MATERIAL AND METHOD

    2.1 Microorganisms and reagents

    Bacillussp. CX01 was isolated from the deep sea of the Western Pacific Ocean. The plasmid pET28(a) was used as the protein expression vector. The strainsEscherichiacoliDH5α andE.coliBL21 (DE3) were used for cloning and protein expression, respectively.The reagents related to gene cloning were all obtained from the company of TransGen Biotech (Beijing,China).P-nitrophenyl (p-NP) esterases were purchased from Sigma (USA). (±)- methyl 2-chloropropionate and enantiomerically pure methyl 2-chloropropionate were purchased from Aladdin Industrial Corporation(Shanghai, China) and TCI Development Corporation(Shanghai, China), respectively. Other chemicals were of analytical grade.

    2.2 Sequence analysis

    The sequence homology of EstC10 was analyzed by the BLASTP program against the protein data banks at NCBI. DNAMAN 7.0 program was used to construct the multiple sequence alignments. Three dimensional model of enzyme was built and analyzed through the online Swiss-Model (https://www.swissmodel.expasy.org/) Phylogenetic tree analysis was conducted using the MEGA software version 5.Expasy (http://web.expasy.org/compute_pi/) were used to estimate the theoretical molecular weight andpIof EstC10.

    2.3 Construction of cloning and expression vector

    The gene encoding esterase EstC10 was cloned from the genome ofBacillussp. CX01 by using PCR with the following primers: 5'-CATGGATCCATGAAAATCGTCAAACCA-3'5'- CATCTCGAGTTATGTCTGCCAATCCAG-3'(the restriction sites ofBamHI andXhoI were underlined). PCR products were purified by using 0.8% agarose gel electrophoresis and cloned into pET28 (a) vectors by T4 ligase.Recombinant plasmids were transformed intoE.coliBL21 (DE3) competent cells for further protein expression.

    2.4 Expression and purification of EstC10

    The recombinantE.coliBL21 (DE3) cells were grown in LB medium supplemented with 50 μg/mL kanamycin at 37°C. To induce the target protein expression, when the OD600reached 0.6–0.8,isopropyl-β-D-thiogalactopyranoside (ITPG) at a final concentration of 0.3 mmol/L was added. After the protein was inducted at 22°C for 16 h, the cells were collected by centrifugation at 4 000 r/min for 20 min. Subsequently, the sediment were washed twice with 50 mmol/L phosphate buff er (pH 8.0),resuspended in 50 mmol/L Tris-HCl buff er (pH 8.0)and then disrupted by sonication on ice for 15 min.The supernatants were collected by centrifugation at 9 000 r/min for 30 min. The purification and desalination of target proteins were carried out by nickel-nitrilotriacetic acid agarose resin (QIAGEN,Hilden, CA, Germany), and PD-10 desalting columns(GE Healthcare Life Science, UK), respectively. The concentrations of purified proteins were measured by the Bradford method with bovine serum albumin as a standard. Purified esterase EstC10 was analyzed by SDS-PAGE. The enzyme powders were preparated by SCIENT-N10 freeze dryer and further stored at-20°C for following experiments.

    2.5 Biochemical characterization of EstC10

    The biochemical characterization was studied by enzymatic reaction systems containng 8 μL substrate(10 mmol/L, dissolved in acetonitrile) and 10-μL(0.16 μg/μL) purified EstC10 in 382 μL Tris-HCl buff er (50 mmol/L, pH 8.0). After the reactions were carried out at room temperature for 5 min, 100 μL ethanol was added to inbibit the reactions. Then the enzymatic activity was determined by measuring the absorbance ofp-NP at the wavelength of 405 nm immediately after the addition of ethanol. One unit of enzyme activity was defined as the amount of esterase required to release 1 μmol ofp-NP per minute.

    Variousp-NP esters (p-NP C2–C12) were used to study the substrate specificity of EstC10 under standard reaction conditions. The optimal pH of of EstC10 was determined by incubating standard reactions in different pH buff ers: 50 mmol/L PBS (pH 6.0–8.0) and 50 mmol/L Tris-HCl (pH 8.0–10.0). The pH stability was researched by detecting the residual hydrolytic activity of EstC10 (same as the method mentioned above that measuring the absorbance ofp-NP at the wavelength of 405 nm) after incubating EstC10 in different pH buff ers for different times.Standard reaction systems were carried out at temperatures ranging from 10–55°C to investigate the optimal temperature of EstC10. The themo-stability of EstC10 was studied by incubating EstC10 for 15 min, 30 min, 45 min and 60 min at temperatures ranging from 30 to 50°C.The effect of organic solvents on the hydrolytic activity of EstC10 was studied by incubating the enzyme at 200 r/min for 3 h in the presence of different organic solvents at a final concentration of 10%, 20% and 50% (V/V),respectively. EstC10 was incubated at room temperature for 3 h in the presence of various metal ions at final concentrations of 2 mmol/L and 5 mmol/L or surfactants at final concentrations of 0.1% and 0.5% (W/V), respectively, to investigate the effect of metal ions and surfactants on the hydrolytic activity of EstC10.

    2.6 Kinetic resolution of racemic methyl 2-chloropropionate by EstC10

    Standard 500-μL reactions containing 10 mg enzyme powders, 80 mmol/L methyl 2-chloropropionate in 50 mmol/L buff er (pH 8.0) were carried out at 37°C for 15 min. Dodecane was added to the reaction samples at a final concentration of 5 mmol/L as an internal standard after the termination of the enzymatic resolution reactions. The products were extracted by adding 500-μL ethyl acetate and the organic phase was analyzed by chiral gas chromatograph (GC) equipped with112-6636 CYCLOSIL-B chiral capillary column(30 m×0.25 mm ID, 0.25 μm df) and H2flame ion detector. The split flow rate of carrier gas, nitrogen,was 1.20 mL/min. The temperature was 100°C, raised to 220°C at a rate of 15°C/min and maintained for 2 min. The temperature of injector and detector were 220 and 250°C, respectively. The enantiomeric excess(e.e.) and yield (Y) were calculated by the formulas described by Chen et al. (1982).

    e.e.=([S-methyl 2-chloropropionate]-[R-methyl 2-chloropropionate]) / ([S-methyl 2-chloropropionate]+[R-methyl 2-chloropropionate]).e. andYrepresent the enantiomeric excess and yield ratio of methyl 2-chloropropionate, respectively.AandA0represent the content ofS-methyl 2-chloropropionate after and before reactions, respectively.

    A series of reactions were performed at different pH buff ers ranging from 6.0 to 9.0 to determine the optimal pH for thepreparing of (R)-methyl 2-chloropropionate. The optimal temperature for preparing (R)-racemic methyl 2-chloropropionate was investigated by incubating the enzymatic reactions at temperatures ranging from 25°C to 50°C.The effect of organic solvents and surfactants on the preparation of (R)-methyl 2-chloropropionate were investigated by incubating the enzymatic reactions in the presence of different organic solvents at a final concentration of 10% (V/V) or various surfactants at a final concentration of 0.1% (W/V). different concentrations of racemic methyl 2-chloropropionate(ranging from 60 mmol/L to 120 mmol/L) were utilized to determine the optimal substrate concentration for the preparing (R)-methyl 2-chloropropionate. The best reaction time for preparing (R)-methyl 2-cholopropionate catalyzed by EstC10 was investigated by incubating the kinetic reactions at 37°C for different times.

    Fig.1 Multiple sequence alignment of EstC10 with some related esterases belonging to Family XIII

    3 RESULT AND DISCUSSION

    3.1 Sequence analysis of EstC10

    A geneEstC10encoding an esterase was identified from the genome ofBacillussp. CX01 isolated from the deep sea of the Western Pacific Ocean. The GenBank accession number ofEstC10is KY478994.The protein sequence of EstC10 was blasted against the NCBI protein database. EstC10 exhibited 99%identity with one putative carboxylesterase(WP_003185416.1) fromBacillus, 98% identity with one putative carboxylesterase YVAK(WP_020452984.1) fromBacillusand 96% identity with one putative carboxylesterase (WP_026588800.1)fromBacillussp. NSP9.1. Although those proteins shared highly similarity with EstC10, the functionalities of those proteins have not been well characterized before. According to the phylogenetic tree analysis, EstC10 could be classified to family XIII of esterases. Multiple sequence alignment analysis indicated that EstC10 shared the pentapeptide GLSLG(Fig.1), which fits the serine α/β hydrolase consensus sequence Sm-X-Nu-X-Sm (Sm: small residue, X: any residue and Nu: nucleophile) (Nardini and Dijkstra,1999). Ser93, Asp192 and His222 were predicted to be the catalytic triad of EstC10 according to the treedimensional model (Rozeboom et al., 2014).

    Fig.2 SDS-PAGE of purified EstC10

    3.2 Expression and purification of EstC10

    The theoretical molecular weight andpIwere calculated to be 31.4kD and 5.1, respectively. The recombinant EstC10 was successfully expressed inE.coliBL21 (DE3) and purified by Ni-NTA affinity chromatography (Fig.2).

    3.3 Substrate specificity of EstC10

    The hydrolytic activity of EstC10 was investigated with differentp-NP esterase as substrates (Fig.3). The experiments showed thatp-NP C2 was the preferred substrate of EstC10, with a specific activity of 310 U/mg. The hydrolytic activity of EstC10 was higher than those of EstA (176 U/mg) (Park et al., 2007),EM2L8 (156 U/mg) (Chu et al., 2008), and Est6(104 U/mg) (Jiang et al., 2012). The relative hydrolytic activity of EstC10 towardp-NP C4 andp-NP C6 were 84% and 56%, respectively. The relative activity towardp-NP (>C6) decreased rapidly. Those results indicated that EstC10 was a true esterase instead of a lipase (Arpigny and Jaeger, 1999).

    Fig.3 Hydrolytic activity of EstC10 toward p-NP esters with different acyl chain lengths (C2–C4)

    3.4 effect of pH on the hydrolytic activity of EstC10

    As shown in Fig.4a, the optimal pH of EstC10 was 8.0. Esterases BSE04211 from Bacillus (Liang et al.,2016a), MT6 (Deng et al., 2016), Est12-7 (Cao et al.,2016) and ScsEst01 from a South China Sea sediment metagenome (Zhang et al., 2015) had similar optimal pH. EstC10 exhibited high hydrolytic activities from pH 7.0 to 9.0. The hydrolytic activity of EstC10 dropped sharply under acidic or alkaline condition.Additionally, the hydrolytic activity of EstC10 in PBS buff er was better than that in Tris-HCl buff er under the same pH. EstC10 exhibited good stability from pH 6.0 to 9.0, with the residual hydrolytic activities being over 50% of the maximum hydrolytic activity(Fig.4b). However, EstC10 did not exhibit good stability at pH lower than 6.0 or higher than 9.0.

    3.5 effect of temperature on the hydrolytic activity of EstC10

    The optimal temperature was 35°C (Fig.4c), which was lower than that of esterases isolated from marine environment, BSE04211 (40°C) (Liang et al., 2016a),BSE01281(50°C) (Liang et al., 2016b) and E29(45°C) (Li, 2016). EstC10 exhibited high stability activities from 30°C to 45°C, at which the relative hydrolytic activities were above 80%. EstC10 performed excellent good stability at temperature between 30°C and 45°C (Fig.4d). After incubating EstC10 at 45°C for 90 min, the residual hydrolytic activity of EstC10 was above 65%. Its thermostability dropped rapidly when the temperature was over 50°C, with 10% of its original hydrolytic activity left after incubation at 50°C for 15 min.

    Fig.4 effect of pH and temperature on the activity of EstC10

    Table 1 effect of organic solvents on the hydrolytic activity of EstC10

    3.6 effect of organic solvents on the hydrolytic activity of EstC10

    As shown in Table 1, 50% (V/V) isooctane and n-hexane were observed to have stimulating effects on its hydrolytic activity. The similar phenomenon was also observed from esterase RppE01 (Ma et al.,2013). However, other organic solvents in this study performed inhibiting effect on the hydrolytic activity of EstC10, especially dimethylbenzene, 1-pentanol,tetrahydrofuran and cyclohexanone.

    3.7 effect of metal ions on the hydrolytic activity of EstC10

    According to the data from Table 2, the hydrolytic activity of EstC10 was not affected by Na+, Ba2+, K+,Mg2+or Ca2+. However, the hydrolytic activity of EstC10 was inhibited by the presence of Cu2+and Zn2+. Esterase EstZF172 (Xu et al., 2015), one esterase fromGeobacillussp. JM6 (Zhu et al., 2015), EstOF4(Rao et al., 2013) and EstCE1 (Elend et al., 2006)exhibited similar results.

    3.8 effect of surfactants on the hydrolytic activity of EstC10

    Fig.5 effect of pH on the preparation of ( R)-methyl 2-chloropropionate (a); effect of temperature on the preparation of ( R)-methyl 2-chloropropionate (b); effect of substrate concentration on the preparation of ( R)-methyl 2-chloropropionate(c); effect of reaction time on the preparation of ( R)-methyl 2-chloropropionate (d)

    Table 2 effect of metal ions on the hydrolytic activity of EstC10

    Results in Table 3 indicated that tween-20 and sodium tripolyphosphate at a concentration of 0.5%had stimulating effect on the hydrolytic activity of EstC10. However, Tween-80 and TritonX-100 exhibited negative effects on its hydrolytic activity. In addition, the hydrolytic activity of EstC10 was strongly inhibited by SDS, SDBS and CTAB.

    Table 3 effect of surfactants on the hydrolytic activity of EstC10

    3.9 effect of pH on the preparation of ( R)-methyl 2-chloropropionate

    The effect of pH on the kinetic resolution of methyl 2-chloropropionate catalyzed by EstC10 was investigated by incubating standard reaction systems at pH ranging from 6.0–8.5 (Fig.5a). The optimal pH for the enzymatic resolution of racemic methyl 2-chloropropionate was found to be pH 8.0,with thee.e. being 83% and a yield being 33%.

    3.10 effect of temperature on the preparation of( R)-methyl 2-chloropropionate by EstC10

    Standard reaction systems were carried out atdifferent temperatures ranging from 25°C to 50°C to study the effect of temperature on the kinetic resolution of racemic methyl 2-chloropropionate catalyzed by EstC10 (Fig.5b). The higheste.e. (83%)was obtained at 37°C with a yield of 33%. So, 37°C was determine to be the optimal temperature for preparing (R)-methyl 2-chloropropionate catalyzed by EstC10.

    Table 4 effect of organic solvents on the preparation of ( R)-methyl 2-chloropropionate

    Table 5 effect of surfactants on the preparation of ( R)-methyl 2-chloropropionate

    3.11 effect of organic solvents on the preparation of ( R)-methyl 2-chloropropionate by EstC10

    different organic solvents were used to study the effect of organic solvents on the kinetic resolution of racemic methyl 2-chloropropionate catalyzed by EstC10 under the optimal temperature and pH (37°C,pH 8.0). As shown in Table 4, all organic solvents exhibited negative effects on the kinetic resolution of racemic methyl 2-chloropropionate by EstC10.Therefore, no organic solvent was added to further optimize the kinetic resolutions.

    3.12 effect of surfactants on the preparation of ( R)-methyl 2-chloropropionate catalyzed by EstC10

    Various surfactants at final concentrations of 0.1%,0.5% and 1% (W/V) were added into the standard resolution reaction systems to investigate the effect of surfactants on the kinetic resolution of racemic methyl 2-chloropropionate (Table 5). All tested surfactants had negative effects on the kinetic resolution of racemic methyl 2-chloropropionate. So no surfactant was utilized to further optimize the kinetic resolutions.

    3.13 effect of substrate concentration on the preparation of ( R)-methyl 2-chloropropionate catalyzed by EstC10

    The effect of substrate concentration on the preparation of ( R)-methyl 2-chloropropionate was investigated by adding substrate of concentrations ranging from 60 mmol/L to 120 mmol/L into the standard reaction systems (Fig.5c). The enantiomeric excess and yield of ( R)-methyl 2-chloropropionate generated could reach up to 93% and 33%,respectively, when the concentration of substrate was set at 80 mmol/L. With the increase of substrate concentration, the yield increased, but the enantiomeric excess dramatically decreased.Therefore, 80 mmol/L was determined to be the optimal substrate concentration for the kinetic resolution of racemic methyl 2-chloropropionate catalyzed by EstC10.

    3.14 effect of reaction time on the preparation of( R)-methyl 2-chloropropionate by EstC10

    The enzymatic kinetic resolution reactions were carried out at 37°C for different times to study the effect of reaction time on the kinetic resolution of racemic methyl 2-chloropropionate catalyzed by EstC10 (Fig.5d). The highest enantiomeric excess was obtained when the enzymatic kinetic resolution reaction was carried out for 30 min, with an enantiomeric excess of 99% and yield of 30%. When the reactions were carried out for longer times, both the enantiomeric excess and the yield basically remained unchanged.Therefore, 30 min was characterized to be the optimal reaction time for enzymatic kinetic resolution of methyl 2-chloropropionate.

    3.15 Comparation of EstC10 and other esterases in the kinetic resolutions of racemic methyl 2-chloropropionate

    Before this study, our research group functionally characterized one novel esterase Est12-7 fromPseudoncardiaantitumoradisand used Est12-7 as a novel biocatalyst in the enzymatic kinetic resolution of methyl 2-chloropropionate. Esterase Est12-7 could hydrolyze racemic methyl 2-chloropropionate and generate (R)-methyl 2-chloropropionate with high enantiomeric excess (e.e.>99%) and conversion (49%)under optimal conditions (50 mmol/L racemic substrate, pH 8.0, 30 min at 20°C) (Cao et al., 2016).In our work, we also functionally characterized one novel deep-sea microbial esterase EstC10. Esterase EstC10 was further used as a novel biocatalyst in the kinetic resolution of racemic methyl 2-chloropropionate and generated (R)-methyl 2-chloropropionate. Under optimal conditions (80 mmol/L racemic substrate, pH 8.0, 30 min at 37°C), the enantiomeric excess could reach 99%. However, the yield of (R)-methyl 2-chloropropionate generated by EstC10 was not quite high (30%) and remained constant even after the enzymatic reactions were incubated for longer time, possibly because the acid generated during kinetic resolutions denatured the biocatalyst.Remarkably, compared to Est12-7, the optimal substrate concentration of EstC10 was much higher,indicating esterase EstC10 is a novel biocatalyst which can bear higher substrate concentration and possesses very good potential in asymmetric synthesis. In addition, the sequence alignment of EstC10 with Est12-7 showed that these two enzymes were of very low sequence identities (10.39%), indicating that EstC10 was a novel promising biocatalyst identified from deep-sea microorganisms. Additionally, one lipase fromCandidacylindraceacould hydrolyze racemic methyl 2-chloropropionate and generate (S)-methyl 2-chloropropionate, with the enantiomeric excess and yield being 95% and 30%, respectively(Dahod and Siuta-Mangano, 1987).

    4 CONCLUSION

    In conclusion, we identified one novel esterase EstC10 fromBacillussp. CX01 isolated from the deep sea of the Western Pacific Ocean and characterized the functionalities of EstC10. At present, the reports about the kinetic resolution of racemic methyl 2-chloropropionate were quite rare.So further developed deep-sea microbial esterase EstC10 as a novel biocatalyst in the kinetic resolution of racemic methyl 2-chloropropionate and generate(R)-methyl 2-chloropropionate with high enantiomeric excess (>99%) after the optimization of process parameters such as pH, temperature, organic cosolvents, surfactants, substrate concentration and reaction time. Notably, the optimal substrate concentration (80 mmol/L) of esterase EstC10 was higher than that of another kinetic resolution catalyzed by esterase Est12-7 (50 mmol/L) (Cao et al., 2016).Thus, deep-sea microbial esterase EstC10 is a promising biocatalyst in the generation of (R)-methyl 2-chloropropionate as well of many other valuable chiral chemicals in industry. Some other technologies such as protein engineering may be necessary to further improve the enzymatic properties of biocatalyst EstC10.

    5 ACKNOWLEDGEMENT

    We thank the R/VKexueof the Chinese Academy of Sciences for collecting samples and WPOS sample center for providing samples.

    Arpigny J L, Jaeger K E. 1999. Bacterial lipolytic enzymes:classification and properties.Biochem.J.,343: 177-183.

    Ballesteros A, Bernabé M, Cruzado C, Martín-Lomas M,Otero C. 1989. Regioselective deacylation of 1,6-anhydroβ-D-galactopyranose derivatives catalyzed by soluble and immobilized lipases.Tetrahedron,45(22): 7 077-7 082,https://doi.org/10.1016/S0040-4020(01)89175-4.

    Breuer M, Ditrich K, Habicher T, Hauer B, Ke?eler M, Stürmer R, Zelinski T. 2004. Industrial methods for the production of optically active intermediates.Angew.Chem.Int.Ed.Engl.,43(7): 788-824.

    Bui V P, Hansen T V, Stenstr?m Y, Hudlicky T. 2000. Direct biocatalytic synthesis of functionalized catechols: a green alternative to traditional methods with high effective mass yield.GreenChemistry,2(6): 263-265.

    Cambou B, Klibanov A M. 1984. Comparison of different strategies for the lipase-catalyzed preparative resolution of racemic acids and alcohols: asymmetric hydrolysis,esterification, and transesterification.Biotechnologyand Bioengineering,26(12): 1 449-1 454.

    Cao Y Y, Deng D, Sun A J, Zhang Y, Hu Y F. 2016. Functional characterization of a novel marine microbial esterase and its utilization in the enantioselective preparation of (R)-methyl 2-chloropropionate.Appl.Biochem.Biotechnol.,180(2): 210-227.

    Carta G, Gainer J L, Benton A H. 1991. Enzymatic synthesis of esters using an immobilized lipase.Biotechnologyand Bioengineering,37(11): 1 004-1 009.

    Chen C S, Fujimoto Y, Girdaukas G, Sih C J. 1982. Quantitative analyses of biochemical kinetic resolutions of enantiomers.J.Am.Chem.Soc.,104(25): 7 294-7 299.

    Chu X M, He H Z, Guo C Q, Sun B L. 2008. Identification of two novel esterases from a marine metagenomic library derived from South China Sea.Appl.Microbiol.Biotechnol.,80(4): 615-625.

    Colton I J, Ahmed S N, Kazlauskas R J. 1995. A 2-propanol treatment increases the enantioselectivity ofCandida rugosalipase toward esters of chiral carboxylic acids.J.Org.Chem.,60(1): 212-217.

    Dahod S K, Siuta-Mangano P. 1987. Carbon tetrachloridepromoted stereo selective hydrolysis of methyl-2-chloropropionate by lipase.Biotechnologyand Bioengineering,30(8): 995-999.

    Deng D, Zhang Y, Sun A J, Liang J Y, Hu Y F. 2016. Functional characterization of a novel marine microbial GDSL lipase and its utilization in the resolution of (±)-1-phenylethanol.Appl.Biochem.Biotechnol.,179(1): 75-93.

    Elend C, Schmeisser C, Leggewie C, Babiak P, Carballeira J D, Steele H L, Reymond J L, Jaeger K E, Streit W R.2006. Isolation and biochemical characterization of two novel metagenome-derived esterases.Appl.Environ.Microbiol.,72(5): 3 637-3 645.

    Jiang X W, Xu X W, Huo Y Y, Wu Y H, Zhu X F, Zhang X Q,Wu M. 2012. Identification and characterization of novel esterases from a deep-sea sediment metagenome.Arch.Microbiol.,194(3): 207-214.

    Koeller M K, Wong C H. 2001. Enzymes for chemical synthesis.Nature,409(6817): 232-240.

    K?hler J E H, Hohla M, Richters M, K?nig W A. 1994. A molecular-dynamics simulation of the complex formation between methyl (R)/(S)-2-chloropropionate and heptakis(3-O-acetyl-2, 6-di-O-pentyl)-β-cyclodextrin.BerichtederdeutschenchemischenGesellschaft,127(1):119-126.

    Kurata A, Kurihara T, Kamachi H, Esaki A N. 2004.Asymmetric reduction of 2-chloroacrylic acid to (S)-2-chloropropionic acid by a novel reductase fromBurkholderiasp. WS.Tetrahedron:Asymmetry,15(18):2 837-2 839, https://doi.org/10.1016/j.tetasy.2004.06.035.

    Li Z Y, Rong Z, Wang Z, Huo Y Y, Meng F X, Wang C S, Cui H L, Xu X W. 2016. Cloning, expression and characterization of a novel esterase (E29) from a marine bacterium Altererythrobacter luteolus SW109T.Microbiology,43(5): 1 051-1 059, https://doi.org/10.13344/j.microbiol.china.150974. (in Chinese with English abstract)

    Liang J Y, Sun A J, Zhang Y, Deng D, Wang Y F, Ma S M, Hu Y F. 2016a. Functional characterization of a novel microbial esterase identified from the Indian Ocean and its use in the stereoselective preparation of (R)-methyl mandelate.Chin.J.Oceanol.Limnol.,34(6): 1 269-1 277.

    Liang J Y, Zhang Y, Sun A J, Deng D, Hu Y F. 2016b.Enantioselective resolution of (±)-1-phenylethanol and(±)-1-phenylethyl acetate by a novel esterase fromBacillussp. SCSIO 15121.Appl.Biochem.Biotechnol.,178(3): 558-575.

    Ma B D, Yu H L, Pan J, Li J Y, Ju X, Xu J H. 2013. A thermostable and organic-solvent tolerant esterase fromPseudomonasputidaECU1011: catalytic properties and performance in kinetic resolution of α-hydroxy acids.BioresourceTechnology,133: 354-360, https://doi.org/10.1016/j.biortech.2013.01.089.

    Moore J C, Pollard D J, Kosjek B, Devine P N. 2007. Advances in the enzymatic reduction of ketones.Acc.Chem.Res.,40(12): 1 412-1 419.

    Nardini M, Dijkstra B W. 1999. α/β Hydrolase fold enzymes:the family keeps growing.CurrentOpinioninStructural Biology,9(6): 732-737, https://doi.org/10.1016/S0959-440X(99)00037-8.

    Otero C, Pastor E, Ballesteros A. 1990. Synthesis of monobutyrylglycerol by transesterification with soluble and immobilized lipases.Appl.Biochem.Biotechnol.,26(1): 36-44.

    Park H J, Jeon H J, Kang S G, Lee J H, Lee S A, Kim H K.2007. Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome.Protein.Expr.Purif.,52(2): 340-347, https://doi.org/10.1016/j.pep.2006.10.010.

    Rao L, Xue Y F, Zheng Y Y, Lu J R, Ma Y H. 2013. A novel alkaliphilicbacillusesterase belongs to the 13thbacterial lipolytic enzyme family.PLoSOne,8(4): e60645, https://doi.org/10.1371/journal.pone.0060645.

    Rozeboom H J, Godinho L F, Nardini M, Quax W J, Dijkstra B W. 2014. Crystal structures of twoBacilluscarboxylesterases with different enantioselectivities.Biochim.Biophys.Acta,1844(3): 567-575, https://doi.org/10.1016/j.bbapap.2014.01.003.

    Schulze B, Wubbolts M G. 1999. Biocatalysis for industrial production of fine chemicals.CurrentOpinionin Biotechnology,10(6): 609-615, https://doi.org/10.1016/S0958-1669(99)00042-7.

    Shen G Y. 2005. 2-chloropropionic acid.FineandSpecialty Chemicals,13(13): 19-20, 30. (in Chinese with English abstract)

    Wang M, Bao W J, Wang J, Wang K, Xu J J, Chen H Y, Xia X H. 2014. A green approach to the synthesis of novel“Desert rose stone”-like nanobiocatalytic system with excellent enzyme activity and stability.Sci.Rep.,4: 6 606.

    Xu F X, Chen S Y, Xu G, Wu J P, Yang L R. 2015. Discovery and expression of aPseudomonassp. esterase as a novel biocatalyst for the efficient biosynthesis of a chiral intermediate of pregabalin.BiotechnologyandBioprocess Engineering,20(3): 473-487.

    Zhang H, Li F C, Chen H X, Zhao J, Yan J F, Jiang P, Li R G,Zhu B L. 2015. Cloning, expression and characterization of a novel esterase from a South China Sea sediment metagenome.Chin.J.Oceanol.Limnol.,33(4): 819-827.

    Zhu Y B, Zheng W G, Ni H, Liu H, Xiao A F, Cai H N. 2015.Molecular cloning and characterization of a new and highly thermostable esterase fromGeobacillussp. JM6.J.BasicMicrobiol.,55(10): 1 219-1 231.

    猜你喜歡
    張?jiān)?/a>
    修德箴言
    Electronic structure study of the charge-density-wave Kondo lattice CeTe3
    《大漠英雄曲》
    民族藝林(2022年4期)2023-01-19 04:06:16
    A Clever Bird
    搭伙
    金秋(2021年5期)2021-07-01 12:29:46
    出山一條路
    上海故事(2021年11期)2021-01-13 06:17:43
    均值—方差分析及CAPM模型的運(yùn)用
    智富時代(2019年4期)2019-06-01 07:35:00
    豪華墓地
    故事會(2017年6期)2017-03-23 18:22:58
    造墳
    大江南北(2015年1期)2015-08-12 07:32:05
    国产精品一区二区免费欧美| 国产av麻豆久久久久久久| 免费av观看视频| 亚洲中文字幕一区二区三区有码在线看| 搡老岳熟女国产| 国产一区二区三区在线臀色熟女| 精品一区二区三区视频在线| 日日夜夜操网爽| 一级黄色大片毛片| 精品国产三级普通话版| 亚洲七黄色美女视频| aaaaa片日本免费| 成人欧美大片| 免费在线观看影片大全网站| АⅤ资源中文在线天堂| 久久久色成人| 可以在线观看毛片的网站| 亚洲中文字幕日韩| 黄色女人牲交| www.www免费av| 老司机福利观看| 9191精品国产免费久久| 亚洲美女黄片视频| 亚洲av熟女| 又爽又黄无遮挡网站| 看黄色毛片网站| 91久久精品电影网| 午夜福利免费观看在线| 非洲黑人性xxxx精品又粗又长| 欧美潮喷喷水| 国产精品久久久久久亚洲av鲁大| 欧美一区二区国产精品久久精品| 观看免费一级毛片| 日日夜夜操网爽| 美女 人体艺术 gogo| 日日干狠狠操夜夜爽| 亚洲狠狠婷婷综合久久图片| 日本a在线网址| a在线观看视频网站| 欧美日韩福利视频一区二区| xxxwww97欧美| 直男gayav资源| 亚洲av成人精品一区久久| 国产精品人妻久久久久久| 国产精品国产高清国产av| 国产午夜福利久久久久久| 久久人人精品亚洲av| 国产蜜桃级精品一区二区三区| 一进一出好大好爽视频| 极品教师在线免费播放| xxxwww97欧美| 国产黄片美女视频| 亚洲av免费在线观看| 亚洲五月婷婷丁香| 欧美最黄视频在线播放免费| 男人舔奶头视频| 亚洲国产欧洲综合997久久,| 热99re8久久精品国产| 99久久精品一区二区三区| 国产精品综合久久久久久久免费| 99在线人妻在线中文字幕| 国产淫片久久久久久久久 | 窝窝影院91人妻| 12—13女人毛片做爰片一| 免费高清视频大片| 两人在一起打扑克的视频| 好看av亚洲va欧美ⅴa在| 亚洲真实伦在线观看| 中文字幕熟女人妻在线| 午夜福利成人在线免费观看| 亚洲最大成人中文| 日本免费一区二区三区高清不卡| 日韩精品青青久久久久久| 精品午夜福利在线看| 淫秽高清视频在线观看| 成人性生交大片免费视频hd| 国产人妻一区二区三区在| 亚洲精品456在线播放app | 九九热线精品视视频播放| 精品一区二区三区视频在线观看免费| av欧美777| 美女xxoo啪啪120秒动态图 | 成人高潮视频无遮挡免费网站| 亚洲专区国产一区二区| av中文乱码字幕在线| 91麻豆精品激情在线观看国产| 黄色视频,在线免费观看| 国产亚洲av嫩草精品影院| 麻豆av噜噜一区二区三区| 国产高清三级在线| 欧美激情久久久久久爽电影| 中文字幕av成人在线电影| 91麻豆精品激情在线观看国产| 制服丝袜大香蕉在线| 麻豆成人午夜福利视频| 亚洲男人的天堂狠狠| 日日夜夜操网爽| 久久国产精品影院| 午夜影院日韩av| 亚洲熟妇中文字幕五十中出| 乱人视频在线观看| 男女之事视频高清在线观看| 精品熟女少妇八av免费久了| 亚洲av不卡在线观看| 中文字幕av在线有码专区| 全区人妻精品视频| 日韩欧美精品v在线| 变态另类丝袜制服| 亚洲精品久久国产高清桃花| 国产单亲对白刺激| 日日摸夜夜添夜夜添小说| 变态另类成人亚洲欧美熟女| 少妇人妻一区二区三区视频| 国产一区二区亚洲精品在线观看| 国产精品久久视频播放| 久久久国产成人精品二区| 99热精品在线国产| 久久国产精品影院| 3wmmmm亚洲av在线观看| 免费看美女性在线毛片视频| 在线免费观看不下载黄p国产 | 韩国av一区二区三区四区| 国内精品美女久久久久久| 国产高清激情床上av| 天堂动漫精品| 69av精品久久久久久| 麻豆av噜噜一区二区三区| 波多野结衣巨乳人妻| 动漫黄色视频在线观看| 亚洲精品日韩av片在线观看| 噜噜噜噜噜久久久久久91| 可以在线观看的亚洲视频| 欧美国产日韩亚洲一区| 久久精品国产亚洲av涩爱 | 亚洲av熟女| 亚洲黑人精品在线| 一进一出抽搐动态| 又爽又黄a免费视频| 中文字幕高清在线视频| 精品一区二区三区视频在线观看免费| 少妇人妻一区二区三区视频| 国产av不卡久久| av在线天堂中文字幕| 日本免费a在线| 三级国产精品欧美在线观看| 成人亚洲精品av一区二区| 亚洲人成网站高清观看| 身体一侧抽搐| 国产91精品成人一区二区三区| 99在线人妻在线中文字幕| 日日干狠狠操夜夜爽| 亚洲成av人片免费观看| 色综合欧美亚洲国产小说| 久久久久久久久中文| 国产一级毛片七仙女欲春2| 男女做爰动态图高潮gif福利片| 国产精品自产拍在线观看55亚洲| 亚洲av免费高清在线观看| 亚洲av电影不卡..在线观看| 国产亚洲精品av在线| 身体一侧抽搐| 我的女老师完整版在线观看| 免费人成视频x8x8入口观看| 女人被狂操c到高潮| 国产精品99久久久久久久久| 亚洲精品一区av在线观看| 色哟哟·www| 欧美xxxx黑人xx丫x性爽| 国产在线精品亚洲第一网站| 美女免费视频网站| 亚洲欧美日韩高清专用| 国产伦在线观看视频一区| 香蕉av资源在线| bbb黄色大片| 国产精品一区二区三区四区免费观看 | 国产欧美日韩一区二区精品| 少妇熟女aⅴ在线视频| 深夜精品福利| 亚洲av不卡在线观看| 不卡一级毛片| 欧美在线一区亚洲| 亚洲欧美日韩高清在线视频| 无遮挡黄片免费观看| 嫁个100分男人电影在线观看| 真人做人爱边吃奶动态| 高清日韩中文字幕在线| 99久久精品国产亚洲精品| 少妇高潮的动态图| 床上黄色一级片| 国产单亲对白刺激| 欧美一区二区精品小视频在线| 91在线精品国自产拍蜜月| 九色国产91popny在线| 波多野结衣高清无吗| 一个人观看的视频www高清免费观看| 成人美女网站在线观看视频| 久久这里只有精品中国| 首页视频小说图片口味搜索| 欧美一区二区精品小视频在线| av视频在线观看入口| 啦啦啦观看免费观看视频高清| 午夜福利18| 美女cb高潮喷水在线观看| 国产亚洲精品综合一区在线观看| 自拍偷自拍亚洲精品老妇| 制服丝袜大香蕉在线| avwww免费| av中文乱码字幕在线| 五月玫瑰六月丁香| 丰满人妻一区二区三区视频av| 久久久久国内视频| 天堂影院成人在线观看| 看十八女毛片水多多多| 一区福利在线观看| 亚洲欧美日韩卡通动漫| 久久99热6这里只有精品| 波多野结衣巨乳人妻| 亚洲精品亚洲一区二区| 精品久久国产蜜桃| 一进一出抽搐动态| 色综合婷婷激情| 悠悠久久av| 国产一区二区激情短视频| 久99久视频精品免费| 啦啦啦韩国在线观看视频| 国产精品一区二区性色av| 天堂√8在线中文| 国产精品嫩草影院av在线观看 | 国产欧美日韩一区二区精品| 国产探花在线观看一区二区| 老熟妇仑乱视频hdxx| 国产精品日韩av在线免费观看| 亚洲国产色片| 欧美潮喷喷水| 亚洲精品日韩av片在线观看| 男女之事视频高清在线观看| 最近最新中文字幕大全电影3| 日本免费一区二区三区高清不卡| 国产黄色小视频在线观看| 蜜桃久久精品国产亚洲av| 动漫黄色视频在线观看| 色哟哟·www| 成年女人毛片免费观看观看9| 在线观看66精品国产| 美女高潮喷水抽搐中文字幕| 精品熟女少妇八av免费久了| 亚洲成av人片在线播放无| eeuss影院久久| 日韩欧美精品免费久久 | 精品久久国产蜜桃| 精品午夜福利视频在线观看一区| 熟女电影av网| 舔av片在线| 欧美日韩亚洲国产一区二区在线观看| 国产视频一区二区在线看| 国产一区二区在线av高清观看| 桃色一区二区三区在线观看| 成人国产综合亚洲| 午夜福利欧美成人| 日日干狠狠操夜夜爽| 亚洲男人的天堂狠狠| 日本成人三级电影网站| 国产不卡一卡二| 男女视频在线观看网站免费| 亚洲久久久久久中文字幕| 久久久久久久久久黄片| 亚洲五月婷婷丁香| 国产精品野战在线观看| 丁香欧美五月| 国产精品久久久久久人妻精品电影| 在线播放国产精品三级| 欧美区成人在线视频| 美女黄网站色视频| 精品人妻熟女av久视频| 99久久99久久久精品蜜桃| 又紧又爽又黄一区二区| 亚洲国产精品999在线| 国产伦人伦偷精品视频| 国产精品久久电影中文字幕| 国产精品一区二区三区四区免费观看 | 日韩中字成人| 久久午夜福利片| 精品一区二区三区av网在线观看| 精品福利观看| 精品午夜福利视频在线观看一区| 在线a可以看的网站| 国产精品,欧美在线| 毛片一级片免费看久久久久 | 免费电影在线观看免费观看| 日韩亚洲欧美综合| 色5月婷婷丁香| 国产一区二区在线观看日韩| 午夜亚洲福利在线播放| 午夜老司机福利剧场| 精品人妻一区二区三区麻豆 | 99久久无色码亚洲精品果冻| 国产精品一区二区免费欧美| 国产乱人视频| 听说在线观看完整版免费高清| 久久精品国产99精品国产亚洲性色| 少妇高潮的动态图| 美女黄网站色视频| 精品一区二区三区视频在线| 欧美高清成人免费视频www| 亚洲成人中文字幕在线播放| 亚洲五月天丁香| 日日摸夜夜添夜夜添小说| 色在线成人网| 最近最新免费中文字幕在线| 久久人妻av系列| 午夜影院日韩av| 99在线视频只有这里精品首页| 久久久久久久久中文| 男人舔女人下体高潮全视频| 69av精品久久久久久| 看黄色毛片网站| 久久精品国产清高在天天线| 少妇人妻精品综合一区二区 | 国产精品亚洲美女久久久| 日韩中字成人| 日韩 亚洲 欧美在线| 变态另类成人亚洲欧美熟女| 亚洲18禁久久av| 人妻久久中文字幕网| 国产精品一区二区性色av| 日日干狠狠操夜夜爽| 国产 一区 欧美 日韩| 日本黄色视频三级网站网址| 日本a在线网址| 性欧美人与动物交配| 国产精品98久久久久久宅男小说| 国产精品免费一区二区三区在线| 午夜精品久久久久久毛片777| 在线观看66精品国产| 麻豆国产97在线/欧美| 成年女人毛片免费观看观看9| 亚洲中文字幕一区二区三区有码在线看| 黄色视频,在线免费观看| 国产美女午夜福利| 中文亚洲av片在线观看爽| 久久6这里有精品| 国产欧美日韩精品一区二区| 国产精品影院久久| 久久久色成人| 欧美不卡视频在线免费观看| 天堂√8在线中文| 一卡2卡三卡四卡精品乱码亚洲| 国产三级中文精品| 97热精品久久久久久| 国产aⅴ精品一区二区三区波| ponron亚洲| 国产精品女同一区二区软件 | 在线播放国产精品三级| 一进一出抽搐gif免费好疼| 日韩av在线大香蕉| 国产色婷婷99| 日韩成人在线观看一区二区三区| 中亚洲国语对白在线视频| 高潮久久久久久久久久久不卡| 午夜福利高清视频| 嫩草影院新地址| 亚洲三级黄色毛片| 久久国产乱子免费精品| 禁无遮挡网站| 赤兔流量卡办理| 久久精品国产亚洲av香蕉五月| 精品一区二区三区视频在线| 在线十欧美十亚洲十日本专区| 五月玫瑰六月丁香| 午夜福利成人在线免费观看| 国产爱豆传媒在线观看| 成人无遮挡网站| 国产成+人综合+亚洲专区| 久久婷婷人人爽人人干人人爱| 最新在线观看一区二区三区| 五月伊人婷婷丁香| 久久伊人香网站| 男女做爰动态图高潮gif福利片| 国产久久久一区二区三区| 久久精品国产亚洲av香蕉五月| 久久精品国产亚洲av天美| av欧美777| 国产久久久一区二区三区| 国产蜜桃级精品一区二区三区| 又爽又黄a免费视频| 久久精品国产清高在天天线| 怎么达到女性高潮| 精品乱码久久久久久99久播| 亚洲avbb在线观看| 日韩国内少妇激情av| 99久久九九国产精品国产免费| 丁香欧美五月| 男人和女人高潮做爰伦理| 特级一级黄色大片| 久久久久久久久大av| 桃色一区二区三区在线观看| 麻豆久久精品国产亚洲av| 18美女黄网站色大片免费观看| 黄色配什么色好看| 国产一区二区激情短视频| 国产精品久久电影中文字幕| 不卡一级毛片| 观看美女的网站| 久久人妻av系列| 亚洲欧美日韩无卡精品| www.www免费av| 国产成+人综合+亚洲专区| 美女高潮喷水抽搐中文字幕| 欧美成人免费av一区二区三区| 久久久久久九九精品二区国产| 国内毛片毛片毛片毛片毛片| 人妻丰满熟妇av一区二区三区| 少妇丰满av| 日本黄色片子视频| 每晚都被弄得嗷嗷叫到高潮| 性插视频无遮挡在线免费观看| 91狼人影院| 香蕉av资源在线| 精品午夜福利视频在线观看一区| 国产真实伦视频高清在线观看 | 国产成人啪精品午夜网站| 精品一区二区免费观看| 啦啦啦观看免费观看视频高清| 成年女人永久免费观看视频| 日日摸夜夜添夜夜添av毛片 | 色av中文字幕| 国产91精品成人一区二区三区| 欧美一级a爱片免费观看看| 女生性感内裤真人,穿戴方法视频| 99久久99久久久精品蜜桃| 性色av乱码一区二区三区2| 757午夜福利合集在线观看| 一二三四社区在线视频社区8| 亚洲男人的天堂狠狠| 老司机深夜福利视频在线观看| 亚洲国产精品合色在线| 精品乱码久久久久久99久播| 免费在线观看亚洲国产| 欧美三级亚洲精品| 亚洲五月天丁香| 内射极品少妇av片p| 免费高清视频大片| 成人亚洲精品av一区二区| av中文乱码字幕在线| 午夜精品久久久久久毛片777| 波野结衣二区三区在线| 国内毛片毛片毛片毛片毛片| 国产精品野战在线观看| 深夜精品福利| 国内久久婷婷六月综合欲色啪| 九色国产91popny在线| 亚洲,欧美精品.| 美女被艹到高潮喷水动态| 极品教师在线免费播放| 日韩精品中文字幕看吧| 亚洲最大成人手机在线| 国产男靠女视频免费网站| 亚洲av电影不卡..在线观看| 欧美黑人欧美精品刺激| 欧美日本视频| 色精品久久人妻99蜜桃| 午夜福利高清视频| 婷婷丁香在线五月| 国产免费av片在线观看野外av| 亚洲欧美日韩高清在线视频| 欧美一区二区亚洲| 小说图片视频综合网站| 国产成人aa在线观看| 午夜福利18| 别揉我奶头 嗯啊视频| 一二三四社区在线视频社区8| 91在线观看av| 久久午夜福利片| 一区二区三区高清视频在线| 男人狂女人下面高潮的视频| 国产精品久久久久久精品电影| 怎么达到女性高潮| 欧美日韩乱码在线| 在线天堂最新版资源| 能在线免费观看的黄片| 欧美在线黄色| 又黄又爽又刺激的免费视频.| 国产三级黄色录像| 国产毛片a区久久久久| 亚洲成av人片免费观看| 久久人人精品亚洲av| 在线播放国产精品三级| 丰满乱子伦码专区| 亚洲国产精品合色在线| 亚洲欧美日韩高清专用| 久久国产乱子免费精品| 男女那种视频在线观看| 久久久精品欧美日韩精品| 国产黄片美女视频| 国产成人aa在线观看| 中文字幕人成人乱码亚洲影| 怎么达到女性高潮| 搡老妇女老女人老熟妇| 亚洲中文字幕一区二区三区有码在线看| 日本撒尿小便嘘嘘汇集6| 草草在线视频免费看| 窝窝影院91人妻| 最近中文字幕高清免费大全6 | 国产精品98久久久久久宅男小说| 简卡轻食公司| 男女下面进入的视频免费午夜| 欧美一级a爱片免费观看看| 国内毛片毛片毛片毛片毛片| 午夜两性在线视频| 久久久久性生活片| 国产免费av片在线观看野外av| 国产激情偷乱视频一区二区| 色播亚洲综合网| 两个人的视频大全免费| 免费看日本二区| 深夜精品福利| 伊人久久精品亚洲午夜| 青草久久国产| 精品人妻视频免费看| 男女那种视频在线观看| 少妇裸体淫交视频免费看高清| 99视频精品全部免费 在线| 很黄的视频免费| 亚洲美女视频黄频| 99久久精品一区二区三区| 免费人成视频x8x8入口观看| 久久久久久九九精品二区国产| 人妻久久中文字幕网| 哪里可以看免费的av片| 波多野结衣巨乳人妻| 中文字幕熟女人妻在线| 成人永久免费在线观看视频| 99久久九九国产精品国产免费| 国产高清有码在线观看视频| 国产精品99久久久久久久久| 亚洲aⅴ乱码一区二区在线播放| h日本视频在线播放| 久久天躁狠狠躁夜夜2o2o| 在线观看66精品国产| 久久久久国内视频| 国产精品久久久久久久电影| 国产精华一区二区三区| 日韩欧美 国产精品| 三级毛片av免费| 自拍偷自拍亚洲精品老妇| 如何舔出高潮| 日韩欧美一区二区三区在线观看| av黄色大香蕉| 亚洲内射少妇av| 韩国av一区二区三区四区| а√天堂www在线а√下载| 久久久久国产精品人妻aⅴ院| 最近最新免费中文字幕在线| 亚洲 欧美 日韩 在线 免费| 一级作爱视频免费观看| 91字幕亚洲| 美女高潮的动态| 五月伊人婷婷丁香| 高清在线国产一区| 久久久久久久久久黄片| 国产av麻豆久久久久久久| 我要看日韩黄色一级片| 两性午夜刺激爽爽歪歪视频在线观看| 99国产精品一区二区蜜桃av| 久久久久国产精品人妻aⅴ院| 一边摸一边抽搐一进一小说| 亚洲精品在线美女| bbb黄色大片| 美女大奶头视频| 天堂影院成人在线观看| 亚洲内射少妇av| 国产成+人综合+亚洲专区| 一区二区三区免费毛片| 一区二区三区高清视频在线| 成年免费大片在线观看| 午夜a级毛片| 国产在线精品亚洲第一网站| 99在线人妻在线中文字幕| 搡老熟女国产l中国老女人| 美女黄网站色视频| 狂野欧美白嫩少妇大欣赏| 久久人人精品亚洲av| 色尼玛亚洲综合影院| 在线免费观看的www视频| 国产精品不卡视频一区二区 | 亚洲国产色片| 我的老师免费观看完整版| 国产中年淑女户外野战色| 少妇丰满av| 又爽又黄无遮挡网站| 美女黄网站色视频| 亚洲成av人片在线播放无| а√天堂www在线а√下载| av福利片在线观看| 99久久精品一区二区三区| 亚洲最大成人av| 国产伦一二天堂av在线观看| 夜夜爽天天搞| 日本黄色视频三级网站网址| 三级毛片av免费| 精品久久久久久,| 亚洲人成网站在线播| 久久久久久久久大av| 可以在线观看毛片的网站| 制服丝袜大香蕉在线| 又紧又爽又黄一区二区| 一级作爱视频免费观看| 国产午夜精品久久久久久一区二区三区 | 无人区码免费观看不卡| 亚洲精品在线观看二区| 亚洲av电影不卡..在线观看| 亚洲片人在线观看| 高清在线国产一区| 久久人人爽人人爽人人片va | 精品人妻视频免费看|