• <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 ?

    Purification and Characterization of a Nonylphenol (NP)-degrading Enzyme from Bacillus cereus. Frankland*

    2011-03-22 10:09:10YANGGe楊革ZHANGYing張營(yíng)andBAIYanfen白艷芬22CollegeofTextilesTianjinPolytechnicUniversityTianjin30060ChinaKeyLabofBiogeologyandEnvironmentalGeologyofMinistryofEducationChinaUniversityofGeosciencesWuhan430074China

    YANG Ge (楊革)**, ZHANG Ying (張營(yíng)) and BAI Yanfen (白艷芬)22 College of Textiles Tianjin Polytechnic University, Tianjin 30060, China Key Lab of Biogeology and Environmental Geology of Ministry of Education, China University of Geosciences,Wuhan 430074, China

    1 INTRODUCTION

    Alkylphenol polyethoxylates (APEOs) were a group of non-ionic surfactants and the hard-degradable polymer that was found widespread use as detergents,emulsifiers, wetting agents, stabilisers, defoaming agents and intermediates in the synthesis of anionic surfactants. It had been reported that these substances were degraded into more toxic products, mainly as NP[1-3]. In particular, concerns had been expressed regarding the possible endocrine disrupting effects of these‘hormone mimicking’ degradation products [4-9].

    In recent years, the development of cleanproduction process for textile industry attracted great interest and therefore, the biodegradation of NP at the desizing stage, which could greatly reduce discharge of NP waste water and minimize damage of cotton fiber in the desizing process, became one of the key points in textile biotechnology [10, 11].Till now, the research about biodegradation of NP was mainly focused on the screening of NP-degrading microorganisms and the characteristics of PVA (polyvinyl alcohol)-degrading enzymes from obtained strains. PVA-degrading enzymes were still not applicable in real industry process due to their low activity and the producing strains of NP-degrading enzyme were limited and they often grew very slowly. Here the recent research of NP biodegradation, including purification and characterization of the NP-degrading enzyme was reported [12-16].

    Bacillus cereus. Frankland No. BCF83 was a newly isolated strain from soil in Shandong of China for its high NP-degrading enzyme activity secreted in culture medium. In this study the extracellular NP-degrading enzyme was purified to homogeneity from the fermented broth ofBacillus cereus. Frankland No.BCF83 to investigate its physico-chemical properties.With the determination of partialN-terminal amino acid sequence and its characteristics, it was demonstrated that the purified enzyme was a novel endo NP-degrading enzyme.

    2 MATERIALS AND METHODS

    2.1 Bacterial strain and culture condition

    Bacillus cereus. Frankland No. BCF83 was isolated from soil in Shandong of China. Cultures were maintained on nutrient agar slants and incubated at 37 °C for 24 h. The cells were then inoculated into a 500-ml Erlenmeyer flask containing 200ml liquid medium, cultured at 37 °C for 24-36 h on a shaker. The medium adjusted to pH 7.0 was composed of (g·L-1):1 NP, 1 NH4NO3, 1 yeast extract, 0.5 KH2PO4, 0.2 MgSO4·7H2O, 0.02 FeSO4·7H2O, and 0.1 CaCl2.

    2.2 Chemicals

    Phenyl-Sepharose CL-4B, DEAE-Sepharose Fast Flow and Sephadex G-150 were purchased from Pharmacia LKB (Uppsala Sweden). SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis) protein markers were purchased from Sigma(Santa Clara, USA). Other chemicals were of analytical grade.

    2.3 Purification of NP-degrading enzyme

    The fermented broth ofBacillus cereus. Frankland No. BCF83 was collected by centrifugation at 8000 g for 10 min, and the proteins fractionated with 50% saturation (NH4)2SO4were collected by centrifugation at 8000 g for 20 min. The protein precipitate was dissolved in 0.8 mol·L-1(NH4)2SO4solution and the insoluble materials were removed by centrifugation at 15000 g for 30 min. The derived supernatant was applied onto a Phenyl-Sepharose CL-4B column (φ1.2 cm×10 cm) preequilibrated with 1 mol·L-1(NH4)2SO4.The column was washed with 1.5 bed volumes of 1 mol·L-1(NH4)2SO4, 2 bed volumes of 0.1 mol·L-1(NH4)2SO4, and eluted with distilled water. The flow rate was maintained at 0.5 ml·min-1. The fractions with NP-degrading enzyme activity were pooled and dialyzed overnight at 4 °C against 10 mmol·L-1tris-HCI buffer with pH 6.2. The dialysate was collected and immediately applied on a DEAE-Sepharose Fast Flow column (φ1.2 cm×4 cm) pre-equilibrated with 10 mmol·L-1tris-HCI buffer with pH 6.2. The flow rate was maintained at 0.25 ml·min-1. The fractions with NP-degrading enzyme activity were pooled, 10 times concentrated and kept at -20 °C until use.

    ForN-terminal amino acid sequencing, the enzyme fraction was further purified by reverse-phase HPLC (High Performance Liquid Chromatography).NP-degrading enzyme fractions derived from DEAE-Sepharose Fast Flow were loaded on a Zorbas 300SBCN column (du Pont,φ250 mm×4.6 mm I. D.),and the column was developed with acetonitrile gradient (0-10% for 20 min) supplemented with 0.1%trifluoroacetic acid (TFA). The elution pattern was monitored by absorbance at 220 nm and 280 nm. The major peak was collected and lyophilized for automatic amino acid sequencing.

    2.4 Enzymatic activity assay

    Two methods for enzymatic activity assay were used in this work. For rapidly tracing NP-degrading enzyme activity during the chromatographic separation processes, 100 μl of each fraction was mixed with 2 ml 10 g·L-1nonylphenol (NP) in 50 mmol·L-1acetate buffer (pH 7.0) and incubated at 50 °C for 10 min.The enzyme activity was calculated from a standard curve obtained with known concentration of nonylphenol (NP). One unit of NP-degrading enzyme activity was defined as the amount of enzyme that liberated 1 μmol NP-degrading per min at pH 7.0 and 50 °C.Negative control tubes contained all components except substrate, and blanks contained all components except the enzyme.

    2.5 Electrophoresis

    SDS-polyacrylamide gel electrophoresis (SDSPAGE) was performed. The proteins were stained with Coomassie brilliant blue R-225. For isoelectric focusing (IEF) experiment, about 5 μg of sample proteins in 20 μl solution were loaded to a precasted capillary gel with 0.75% Ampholine (pH range 3.5-10), and run under 200 V for 5 h. Amyloglucosidase (pl 3.6), trypsin inhibitor (pI 4.6), β-lactoglubin A (pI 5.1), conalbumin (pI 6.0), myoglobin (pI 6.8, 7.2), lentil lectin(pI 8.2, 8.6, 8.8), and trypsinogen (pI 9.3) were used as markers.

    2.6 Zymogram

    To identify the protein of NP-degrading enzyme,zymographic approach was applied on samples derived from DEAE Fast Flow chromatography. Samples were separated on 10% polyacrylamide gel electrophoresis at pH 8.3. As soon as the electrophoresis was finished, the gel was immediately placed on an agarose slab gel containing 10 g·L-1nonylphenol. After incubation for 1.5 h at 37 °C, a transparent band could be seen on the agarose slab. The sections of the polyacrylamide gel overlapping with the transparent band were carefully cut out and pestled with the transparent band were carefully cut out and pestled with sample buffer in an Eppendorf tube. The derived paste was analyzed on 10% SDS-PAGE.

    2.7 Gel filtration chromatography

    Gel filtration chromatography was used for determination of molecular weight of molecular weight of NP-degrading enzyme. Sephadex G-150 was packaged in a 1.2 cm×60 cm column and equilibrated with 10 mmol·L-1tris-HCI buffer (pH 6.2) at a flow rate of 0.15 ml·min-1. About 0.4 ml concentrated enzyme obtained from the DEAE Fast Flow column was applied to the column and eluted with the same buffer.Protein profile was monitored at 280 nm. The molecular weight was estimated from a standard curve obtained from the proteins with their relative molecular mass known [17].

    2.8 N-Terminal amino acid sequencing

    Samples obtained from reverse-phase HPLC were lyophilized and subjected toN-terminal amino acid sequencing on an automatic protein sequencer( Model 473A, Applied Biosystems Inc., USA).

    3 RESULTS AND DISCUSSION

    3.1 Purification of NP-degrading enzyme

    A NP-degrading enzyme secreted by this new strain ofBacillus cereus. Frankland No. BCF83 was purified for further study. Proteins in the fermented broth were recovered with (NH4)2SO4precipitation at 50% saturation. The protein precipitates were dissolved in 0.8 mol·L-1(NH4)2SO4solution and separated by Phenyl-Sepharose CL-4B hydrophobic interaction chromatography. In a typical separation, 30 ml of the sample solution containing 68.4 mg of crude proteins was applied to a 1.2 cm×10 cm column, and developed as described in Section 2. Four protein peaks were detected at 280 nm as shown in Figs. 1 and 2.NP-degrading enzyme activity was only found in the last peak eluted with distilled H2O.

    Figure 1 Elution profile of NP-degrading enzyme on Phenyl-Sepharose CL-4B column

    Figure 2 Elution profile of NP-degrading enzyme on DEAESepharose Fast Flow column chromatography

    The fractions with NP-degrading enzyme activity were pooled and dialyzed against 10 mmol·L-1tris-HCl buffer (pH 6.2) overnight. The dialysate containing 23.46 mg proteins was then applied onto a DEAESepharose Fast Flow column for anion-exchange chromatography described as Section 2. The NP-degrading enzyme activity was detected in the third peak as shown in Fig. 2. When this peak was analyzed on 10%SDS-PAGE, a protein band with relative molecular mass of 58.3 kDa (Fig. 3) was shown. Meanwhile,zymographic approach was applied to identify the protein with NP-degrading enzyme activity. Table 1 is the summary of purification.

    Figure 3 SDS-PAGE analysis of NP-degrading enzyme under various conditions

    3.2 N-terminal amino acid sequence

    ForN-terminal sequencing, the enzyme fractions from DEAE-Sepharose Fast Flow were further purified by reverse-phrase HPLC. As shown in Fig. 4,only one protein peak was detected. The protein peak was collected, lyophilized and subjected to amino acid sequencing. The first 10 amino acids in theN-terminal sequence were determined to be ASVNSIKIGY. The sequence was blasted against GenBank, however, no NP-degrading enzyme known showed significant similarity with this sequence.

    Figure 4 Elution profile of NP-degrading enzyme on reverse-phase HPLC

    Table 1 Purification of NP-degrading enzyme from Bacillus cereus. Frankland No. BCF83

    3.3 Characteristics of the purified NP-degrading enzyme

    The difference between relative molecular mass of proteins in DEAE-Sepharose Fast Flow fractions and HPLC fractions implied a dimer structure of NP-degrading enzyme. A series of experiments or further characterization of this protein was performed.The purified NP-degrading enzyme was subjected to isoelectric focusing analysis and the pI of the NP-degrading enzyme was found to be 5.5. On the result of RPC (Reversed Phase Chromatography), the molecular weight of NP-degrading enzyme was determined to be around 56 kDa (Fig. 4), and other two peaks were not an enzyme activity. The relative molecular mass of NP-degrading enzyme protein on SDS-PAGE differed depending on conditions. If the NP-degrading enzyme in DEAE fractions was heated in boiling sample buffer before SDS-PAGE analysis,the protein band on SDS-PAGE was at the position of 58.3 kDa. After treatment of the enzyme with 4%2-mercaptoethanol, the relative molecular mass of the purified enzyme was still 58.3 kDa on SDS-PAGE,implying that disulfide bond was not involved in the formation of NP-degrading enzyme with 8 mol·L-1urea at 50 °C for 30 min and could cause a total loss of enzymatic activity and a shift of the protein band position from 58.3 kDa to 28.5 kDa on SDS-PAGE(Fig. 3). After dialysis of the enzymes depolymerized by 8 mol·L-1urea, heating at 100 °C or 0.1% TFA treatment against 10 mmol·L-1tris-HCl buffer (pH6.2),the enzymatic activity recovered by 79%, 75% and 87%, respectively. The dimer was found to be the major component revealed by SDS-PAGE (data not shown).These results strongly suggest that the NP-degrading enzyme had a homodimer structure based on hydrophobic interaction. After incubation the NP-degrading enzyme with 8 mol·L-1urea and then dialyzing it against 40% alcohol, the free NP-degrading enzyme subunits were obtained, which utterly lost the activity(the NP-degrading enzyme in 40% alcohol still exhibited hydrolytic activity). It was thus concluded that the compact structure of the dimer is necessary for NP-degrading enzyme activity.

    Up to now, no NP-degrading enzyme with dimer structure from bacteria had been reported. In other species only an insect inhibitor/endo NP-degrading enzyme from plant origin was shown to have a structure of dimer. By this fact, as well as the result of theN-terminal amino acid sequence, it was concluded that the NP-degrading enzyme produced byBacillus cereus. Frankland No. BCF83 was a novel NP-degrading enzyme with an unusual structure.

    Figure 5 Effect of pH (◆) and temperature (●) on enzymatic activity of the purified NP-degrading enzyme

    Figure 6 Effect of temperature on stability of the purified NP-degrading enzyme

    Figure 7 Effect of Cu2+ion on enzymatic activity of the purified NP-degrading enzyme

    The optimal conditions for enzymatic reaction were studied systemically. 6μg purified NP-degrading enzyme was used to determine its characteristic. The optimal pH of the NP-degrading enzyme was 6.0 (Fig. 5),and the NP-degrading enzyme was stable and could hydrolyze collidal nonylphenol at a wide pH range(from pH 4.0 to pH 8.0). The NP-degrading enzyme exhibited the highest activity at 60 °C and retained high activity even over 80 °C (Fig. 5). However, in the absence of substrate the NP-degrading enzyme lost its activity markedly above 60 °C (Fig. 6), inferring that the substrate could protect the active center of the NP-degrading enzyme from denaturation.

    TheBacillus cereus. Frankland No. BCF83 NP-degrading enzyme could be inactivated by Cu2+ion (Fig. 7). After incubation with 0.5 mmol·L-1Cu2+at pH 6.0 and 30 °C for 30 min, only 60% of the enzyme activity remained.

    4 CONCLUSIONS

    TheBacillus cereus. Frankland No. BCF83 NP-degrading enzyme was highly stable (retaining higher than 80% activity) in a wide range of pH (pH 6.0 to 10.0) and temperature (from 35 °C to 72 °C). In comparison, the NP-degrading enzyme fromBacillus cereus. Frankland No. BCF83 and other strains [18-21]exhibit their enzymatic activities in a more narrow temperature range and are less stable. Furthermore, the purified NP-degrading enzyme fromBacillus cereus.Frankland No. BCF83 was strongly resistant to the hydrolysis by trypsin. A common condition was not sufficient for trypsin digestion of the NP-degrading enzyme. The NP-degrading enzyme in fermented broth could be kept at 4 °C for at least two months without loss of enzymatic activity. The crude fermented broth of theBacillus cereus. Frankland No.BCF83 NP-degrading enzyme could be widely applied as a new tool for clean-production process of textile.

    1 Ferguson, P.L., Brownawell, B.J., “Degradation of nonylphenol ethoxylates in estuarine sediment under aerobic and anaerobic conditions”,Environ.Toxicol.Chem., 22, 1189-1199 (2003).

    2 Xiao, C.B., Ning, J., Yan, H., Sun, X.D., Hu, J.Y., “Biodegradation of aniline by a newly isolatedDelftiasp. XYJ6”,Chin.J.Chem.Eng., 17 (3), 500-505(2009).

    3 M?nsson, N., S?rme L., Wahlberg, C., Bergb?ck, B., “Sources of alkylphenols and alkylphenol ethoxylates in wastewater—a substance flow analysis in Stockholm, Sweden”,Water,Air,& Soil Pollut:Focus, 8, 445-456 (2008).

    4 Ohtsubo, Y., Kudo, T., Tsuda, M., Nagata, Y., “Strategies for bioremediation of polychlorinated biphenyls”,Appl.Microbiol.Biotechnol., 65, 250-258 (2004).

    5 Wackett, L.P., Sadosky, M.J., Martinez, B., Shapir, N., “Biodegradation of atrazine and related striazine compounds from enzymes to field studies”,Appl.Microbiol.Biotechnol., 58, 39-45 (2002).

    6 Cravotto, G., Carlo, S.D., Binello, A., Mantegna, S., Girlanda, M.,Lazzari, A., “Integrated sonochemical and microbial treatment for decontamination of nonylphenol-polluted water”,Wate,Air,& Soil Pollution, 187 (1-4), 353-359 (2008).

    7 Jontofsohn, M., Pfister, G., Severin, G., Schramm, K.W., Hartmann,A., Schloter, M., “Bacterial community structure in lake sediments of microcosms contaminated with nonylphenol”,Journal of Soils and Sediments, 2 (4), 211-215 (2002).

    8 Mai, H., EI-Dakdoky, Mona, A.M., HelaI, “Reproductive toxicity of male mice after exposure to nonylphenol”.Bulletin of Environmental Contamination and Toxicology, 79 (2), 188-191 (2007).

    9 Beklioglu, M., Banu Akkas, S., Elif Ozcan, H., Bezirci, G., Togan, I.,“Effects of 4-nonylphenol, fish predation and food availability on survival and life history traits ofDaphnia magnastraus”,Ecotoxicology, 19 (5), 901-910 (2010).

    10 Hermuth, K., Leuthner, B., Heider, J., “Operon structure and expression of the genes for benzylsuccinate synthase in Thauera aromatica strain K172”,Arch.Microbiol., 177, 132-138 (2002).

    11 Krieger, J., Roseboom, W., Albracht, S.P., Spormann, A.M., “A stable organic free radical in anaerobic benzylsuccinate synthase ofAzoarcussp. strain T”,J.Biol.Chem., 276, 12924-12927 (2001).

    12 Song, B., Palleroni, N.J., Haggblom, M.M., “Isolation and characterization of diverse halobenzoate-degrading denitrifying bacteria from soils and sediments”,Appl.Environ.Microbiol., 66, 3446-3453(2000).

    13 Lu, J., He, Y.L., Wu, J., Jin, Q., “Aerobic and anaerobic biodegradation of nonylphenol ethoxylates in estuary sediment of Yangtze River,China”,Environmental Geology, 57 (1), 1-8 (2009).

    14 Liu, X., Tani, A., Kimbara, K., Kawai, F., “Metabolic pathway of xenoestrogenic short ethoxy chain-nonylphenol to nonylphenol by aerobic bacteria,Ensifersp. strain AS08 andPseudomonassp. strain AS90”,Applied Microbiology and Biotechnology, 72 (3), 552-559(2006).

    15 Latorre, A., Lacorte, A., Barceló, D., “Presence of nonylphenol, octyphenol and bisphenol a in two aquifers close to agricultural, industrial and urban areas”,Chromatographia, 57 (1-2), 111-116 (2003).

    16 Park, S.Y., Choi, J., “Genotoxic effects of nonylphenol and bisphenol a exposure in aquatic biomonitoring species: freshwater crustacean,daphnia magna, and aquatic midgechironomus riparius”,Bulletin of Environmental Contamination and Toxicology, 83 (4),463-468 (2009).

    17 Chen, M., Yao, S.J., Zhang, H., Liang, X.L., “Purification and characterization of a versatile peroxidase from edible mushroom Pleurotus eryngii”,Chin.J.Chem.Eng, 18 (5), 824-829 (2010).

    18 Morgan, P., Watkinson, R.J., “Microbiological methods for the clean up of soil and groundwater contaminated with halogenated organic compounds”,FEMS Microbiol.Rev. 63, 277-300 (1989).

    19 Takasu, T., Iles, A., Hasebe, K., “Determination of alkylphenols and alkylphenol polyethoxylates by reversed-phase high-performance liquid chromatography and solid-phase extraction”,Anal.Bioanal.Chem., 372, 554-561 (2002).

    20 Zhang, X., Young, L.Y., “Carboxylation as an initial reaction in the anaerobic metabolism of naphthalene and phenanthrene by sulfidogenic consortia”,Appl.Environ.Microbiol., 63, 4759-4764 (1997).

    21 Zhang, X., Sullivan, E.R., Young, L.Y., “Evidence for aromaticring reduction in the biodegradation pathway of carboxylated naphthalene by a sulfate-reducing consortium”,Biodegradation, 11, 117-124(2002b).

    国产黄色视频一区二区在线观看| 国产一级毛片在线| 欧美日韩亚洲高清精品| 精品少妇黑人巨大在线播放| 日本午夜av视频| 国产亚洲最大av| av国产久精品久网站免费入址| 亚洲精品在线美女| 伦精品一区二区三区| 男女边摸边吃奶| 国产亚洲欧美精品永久| 亚洲av免费高清在线观看| 97在线人人人人妻| 99国产综合亚洲精品| 久久精品久久久久久噜噜老黄| 中文字幕最新亚洲高清| 9热在线视频观看99| 波多野结衣av一区二区av| 久久精品人人爽人人爽视色| 国产 一区精品| 中国三级夫妇交换| 亚洲经典国产精华液单| 汤姆久久久久久久影院中文字幕| 久久久久久久久久人人人人人人| 午夜免费观看性视频| 新久久久久国产一级毛片| 国产淫语在线视频| 欧美最新免费一区二区三区| 日本免费在线观看一区| 久久精品国产亚洲av高清一级| 老鸭窝网址在线观看| 香蕉国产在线看| 成人国语在线视频| 天堂8中文在线网| 校园人妻丝袜中文字幕| 啦啦啦中文免费视频观看日本| 人妻人人澡人人爽人人| 欧美激情高清一区二区三区 | 婷婷色综合大香蕉| 美女福利国产在线| 老汉色∧v一级毛片| 高清视频免费观看一区二区| 久久99蜜桃精品久久| 国产高清国产精品国产三级| 亚洲av综合色区一区| 黄片小视频在线播放| a级片在线免费高清观看视频| 天天操日日干夜夜撸| 国产成人精品婷婷| 国产免费一区二区三区四区乱码| 午夜激情av网站| 亚洲av免费高清在线观看| 国产成人欧美| 久久这里有精品视频免费| 黑丝袜美女国产一区| 欧美精品一区二区免费开放| 桃花免费在线播放| 自拍欧美九色日韩亚洲蝌蚪91| 久久久久久久精品精品| 少妇猛男粗大的猛烈进出视频| 久久青草综合色| av电影中文网址| 国语对白做爰xxxⅹ性视频网站| 午夜老司机福利剧场| av女优亚洲男人天堂| 秋霞在线观看毛片| 亚洲国产欧美日韩在线播放| 青春草视频在线免费观看| 国产伦理片在线播放av一区| 精品少妇黑人巨大在线播放| 男女免费视频国产| 成人手机av| 99久久中文字幕三级久久日本| 丰满乱子伦码专区| 久久国产亚洲av麻豆专区| 午夜福利影视在线免费观看| 久久精品久久久久久久性| 老女人水多毛片| 欧美激情 高清一区二区三区| 亚洲av日韩在线播放| 亚洲精品日韩在线中文字幕| 精品国产乱码久久久久久小说| 国产国语露脸激情在线看| 日本wwww免费看| 欧美中文综合在线视频| 亚洲国产欧美网| 下体分泌物呈黄色| 久久精品国产a三级三级三级| 日韩在线高清观看一区二区三区| 成人亚洲欧美一区二区av| 91aial.com中文字幕在线观看| 亚洲av.av天堂| 久久久久久久精品精品| 国产黄色视频一区二区在线观看| 涩涩av久久男人的天堂| 亚洲国产日韩一区二区| 亚洲激情五月婷婷啪啪| 桃花免费在线播放| 黄色毛片三级朝国网站| 少妇熟女欧美另类| 亚洲第一av免费看| 久久久国产精品麻豆| 18在线观看网站| 女人精品久久久久毛片| 久久97久久精品| 97精品久久久久久久久久精品| 久久久久久人妻| 亚洲精品一二三| 免费观看在线日韩| 久久久亚洲精品成人影院| 亚洲伊人久久精品综合| 日本爱情动作片www.在线观看| 国产精品久久久久久精品电影小说| 婷婷色av中文字幕| 韩国精品一区二区三区| 高清视频免费观看一区二区| 麻豆av在线久日| av又黄又爽大尺度在线免费看| videossex国产| 亚洲国产精品成人久久小说| 国产乱人偷精品视频| 久久久久国产网址| 视频在线观看一区二区三区| 国产精品人妻久久久影院| 看免费av毛片| 国产日韩欧美视频二区| 久久精品aⅴ一区二区三区四区 | 国产国语露脸激情在线看| 色视频在线一区二区三区| 狂野欧美激情性bbbbbb| 蜜桃国产av成人99| 国产亚洲av片在线观看秒播厂| 国产精品.久久久| 国产精品国产三级专区第一集| 国产男人的电影天堂91| 一本久久精品| 免费黄网站久久成人精品| 精品人妻熟女毛片av久久网站| 视频在线观看一区二区三区| 一本色道久久久久久精品综合| 丰满少妇做爰视频| 国产乱人偷精品视频| 99精国产麻豆久久婷婷| 哪个播放器可以免费观看大片| 97在线视频观看| 婷婷色综合www| 性色av一级| 热99久久久久精品小说推荐| 男女下面插进去视频免费观看| 十分钟在线观看高清视频www| 热re99久久国产66热| 黑人巨大精品欧美一区二区蜜桃| 人妻一区二区av| 亚洲国产精品一区二区三区在线| av又黄又爽大尺度在线免费看| 色婷婷久久久亚洲欧美| 国产av一区二区精品久久| 中文字幕另类日韩欧美亚洲嫩草| 桃花免费在线播放| 毛片一级片免费看久久久久| av国产久精品久网站免费入址| 999精品在线视频| a级毛片黄视频| 男女无遮挡免费网站观看| 五月开心婷婷网| 国产精品国产三级专区第一集| 一本色道久久久久久精品综合| 国产激情久久老熟女| 9191精品国产免费久久| 建设人人有责人人尽责人人享有的| 在线观看人妻少妇| av视频免费观看在线观看| 久热这里只有精品99| 亚洲经典国产精华液单| 女人久久www免费人成看片| 女性生殖器流出的白浆| 久久这里只有精品19| 黄色毛片三级朝国网站| 日韩制服丝袜自拍偷拍| 日韩熟女老妇一区二区性免费视频| 成人手机av| 丰满乱子伦码专区| 999精品在线视频| 大香蕉久久网| 欧美国产精品一级二级三级| 高清在线视频一区二区三区| 一区二区三区精品91| 丝袜美足系列| 亚洲国产成人一精品久久久| 黄色一级大片看看| 哪个播放器可以免费观看大片| 91久久精品国产一区二区三区| a 毛片基地| 国产精品国产三级国产专区5o| 纯流量卡能插随身wifi吗| 男人添女人高潮全过程视频| 国产熟女欧美一区二区| 亚洲av国产av综合av卡| 啦啦啦在线观看免费高清www| 一区二区三区乱码不卡18| 老汉色∧v一级毛片| 人人妻人人爽人人添夜夜欢视频| 男女边摸边吃奶| 欧美日韩精品成人综合77777| 久久影院123| 亚洲欧美中文字幕日韩二区| 免费少妇av软件| 大话2 男鬼变身卡| 亚洲国产av新网站| 男女高潮啪啪啪动态图| 国产成人精品婷婷| 中文乱码字字幕精品一区二区三区| 久久久国产欧美日韩av| 免费大片黄手机在线观看| 如日韩欧美国产精品一区二区三区| 97在线视频观看| 黄色视频在线播放观看不卡| 国产欧美亚洲国产| 一边亲一边摸免费视频| 亚洲少妇的诱惑av| 国产 一区精品| 99九九在线精品视频| 国产又爽黄色视频| 国产一区二区三区av在线| 精品卡一卡二卡四卡免费| 在线观看免费视频网站a站| 国产一区二区三区av在线| 一区二区三区乱码不卡18| 日韩在线高清观看一区二区三区| 亚洲av福利一区| 激情视频va一区二区三区| 亚洲经典国产精华液单| 久久综合国产亚洲精品| 男女无遮挡免费网站观看| 日韩精品有码人妻一区| 欧美亚洲日本最大视频资源| 高清黄色对白视频在线免费看| 午夜免费鲁丝| 国产精品国产av在线观看| 菩萨蛮人人尽说江南好唐韦庄| 国产精品免费视频内射| 欧美最新免费一区二区三区| 黄片播放在线免费| 一个人免费看片子| 久久这里只有精品19| 亚洲三区欧美一区| 国产精品 国内视频| 99久久综合免费| 国产又色又爽无遮挡免| 又大又黄又爽视频免费| 久久精品夜色国产| 日韩熟女老妇一区二区性免费视频| 日韩av免费高清视频| 狠狠精品人妻久久久久久综合| 午夜日本视频在线| 女性被躁到高潮视频| 美女高潮到喷水免费观看| 有码 亚洲区| 国产精品免费视频内射| 免费大片黄手机在线观看| 一级片免费观看大全| 大码成人一级视频| 不卡av一区二区三区| 久久久久久人人人人人| 国产探花极品一区二区| 精品一区二区三区四区五区乱码 | 欧美+日韩+精品| 菩萨蛮人人尽说江南好唐韦庄| 在线观看免费高清a一片| 韩国精品一区二区三区| 女性生殖器流出的白浆| 久久这里只有精品19| 久久午夜福利片| 女人高潮潮喷娇喘18禁视频| 免费高清在线观看日韩| 亚洲av.av天堂| 午夜日韩欧美国产| 久久精品国产鲁丝片午夜精品| 色网站视频免费| 免费不卡的大黄色大毛片视频在线观看| 日韩欧美精品免费久久| 亚洲国产毛片av蜜桃av| 中文字幕人妻丝袜制服| 各种免费的搞黄视频| 在现免费观看毛片| 午夜福利一区二区在线看| 亚洲精品,欧美精品| 日日爽夜夜爽网站| 老熟女久久久| 人人妻人人澡人人爽人人夜夜| 久久久精品国产亚洲av高清涩受| 丝袜美足系列| 国产免费又黄又爽又色| 2022亚洲国产成人精品| 多毛熟女@视频| 国产无遮挡羞羞视频在线观看| 国产白丝娇喘喷水9色精品| 少妇人妻久久综合中文| 午夜激情久久久久久久| 侵犯人妻中文字幕一二三四区| 亚洲国产日韩一区二区| 久久久久视频综合| 国产无遮挡羞羞视频在线观看| 人体艺术视频欧美日本| av天堂久久9| 精品亚洲乱码少妇综合久久| 精品人妻偷拍中文字幕| 久久99热这里只频精品6学生| 午夜福利视频在线观看免费| 欧美精品人与动牲交sv欧美| 亚洲情色 制服丝袜| 亚洲精品一区蜜桃| 亚洲一区二区三区欧美精品| 国产成人免费无遮挡视频| 亚洲,欧美精品.| 18禁国产床啪视频网站| 男人操女人黄网站| 精品少妇内射三级| 国产精品香港三级国产av潘金莲 | av在线老鸭窝| 亚洲精品视频女| 丝袜美足系列| 久久毛片免费看一区二区三区| 亚洲欧美成人综合另类久久久| 在现免费观看毛片| 国产精品二区激情视频| 国产97色在线日韩免费| 激情视频va一区二区三区| 国产有黄有色有爽视频| 国产一区二区 视频在线| 老女人水多毛片| 在线观看免费日韩欧美大片| 欧美日韩国产mv在线观看视频| 免费看不卡的av| 日韩一卡2卡3卡4卡2021年| 在线免费观看不下载黄p国产| 久久女婷五月综合色啪小说| 97在线视频观看| 在线免费观看不下载黄p国产| 精品酒店卫生间| 亚洲av福利一区| 亚洲精品中文字幕在线视频| 久久人妻熟女aⅴ| 人人澡人人妻人| 国产亚洲av片在线观看秒播厂| 国产黄频视频在线观看| 天天躁日日躁夜夜躁夜夜| 欧美老熟妇乱子伦牲交| 日韩不卡一区二区三区视频在线| 亚洲一区中文字幕在线| 成人免费观看视频高清| 97在线视频观看| 国产亚洲午夜精品一区二区久久| 午夜激情久久久久久久| 亚洲av综合色区一区| 亚洲,欧美,日韩| 18禁裸乳无遮挡动漫免费视频| 国产无遮挡羞羞视频在线观看| 国产免费福利视频在线观看| 人人妻人人添人人爽欧美一区卜| 一个人免费看片子| av免费观看日本| 黄色视频在线播放观看不卡| 夫妻性生交免费视频一级片| 欧美日韩亚洲国产一区二区在线观看 | 国产精品无大码| 亚洲av免费高清在线观看| 亚洲精品美女久久av网站| av网站在线播放免费| 欧美成人精品欧美一级黄| 日本免费在线观看一区| 久久久久久久久免费视频了| 国产又爽黄色视频| 免费播放大片免费观看视频在线观看| 伊人亚洲综合成人网| 午夜福利在线观看免费完整高清在| 国产男女内射视频| 国产深夜福利视频在线观看| av在线观看视频网站免费| 高清欧美精品videossex| 亚洲,欧美,日韩| 亚洲内射少妇av| 国产深夜福利视频在线观看| av在线观看视频网站免费| 2021少妇久久久久久久久久久| 老汉色av国产亚洲站长工具| www.精华液| 亚洲少妇的诱惑av| 如日韩欧美国产精品一区二区三区| 大片免费播放器 马上看| a级片在线免费高清观看视频| 国产97色在线日韩免费| 超碰97精品在线观看| 国产女主播在线喷水免费视频网站| 日日摸夜夜添夜夜爱| 中文字幕人妻丝袜一区二区 | 成年女人毛片免费观看观看9 | 久久99精品国语久久久| 一本久久精品| 国产av一区二区精品久久| 亚洲美女视频黄频| 建设人人有责人人尽责人人享有的| 满18在线观看网站| 亚洲欧美一区二区三区久久| 欧美日本中文国产一区发布| 丰满乱子伦码专区| 日本欧美视频一区| 夫妻性生交免费视频一级片| 亚洲精品国产一区二区精华液| 亚洲图色成人| 街头女战士在线观看网站| 国产精品嫩草影院av在线观看| 免费大片黄手机在线观看| 激情五月婷婷亚洲| 一级爰片在线观看| 国产精品秋霞免费鲁丝片| 国产精品国产三级国产专区5o| 永久网站在线| 好男人视频免费观看在线| 91精品三级在线观看| 多毛熟女@视频| 国产精品麻豆人妻色哟哟久久| 最黄视频免费看| 国产乱人偷精品视频| 五月伊人婷婷丁香| 三上悠亚av全集在线观看| 精品一区二区免费观看| 国产又爽黄色视频| 制服人妻中文乱码| 日本黄色日本黄色录像| 午夜日韩欧美国产| 满18在线观看网站| 精品少妇一区二区三区视频日本电影 | 老汉色av国产亚洲站长工具| videossex国产| 永久网站在线| 一级黄片播放器| 哪个播放器可以免费观看大片| www.自偷自拍.com| 成年女人毛片免费观看观看9 | 一级a爱视频在线免费观看| 亚洲精品日韩在线中文字幕| 女人被躁到高潮嗷嗷叫费观| 国产97色在线日韩免费| 精品人妻在线不人妻| 黑人猛操日本美女一级片| 国产精品不卡视频一区二区| 一区二区三区四区激情视频| 精品国产露脸久久av麻豆| 久久久久久久久久久免费av| 一区二区三区乱码不卡18| 国产成人精品久久二区二区91 | 熟妇人妻不卡中文字幕| 大码成人一级视频| 亚洲,欧美精品.| 免费观看无遮挡的男女| 亚洲人成77777在线视频| 欧美国产精品va在线观看不卡| 亚洲激情五月婷婷啪啪| 中文字幕亚洲精品专区| 日韩大片免费观看网站| 日韩一区二区视频免费看| 黄片播放在线免费| 欧美日韩综合久久久久久| 国产精品久久久久久精品电影小说| 久久99精品国语久久久| av线在线观看网站| 啦啦啦在线观看免费高清www| 日韩视频在线欧美| 国产精品人妻久久久影院| 亚洲国产毛片av蜜桃av| 免费观看a级毛片全部| 日韩中文字幕视频在线看片| 亚洲精品第二区| 伊人亚洲综合成人网| 亚洲欧美精品综合一区二区三区 | 看非洲黑人一级黄片| 晚上一个人看的免费电影| 日本午夜av视频| 青春草国产在线视频| 婷婷色麻豆天堂久久| 男人爽女人下面视频在线观看| 一级毛片黄色毛片免费观看视频| 美女福利国产在线| 国产在线视频一区二区| 久久这里只有精品19| 18禁动态无遮挡网站| 麻豆av在线久日| 下体分泌物呈黄色| 永久网站在线| 亚洲精品国产av成人精品| 免费大片黄手机在线观看| 久久人人爽av亚洲精品天堂| 男人添女人高潮全过程视频| 中国三级夫妇交换| 久久久久久免费高清国产稀缺| 日韩一本色道免费dvd| 9热在线视频观看99| 亚洲欧美一区二区三区黑人 | 99精国产麻豆久久婷婷| 一区二区三区四区激情视频| 久久精品国产亚洲av涩爱| 性色avwww在线观看| 电影成人av| 如何舔出高潮| 日韩中文字幕视频在线看片| 欧美人与善性xxx| av电影中文网址| 2022亚洲国产成人精品| 国产乱人偷精品视频| 97在线视频观看| 五月开心婷婷网| 男人爽女人下面视频在线观看| 国产一区二区三区av在线| 97在线人人人人妻| 国产精品一区二区在线不卡| 人人妻人人澡人人爽人人夜夜| 97人妻天天添夜夜摸| 免费观看a级毛片全部| 天天影视国产精品| xxxhd国产人妻xxx| 日本-黄色视频高清免费观看| 国产亚洲一区二区精品| av免费在线看不卡| 午夜福利,免费看| 亚洲综合精品二区| 久久99精品国语久久久| 国产日韩欧美亚洲二区| 成人影院久久| 美女脱内裤让男人舔精品视频| 麻豆乱淫一区二区| 色吧在线观看| 宅男免费午夜| 婷婷色综合www| 中文欧美无线码| 一个人免费看片子| 日本av手机在线免费观看| 国产成人精品久久久久久| 老汉色∧v一级毛片| 亚洲av福利一区| 久久鲁丝午夜福利片| 老司机亚洲免费影院| 亚洲天堂av无毛| 欧美97在线视频| 亚洲精品av麻豆狂野| 亚洲av欧美aⅴ国产| 如何舔出高潮| 午夜久久久在线观看| 女性生殖器流出的白浆| 久久久久国产精品人妻一区二区| 少妇人妻 视频| 午夜免费男女啪啪视频观看| 天天躁日日躁夜夜躁夜夜| 国产黄频视频在线观看| 国产日韩欧美在线精品| 一区二区三区四区激情视频| 久久久久久久久久久久大奶| 久久精品国产亚洲av天美| 一级毛片电影观看| 最近中文字幕2019免费版| 韩国av在线不卡| 十分钟在线观看高清视频www| 伦精品一区二区三区| 日本欧美国产在线视频| 久久精品国产自在天天线| 青春草视频在线免费观看| 少妇猛男粗大的猛烈进出视频| 麻豆av在线久日| videos熟女内射| 色视频在线一区二区三区| 国产成人精品久久二区二区91 | 日韩视频在线欧美| 久久国产亚洲av麻豆专区| 亚洲精品视频女| 午夜福利视频在线观看免费| 久久99热这里只频精品6学生| 欧美精品一区二区大全| 韩国精品一区二区三区| 亚洲国产精品一区三区| 男女免费视频国产| 九九爱精品视频在线观看| 在线观看国产h片| 免费观看在线日韩| 国产欧美日韩一区二区三区在线| 有码 亚洲区| 欧美日韩综合久久久久久| 成人二区视频| 国产精品三级大全| 叶爱在线成人免费视频播放| 婷婷色av中文字幕| 欧美中文综合在线视频| 国产男女超爽视频在线观看| 超碰97精品在线观看| 日本猛色少妇xxxxx猛交久久| 夫妻性生交免费视频一级片| 热re99久久精品国产66热6| 免费久久久久久久精品成人欧美视频| 人人妻人人澡人人爽人人夜夜| 激情视频va一区二区三区| 大片电影免费在线观看免费| 亚洲人成77777在线视频| 另类精品久久| 色哟哟·www| 在线天堂最新版资源| 如何舔出高潮| 亚洲欧洲精品一区二区精品久久久 | 人妻一区二区av| 久久久久精品性色| 日韩制服骚丝袜av| 人妻一区二区av| 97人妻天天添夜夜摸| 成人国产av品久久久| 最近最新中文字幕大全免费视频 | 美女视频免费永久观看网站| 欧美国产精品一级二级三级|