• <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 ?

    Expression and Characterization of a Novel λ-Carrageenase Cgl150A_Wa from Wenyingzhuangia aestuarii

    2024-03-12 11:14:24SUNYuhaoCAOSiqiZHANGYuyingXUEChanghuXIAOHangandCHANGYaoguang
    Journal of Ocean University of China 2024年1期

    SUN Yuhao , CAO Siqi ZHANG Yuying XUE Changhu , XIAO Hang,and CHANG Yaoguang ,

    1) College of Food Science and Engineering, Ocean University of China, Qingdao 266003,China

    2) Marine Life Research Center, Laoshan National Laboratory, Qingdao 266237, China

    3) Department of Food Science, University of Massachusetts Amherst, MA 01003, United States

    Abstract λ-Carrageenan is a highly sulfated polysaccharide alternating of 1,4-O-α-D-galactopyranose-2,6-sulfate (D2S,6S) and 1,3-O-β-D-galactopyranose-2-sulfate (G2S). λ-Carrageenases are desirable tools for λ-carrageenan degradation. Based on the genome mining, a novel λ-carrageenase Cgl150A_Wa was cloned from the bacterium Wenyingzhuangia aestuarii and expressed in Escherichia coli. Cgl150A_Wa was an endo-acting enzyme and exhibited its maximum activity at 30℃ and pH 8.0. By employing a glycomics strategy that combined ultra-performance liquid chromatography-mass spectrometry analysis and glycoinformatics, Cgl150A_Wa was proven to degrade λ-carrageenan octaose and hexaose, and the major hydrolysis product of Cgl150A_Wa was λ-carrageenan tetrose.In addition to the typical λ-carrageenan motifs, the active center of Cgl150A_Wa might tolerate desulfated λ-carrageenan motifs.Cgl150A_Wa is a potential biotechnological tool for preparing λ-carrageenan oligosaccharides and structural investigation.

    Key words carrageenan; λ-carrageenase; LC-MS; oligosaccharide; GH150

    1 Introduction

    Carrageenan, a class of natural polysaccharides in the cell walls of red algae, mainly consists of 1,3-O-β-D-galactopyranose (G) and 1,4-O-α-D-galactopyranose residues (D)or 1,4-O-3,6-anhydroα-D-galactopyranose residues (DA)repeating disaccharide units (Zhuet al., 2018). According to the difference of the position and number of sulfate groups(S) on galactose as well as the amount of DA, the carrageenan disaccharide units can be divided into different types(Sedayuet al., 2019).λ-Carrageenan, which is primarily generated from the tetrasporic phase ofGigartinceae, consists of D2S,6S-G2S and contains the largest content of sulfate groups (Chauhanet al., 2016; Guoet al., 2022). Due to the absence of DA,λ-carrageenan is more hydrophilic thanκ-andι-carrageenan, although its aqueous solution is very viscous. Consequently,λ-carrageenan is utilized as thickening and stabilizer agents in food, cosmetics and pharmaceutical industries (Ghanbarzadehet al., 2018). More importantly,λ-carrageenan, especially low molecular weightλ-carrageenan andλ-carrageenan oligosaccharides possess versatile biological activities, including anti-tumor, anticoagulant and immunomodulation activities (Zhouet al.,2004; Zhouet al., 2006; Groultet al., 2019; Guoet al.,2022), which have generated increasing interest and showed promising applications in the food and medical fields.

    By the virtues of the specificity and efficiency, enzymes are considered as desirable tools for polysaccharides degradation and oligosaccharides preparation. To the best of our knowledge, few studies have reported the cloning and characterization ofλ-carrageenases. Ohta and Hatada (2006)isolated and purified the firstλ-carrageenase CglA fromPseudoalteromonassp.CL19 and they clarified the gene sequence of CglA in 2006. Guibetet al. (2007) heterologously expressed anotherλ-carrageenase CglA, investigated its hydrolysis pattern and comfirmed that the enzyme contains at least 8 subsides; moreover, created a new category forλ-carrageenase. Recently,λ-carrageenase OUC-CglA was successfully expressed, which showed cold-adaption in the degradation process (Luet al., 2022). Even though threeλ-carrageenases have been successfully cloned, there are no available commercial enzymes for degradingλ-carrageenan yet.

    In order to enrich theλ-carrageenase species, recently, we isolated and screened a marine bacterium namedWenyingzhuangia aestuarii, and revealed a putativeλ-carrageenase gene sequence (GenBank accession no. OP730521) in the genome of this strain by using BLASTP algorithm against the Swiss-Prot database. In this study, the sequence was cloned and heterologously expressed inEscherichia coliBL21(DE3), and a newλ-carrageenase Cgl150A_Wa was produced. The biochemical characteristics of the enzyme were investigated and its hydrolysis process and products were determined by a glycomics strategy.

    2 Material and Methods

    2.1 Bioinformatics Analysis

    dbCAN (Yinet al., 2012) and SignalP 4.1 (Petersenet al.,2011) were used to predict the domain of Cgl150A_Wa.The sequence similarity was examined by using BLASTP(Altschulet al., 1997). The isoelectric point and theoretical molecular weight (Mw) of Cgl150A_Wa were estimated by ExPASy (Artimoet al., 2012). Evolutionary relationship between Cgl150A_Wa and all the enzymes of the GH150 family in CAZy was analyzed by ClustalX2 and MEGA6 (Thompsonet al., 1997). The ClustalX2 was used to performed the multiple sequence alignment, and the phylogenetic tree was constructed by using MEGA6 based on neighbor-joining algorithm.

    2.2 Plasmid Construction and Protein Purification

    W. aestuariiwas collected and the genomic DNA was extracted as previously described (Zhanget al., 2019). The geneCgl150A_Wawithout signal peptide sequence was amplified by polymerase chain reaction (PCR), and a GST tag was added to the N-terminus of the encoded sequence to improve protein solubility. The forward primer was 5’-GACACGGATCCCAAAAGGTAGATACAAAATCAGC TTTG-3’ and the reverse primer was 5’-GACACCTCGA GTTATTTAAGTGGTTTGCTCAACTCAACATC-3’. The PCR amplification products and PGEX-4T-1 vector were digested with the restriction enzymesBamHI/XhoI, and the target fragment was inserted into the vector. The recombinant vector was transformed into BL21 (DE3) competent cells. The recombinant strains were cultivated in LB medium containing 60 ng mL?1ampicillin with a shaking at 170 r min?1and 37℃. Isopropylβ-D-thiogalactoside was added to induce Cgl150A_Wa expression with a concentration of 0.1 mmol L?1at 17℃ for 12 h. Ultimately, the cells were harvested and resuspended in 20 mmol L?1PBS (pH 8.0), disrupted by sonication, and centrifuged to obtain the crude Cgl150A_Wa solution.

    The purification procedures were performed on the AKTA Prime System (GE Healthcare, Sweden). The target proteins were primarily purified by GSTrap columns (GE Healthcare, Sweden) and then with HiPrep SP Fast Flow columns (GE Healthcare, Sweden). After removing GST tag by using thrombin, Cgl150A_Wa was further purified by Superdex 75 Increase 10/300GL column (Cytiva, American), then analyzed by SDS-PAGE. The gel consisted of a stacking gel (5% polyacrylamide) and a separating gel (10%polyacrylamide). The purified enzyme concentration was examined by the Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime Biotechnology, China).

    2.3 Enzyme Activity Assay

    To determine theλ-carrageenase activity, 100 μL enzyme solution was incubated with 250 μL 2 mg mL?1λ-carrageenan (Shanghai Yuanye Bio-Technology, China) and 275 μL PBS at 30℃ for 10 min. The para-hydroxybenzoic acid hydrazide (pHBH) assay (Lever, 1972) was used to measure the amount of the reducing sugar. Activities of Cgl150A_Wa againstκ-carrageenan,ι-carrageenan or furcelleran were comparatively measured. One unit of Cgl150A_Wa activity(1 U) was defined as the amount of Cgl150A_Wa in hydrolyzing substrate to produce 1 nmol reducing sugar (equivalent to D-galactose) per minute.

    2.4 Biochemical Characterization

    The solution containing the enzyme and substrate was incubated at different temperatures from 20℃ to 60℃ to estimate the effect of temperature on Cgl150A_Wa activity.The thermal stability was detected by placing Cgl150A_Wa at temperatures ranging from 4℃ to 50℃ for 24 h,and enzyme solutions were taken at different time intervals for enzyme activity assay.

    Cgl150A_Wa was mixed withλ-carrageenan solution at pH 4 to 10, then the enzyme activity was measured to determine the effect of pH on enzyme activity. To estimate the pH stability of Cgl150A_Wa, the enzyme was mixed in various buffers with pH 4 to 10 and incubated at 4℃for 1 h before the residual activity assay.

    Cgl150A_Wa was incubated withλ-carrageenan solution at a NaCl final concentrations of 0 to 0.5 mol L?1for 10 min, respectively, to study the impact of Na+. In order to evaluate the effects of metal ions and chemical reagents on the enzyme activity, CuSO4, CaCl2, KCl, MgCl2, HgCl2,EDTA (Sinopharm Chemical Reagent Co., Ltd., China),SDS andβ-Mercaptoethanol (Sigma-Aldrich, Germany)were supplemented respectively to the reaction solution at final concentrations of 1 mmol L?1and 5 mmol L?1for 10 min.

    2.5 Hydrolysis Pattern Analysis

    To elucidate the hydrolysis pattern of Cgl150A_Wa, 150 U Cgl150A_Wa was incubated with 10 mL 2 mg mL?1λcarrageenan solution at 30℃ for 24 h. High-performance liquid chromatography (HPLC) and a refractive index detector (RID) were used to analyze the samples that were collected at various time intervals. TSK-GEL SuperAW-4000 column (Tosoh Corporation, Japan) and elution buffer 0.2 mol L?1NaCl with a flow rate of 0.2 mL min?1were employed to estimate the global profile of the hydrolysis products.

    The final products of Cgl150A_Wa were prepared by incubating 1500 U enzyme and 10 mLλ-carrageenan solution whose concentration was 10 mg mL?1at 30℃ for 12 h.Thereafter, another 1500 U enzyme was added to ensure the substrates were hydrolyzed completely. After inactivation,the solution was lyophilized. The lyophilizes were dissolved in 20 mg mL?1, and centrifuged. The supernatant was further purified by AKTA Prime Plus (GE Healthcare, Sweden) and HiLoad 26/60 Superdex 30 PG (GE Healthcare,Sweden) with 5 mmol L?1ammonium formate at a flow rate of 2.6 mL min?1served as the mobile phase. The separated components were identified by liquid chromatographymass spectrometry (LC-MS). Single components with the samem/zwere combined for1H NMR detection.

    The hydrolysis processing of Cgl150A_Wa was investigated by adding 3, 6, 9, 12 and 15 U Cgl150A_Wa with 1 mgλ-carrageenan to aλ-carrageenan final concentration of 1 mg mL?1, respectively, and incubated at 30℃ for 24 h.The supernatant was analyzed by ultra-performance size exclusion chromatography combined with high-resolution mass spectrometry (UPSEC-HRMS). The system was equipped with an ultra-performance liquid chromatography(UPLC) unit (Dionex Ultimate 3000, Thermo Fisher Scientific, San Jose, CA) connected to Thermo Scientific QExactive Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) and an Acquity BEH 125 SEC column (4.6 mm × 150 mm, Waters, Milford, MA). The mobile phase was composed of 20% (v/v) methanol with 10 mmol L?1ammonium acetate, and the flow rate was set as 0.2 mL min?1(Zhanget al., 2020). Mass spectrometer parameters were set as follows: mass rangem/z150 – 2000;negative ionization mode; S-lens RF level 50 V; spray voltage 2000 V; sheath gas pressure 40 psi; capillary temperature 300℃.

    The generated oligosaccharides were identified by a glycoinformatics protocol. The raw data of mass spectrometry was deconvolved using DeconTools (Liet al., 2012).The outputs were processed by GlycoResoft to analyze the composition of oligosaccharides and generate the semiquantitative information (Maxwellet al., 2012).

    2.6 Statistical Analysis

    All experiments were performed at least three times. All the data were expressed as average ± SD. SPSS Statistics 19.0 (SPSS Inc., Chicago, IL, USA) was utilized to perform Tukey’s post-hoc test [analysis of variance (ANOVA)]. TheP-value under 0.05 was considered statistically significant.

    3 Results and Discussion

    3.1 Bioinformatics Analysis

    The geneCgl150A_Waencodes a protein consisting of 950 amino acids. Cgl150A_Wa was predicted to contain a putative signal peptide (residues 1-35) and a GH150 family structural domain (residues 41-947). After removing the signal peptide, the molecular weight of Cgl150A_Wa was calculated as 104 kDa and the isoelectric point was supposed to be 9.24. The phylogenetic tree consisting of all GH150 family proteins recorded in the CAZy database is shown in Fig.1. Cgl150A_Wa is in a separated branch in distance of the previously reportedλ-carrageenases.BLASTP showed that Cgl150A_Wa sequence only shared 47.31%, 46.98% and 53.11% similarity with the characterizedλ-carrageenase CL19, CglA and OUC-CglA isolated fromPseudoalteromonas sp., indicating the novelty of Cgl150A_Wa.

    Fig.1 Phylogenetic tree of Cgl150A_Wa (highlighted by pentacle), previously characterized λ-carrageenases (highlighted by triangle) and other GH150 proteins.

    3.2 Cloning and Expression of Cgl150A_Wa

    After purification by using the GSTrap and HiPrep SP Fast Flow columns, the recombinant Cgl150A_Wa presented a single band of 110 kDa in SDS-PAGE analysis (Fig.2),which was in a good agreement with the predicted value.The purified Cgl150A_Wa was active onλ-carrageenan with the activity of 236 U mg?1, while it was incapable of degradingκ-andι-carrageenan and furcellaran. The results above indicated that Cgl150A_Wa was a novelλ-carrageenase.

    Fig.2 SDS-PAGE analysis of purified Cgl150A_Wa. Line 1, purified Cgl150A_Wa.

    3.3 Biochemical Characteristics of Cgl150A_Wa

    The optimum reaction temperature and pH of Cgl150A_Wa were 30℃ (Fig.3A) and pH 8 (Fig.3B). More than 90%activity could be retained at 4℃ for 24 h; however, the activity decreased significantly when Cgl150A_Wa was maintained at 20℃ and above (Fig.3C). As for pH stability,Cgl150A_Wa retained more than 80% activity between pH 5.5 to 8.5, and exhibited the highest activity at pH 8 (Fig.3D). NaCl could significantly promote the activity of Cgl-150A_Wa (Fig.3E), which was increased about 3.5 times except for 0.5 M NaCl solution. The notable effect of NaCl on Cgl150A_Wa indicated that Cgl150A_Wa activity was extremely enhanced by the salts and the maximum activity was reached at the presence of 0.1 mol L?1NaCl.

    Fig.3 Biochemical characterization of Cgl150A_Wa. (A), effect of temperature on enzyme activity; (B), effect of pH value on enzyme activity; (C), thermal stability; (D), pH stability; (E), effect of NaCl concentration on enzyme activity.

    The influence of organic reagents and metal ions on Cgl-150A_Wa are shown in Table 1. 1 mmol L?1Cu2+, Hg2+and SDS inhibited Cgl150A_Wa activity remarkably, while 5 mmol L?1Mg2+and EDTA could promote the activity of the enzyme. Ca2+, K+, andβ-Mercaptoethanol did not have significant effect on the Cgl150A_Wa activity.

    Table 1 Effects of metal ions and organic reagents on Cgl150A_Wa activity

    3.4 Hydrolysis Pattern of Cgl150A_Wa

    To estimate the acting type of Cgl150A_Wa, the enzyme reaction was monitored by utilizing HPSEC-RID (Fig.4,the complete diagram was shown in Fig.5). A significant late evaluation was observed after 10 min of reaction. It confirmed that Cgl150A_Wa was an endo-acting enzyme,which was consistent with CglA and OUC-CglA. Until now,all theλ-carrageenases that have been identified were endotypeλ-carrageenases.

    Fig.4 HPSEC-RID analysis of the hydrolysis products of Cgl150A_Wa on λ-carrageenan at different reaction times.

    Fig.5 Complete diagram of HPSEC-RID analysis.

    To study the enzymatic reaction process, Cgl150A_Wa with different dosages (3 – 15 U) was incubated withλ-carrageenan for 24 h, and the hydrolyzed products were analyzed by a glycomics workflow. [M-2H]2?withm/zvalue of 289.9855 and 571.9735, [M-3H]3?withm/zvalue of 568.9717 and 756.9638 were observed, which correspond respectively to theλ-carrageenan biose (D2S,6S-G2S)1,tetraose (D2S,6S-G2S)2, hexaose (D2S,6S-G2S)3and octaose (D2S,6S-G2S)4. Peak areas of the extraction ion chromatograms of each component were integrated, and the results were illustrated in Fig.6A. The putativeλ-carrageenan tetraose (D2S,6S-G2S)2was the predominant component in all samples. To further identify the component, the putative tetraose was isolated from the end products for1H-NMR identification and the chemical shifts were assigned according to the previous studies (Guibetet al., 2006;2007). Signals of δ5.51 and δ5.49 were attributed to the anomeric protons inα-configuration at the reducing end (α-G2Sr H1) and the non-reducing end (D2S,6Snr H1) (Fig.7).The internal protons (α-D2S,6S H1 andβ-D2S,6S H1) were assigned at δ5.56 and δ5.57. Combining the mass spectrometry and NMR data, the main product was confirmed to be (D2S,6S-G2S)2.

    Fig.6 (A) Peak areas of extraction ion chromatograms of each oligosaccharide prepared by incubating λ-carrageenan with different dosages of Cgl150A_Wa; (B) Extraction ion chromatograms of the products hydrolyzed by 15 U Cgl150A_Wa.

    Fig.7 1H-NMR spectrum of purified λ-carrageenan tetraose D2S,6Snr-G2S-D2S,6S-G2Sr. r, reducing end; nr, non-reducing end.

    With the increasing dosages of Cgl150A_Wa, the amounts of (D2S,6S-G2S)2increased sharply, coupling with a slow production of (D2S,6S-G2S)1. While the amounts of(D2S,6S-G2S)3and (D2S,6S-G2S)4increased firstly and then decreased. This indicated that Cgl150A_Wa could degradeλ-carrageenan hexaose and octaose, but the biose and tetraose could not be degraded by Cgl150A_Wa. CglA fromPseudoalteromonas carrageenovorawas capable of degrading octaose and hexaose, and generated tetrose as the main product (Guibetet al., 2007), which was consistent with Cgl150A_Wa. The results proved that Cgl150A_Wa consists of at least eight subsites. Considering that tetraose could not be degraded by Cgl150A_Wa, we presumed that the number of the active subsites is more than four, while the exact number needs to be further elucidated. As the products of OUC-CglA in the hydrolysis process were much more complex than those of Cgl150A_Wa and CglA, the biose, tetrose, hexaose, octaose and decaose were all accumulated as the degradation time increased (Luet al., 2022),causing great obstacle to clarify the degradation pattern and the number of subsites.

    Intriguingly, in addition to the typicalλ-carrageenan oligosaccharides,λ-carrageenan tetrasaccharide desulfation products (D2S,6S-G2S)2DeS (m/z531.9951, [M-2H]2?,(D2S,6S-G2S)2lost one sulfate group) and (D2S,6S-G2S)2De2S (m/z492.0167, [M-2H]2?, (D2S,6S-G2S)2lost two sulfate groups, or (D2S,6S-G2S)2DeS lost one sulfate group)were also observed in the 15 U Cgl150A_Wa hydrolysis products (Fig.6B). (D2S,6S-G2S)2DeS showed two peaks at retention time of 13.10 min and 12.75 min. Desulfation of highly sulfated polysaccharides in high-energy magnetic fields of the mass spectrum is common and is an inevitable phenomenon (Anastyuket al., 2015). The peak of(D2S,6S-G2S)2DeS at 12.75 min exhibited the same retention time with (D2S,6S-G2S)2. Therefore, the ion ofm/z531.9951 at 12.75 min was supposed to be the (D2S,6S-G2S)2desulfated product in the MS detection process. Meanwhile, the peak of (D2S,6S-G2S)2DeS at 13.10 min was later than that of (D2S,6S-G2S)2, which was in accordance with the separation principle of SEC. So, it should be(D2S,6S-G2S)2DeS in the hydrolysis products. Similarly,the peak of (D2S,6S-G2S)2De2S at 13.60 min was speculated to be a product obtained from the enzyme degradation. It indicated that Cgl150A_Wa can hydrolyze desulfatedλ-carrageenan and generate corresponding desulfated oligosaccharides.

    According to the above results, it was speculated that Cgl150A_Wa might be able to accommodate less sulfate groups carrageenan motifs in addition to the typicalλ-carrageenan motifs. Sinceλ-carrageenan contains more sulfate groups thanκ- andι-carrageenan,etc.,λ-carrageenase was considered to express a larger cavity than the other carrageenases to accommodate additional sulfate groups. Theoretically, the desulfated substrate possesses less steric hindrance and is not preferably accommodated into the active sites. This is the first report on the sequence ofλ-carrageenase in hydrolyzing desulfatedλ-carrageenan moieties. The study provided novel understanding of the potential degradation pattern of the GH150 family. Further identification of desulfatedλ-carrageenan oligosaccharides will lead to a better understanding of the subsite specificity of Cgl150A_Wa, which is worthy of additional investigation.

    4 Conclusions

    In conclusion, a novelλ-carrageenase Cgl150A_Wa was successfully cloned and well characterized. The enzyme shared the highest 53.11% identity with the characterizedλ-carrageenase, and exhibited its maximum activity at 30℃and pH 8.0. It hydrolyzedλ-carrageenan in an endo-acting manner, and was capable of degrading octaose and hexaose,and producedλ-carrageenan tetraose as the main product.Furthermore, in addition to recognizing the typicalλ-carrageenan structure, the active sites of Cgl150A_Wa might also accommodate desulfatedλ-carrageenan motifs. The characterized biochemical properties and special hydrolysis pattern suggested that Cgl150A_Wa is a promising tool to facilitate full degradation ofλ-carrageenan and oligosaccharides preparation.

    Acknowledgements

    This work was supported by the Fundamental Research Funds for the Central Universities (No. 202012020), and the National Key R&D Program of China (No. 2018YFC 0311203).

    国产亚洲精品久久久久久毛片| 校园春色视频在线观看| 久久久a久久爽久久v久久| 亚洲人成网站高清观看| 欧美日韩国产亚洲二区| 一区二区三区高清视频在线| 亚洲欧洲国产日韩| 超碰av人人做人人爽久久| 国产精华一区二区三区| 婷婷色av中文字幕| 黄色欧美视频在线观看| 色综合色国产| 深夜a级毛片| 国产精品一及| 亚洲电影在线观看av| 成年女人看的毛片在线观看| 久久午夜福利片| 日韩欧美一区二区三区在线观看| 国产不卡一卡二| 97人妻精品一区二区三区麻豆| 欧美日韩在线观看h| 国产精品1区2区在线观看.| 亚洲aⅴ乱码一区二区在线播放| 丰满人妻一区二区三区视频av| 亚洲国产精品sss在线观看| 国产精品福利在线免费观看| 国产精品综合久久久久久久免费| 精品人妻一区二区三区麻豆| 久久精品91蜜桃| 18+在线观看网站| 成人欧美大片| 成人漫画全彩无遮挡| 婷婷色综合大香蕉| 美女大奶头视频| 亚洲综合色惰| 色综合亚洲欧美另类图片| av在线观看视频网站免费| 99国产极品粉嫩在线观看| 国产亚洲5aaaaa淫片| 2021天堂中文幕一二区在线观| 此物有八面人人有两片| 中文在线观看免费www的网站| 听说在线观看完整版免费高清| 99久久中文字幕三级久久日本| 亚洲精品色激情综合| 热99在线观看视频| 久久久久久伊人网av| 久久亚洲精品不卡| 日日撸夜夜添| 久久久久久久久久黄片| 亚洲va在线va天堂va国产| 亚洲精品乱码久久久v下载方式| 搞女人的毛片| 少妇被粗大猛烈的视频| 欧美zozozo另类| 蜜桃亚洲精品一区二区三区| 精品少妇黑人巨大在线播放 | 亚洲,欧美,日韩| 一区二区三区高清视频在线| 日韩中字成人| 狂野欧美激情性xxxx在线观看| 久久国内精品自在自线图片| 直男gayav资源| 尤物成人国产欧美一区二区三区| 久久久欧美国产精品| 久久亚洲国产成人精品v| 久久久久久九九精品二区国产| 97超视频在线观看视频| 麻豆国产av国片精品| 亚洲在线自拍视频| 国产女主播在线喷水免费视频网站 | 麻豆精品久久久久久蜜桃| 亚洲丝袜综合中文字幕| 亚洲精品乱码久久久久久按摩| 国产av不卡久久| 日本一二三区视频观看| 中文字幕久久专区| 久久欧美精品欧美久久欧美| 少妇裸体淫交视频免费看高清| 久久久久性生活片| 国产精品野战在线观看| 亚洲精品久久久久久婷婷小说 | 中国国产av一级| 亚洲av免费在线观看| 五月伊人婷婷丁香| 又粗又爽又猛毛片免费看| 久久99精品国语久久久| 亚洲欧美精品专区久久| 国内揄拍国产精品人妻在线| 成人国产麻豆网| 国产国拍精品亚洲av在线观看| 美女xxoo啪啪120秒动态图| 18禁在线播放成人免费| 91在线精品国自产拍蜜月| 春色校园在线视频观看| 在线免费观看不下载黄p国产| 性欧美人与动物交配| 亚洲在线自拍视频| 亚洲经典国产精华液单| 国产高清不卡午夜福利| 99久久中文字幕三级久久日本| 久久久久九九精品影院| 免费观看精品视频网站| 国产乱人视频| 日本爱情动作片www.在线观看| 精品无人区乱码1区二区| 毛片一级片免费看久久久久| 又爽又黄无遮挡网站| 嫩草影院精品99| 欧美在线一区亚洲| 日韩欧美精品免费久久| 一进一出抽搐动态| 老女人水多毛片| 免费看光身美女| 一级黄色大片毛片| 亚洲av男天堂| 国产成人aa在线观看| 欧美变态另类bdsm刘玥| 亚洲国产精品sss在线观看| 亚洲av第一区精品v没综合| 国产亚洲欧美98| 欧美人与善性xxx| 此物有八面人人有两片| 99久久无色码亚洲精品果冻| 国产大屁股一区二区在线视频| 国产精品.久久久| 九九久久精品国产亚洲av麻豆| 久久午夜福利片| 亚洲国产精品久久男人天堂| 国产精品电影一区二区三区| 欧美潮喷喷水| 爱豆传媒免费全集在线观看| 亚洲av.av天堂| 色哟哟·www| 成人午夜高清在线视频| 性插视频无遮挡在线免费观看| 嫩草影院精品99| 69人妻影院| 国产成年人精品一区二区| 亚洲,欧美,日韩| 国产av麻豆久久久久久久| 日韩,欧美,国产一区二区三区 | 精品久久久久久久久久久久久| 亚洲美女搞黄在线观看| 国产乱人偷精品视频| 国产亚洲精品av在线| 亚洲经典国产精华液单| 男人舔奶头视频| 少妇的逼好多水| 欧美xxxx性猛交bbbb| kizo精华| 婷婷六月久久综合丁香| 99久久精品国产国产毛片| 国产成人精品一,二区 | 最后的刺客免费高清国语| 国产高清三级在线| 亚洲欧美精品专区久久| 亚洲五月天丁香| 午夜久久久久精精品| 日本-黄色视频高清免费观看| 少妇的逼好多水| 日韩 亚洲 欧美在线| 久久精品国产亚洲网站| 成年av动漫网址| 亚洲人成网站在线播| 一级毛片aaaaaa免费看小| 九草在线视频观看| 不卡一级毛片| 久久精品久久久久久噜噜老黄 | 国产成人a∨麻豆精品| 高清在线视频一区二区三区 | 神马国产精品三级电影在线观看| 美女内射精品一级片tv| 一级毛片aaaaaa免费看小| 亚洲欧美精品专区久久| 精品久久久久久久末码| 精品久久久久久久人妻蜜臀av| 国产真实伦视频高清在线观看| 日日摸夜夜添夜夜爱| 国产精品永久免费网站| 国产乱人视频| 97热精品久久久久久| 联通29元200g的流量卡| 国产一区二区在线观看日韩| 久99久视频精品免费| 国产午夜精品论理片| 久久99热这里只有精品18| 毛片女人毛片| 国产高清有码在线观看视频| 亚洲国产色片| 国产亚洲精品av在线| 亚洲精品日韩在线中文字幕 | 成人鲁丝片一二三区免费| 国产探花在线观看一区二区| 网址你懂的国产日韩在线| 国产一级毛片七仙女欲春2| 国产高清激情床上av| 成年女人永久免费观看视频| 麻豆国产av国片精品| 国产av麻豆久久久久久久| 欧美区成人在线视频| 日韩av不卡免费在线播放| 91av网一区二区| 国产av不卡久久| 天天躁日日操中文字幕| 校园春色视频在线观看| 欧美区成人在线视频| 国产一区二区在线av高清观看| 婷婷色av中文字幕| 边亲边吃奶的免费视频| 18禁在线无遮挡免费观看视频| 99热精品在线国产| 欧美日韩精品成人综合77777| 性色avwww在线观看| a级一级毛片免费在线观看| 亚洲精品成人久久久久久| 一本久久中文字幕| 国产精品99久久久久久久久| 国产一区亚洲一区在线观看| 国产视频内射| 国产一级毛片七仙女欲春2| 一级毛片久久久久久久久女| 欧美一级a爱片免费观看看| 91aial.com中文字幕在线观看| 一夜夜www| 亚洲欧美日韩卡通动漫| 免费av不卡在线播放| 日本黄大片高清| 亚洲中文字幕日韩| 日韩人妻高清精品专区| 我的女老师完整版在线观看| 91久久精品国产一区二区成人| 成人二区视频| 国内精品久久久久精免费| 国产 一区精品| 亚洲精品国产av成人精品| 午夜免费男女啪啪视频观看| 国产精品免费一区二区三区在线| 欧美成人精品欧美一级黄| 欧美三级亚洲精品| 日日干狠狠操夜夜爽| 欧美一级a爱片免费观看看| 亚洲精品日韩av片在线观看| 给我免费播放毛片高清在线观看| 欧美激情久久久久久爽电影| 久久久久久大精品| 精品久久久噜噜| 亚洲国产精品sss在线观看| 日韩亚洲欧美综合| 成年版毛片免费区| a级毛片免费高清观看在线播放| 亚洲国产精品久久男人天堂| 深夜a级毛片| 99久国产av精品国产电影| 亚洲欧美日韩东京热| 在线免费观看的www视频| 欧美3d第一页| 91午夜精品亚洲一区二区三区| 性插视频无遮挡在线免费观看| 久久久久久久久中文| 我的女老师完整版在线观看| 欧美另类亚洲清纯唯美| 久久人人精品亚洲av| 日韩亚洲欧美综合| 成年版毛片免费区| 日韩强制内射视频| 啦啦啦韩国在线观看视频| 搡女人真爽免费视频火全软件| 久99久视频精品免费| 欧美最新免费一区二区三区| 亚洲最大成人av| 国产乱人偷精品视频| 久久99蜜桃精品久久| 亚洲最大成人手机在线| 一个人看的www免费观看视频| 亚洲最大成人av| 国产高清三级在线| 国内揄拍国产精品人妻在线| 日本黄色片子视频| 日韩精品青青久久久久久| 亚洲欧美清纯卡通| av福利片在线观看| 亚洲七黄色美女视频| 欧美变态另类bdsm刘玥| 床上黄色一级片| 亚洲久久久久久中文字幕| 大型黄色视频在线免费观看| 97热精品久久久久久| 欧美激情国产日韩精品一区| 99在线人妻在线中文字幕| 日本在线视频免费播放| 婷婷色综合大香蕉| 女人被狂操c到高潮| 日韩人妻高清精品专区| 麻豆国产av国片精品| 三级男女做爰猛烈吃奶摸视频| 免费看美女性在线毛片视频| 91精品一卡2卡3卡4卡| 日韩三级伦理在线观看| 精品久久久久久成人av| 3wmmmm亚洲av在线观看| 久久精品久久久久久久性| 色哟哟哟哟哟哟| av视频在线观看入口| 免费av观看视频| 亚洲自偷自拍三级| 丝袜喷水一区| 99久久精品热视频| 亚洲不卡免费看| 国产av在哪里看| 国产真实乱freesex| av福利片在线观看| 亚洲精品影视一区二区三区av| 久久久久久久久久成人| 综合色av麻豆| 亚洲av中文字字幕乱码综合| 亚洲熟妇中文字幕五十中出| 久久久精品大字幕| av又黄又爽大尺度在线免费看 | 亚洲国产色片| 两个人的视频大全免费| 日韩亚洲欧美综合| 你懂的网址亚洲精品在线观看 | 亚洲一区高清亚洲精品| 久久99精品国语久久久| 三级毛片av免费| 成人特级黄色片久久久久久久| 一区二区三区四区激情视频 | 精品久久久久久久末码| 岛国毛片在线播放| 成人午夜高清在线视频| 国内精品一区二区在线观看| 国产视频首页在线观看| 国产激情偷乱视频一区二区| 久久精品影院6| 国产乱人偷精品视频| 日韩中字成人| 中文字幕制服av| 蜜臀久久99精品久久宅男| 99在线视频只有这里精品首页| 国产亚洲精品久久久久久毛片| 高清在线视频一区二区三区 | 成人三级黄色视频| 青春草国产在线视频 | 99riav亚洲国产免费| 久久亚洲国产成人精品v| АⅤ资源中文在线天堂| 精品久久久噜噜| 美女黄网站色视频| 久久久久久久久久久丰满| 色噜噜av男人的天堂激情| 国产单亲对白刺激| 深夜a级毛片| 人妻系列 视频| 91久久精品电影网| 精品一区二区三区视频在线| 人妻夜夜爽99麻豆av| 久久九九热精品免费| 成人亚洲欧美一区二区av| 男女视频在线观看网站免费| 亚洲色图av天堂| 嫩草影院新地址| 欧美日韩国产亚洲二区| 听说在线观看完整版免费高清| 小蜜桃在线观看免费完整版高清| 91久久精品国产一区二区三区| 国产欧美日韩精品一区二区| 久久久精品94久久精品| 国产午夜精品一二区理论片| 成人特级av手机在线观看| 国产探花在线观看一区二区| 国产一区二区激情短视频| 亚洲在线自拍视频| 精品午夜福利在线看| 级片在线观看| 日韩三级伦理在线观看| 啦啦啦观看免费观看视频高清| 在线免费观看的www视频| 午夜久久久久精精品| 国产成人91sexporn| 亚洲第一电影网av| 亚洲av.av天堂| 精品人妻一区二区三区麻豆| 爱豆传媒免费全集在线观看| 亚洲三级黄色毛片| 欧美一区二区国产精品久久精品| 亚洲精华国产精华液的使用体验 | 亚洲色图av天堂| 丝袜美腿在线中文| 亚洲色图av天堂| 青青草视频在线视频观看| 国产在线精品亚洲第一网站| 91aial.com中文字幕在线观看| 好男人视频免费观看在线| 久久精品国产亚洲av天美| 成人毛片60女人毛片免费| 少妇裸体淫交视频免费看高清| 国产 一区 欧美 日韩| 亚洲精品亚洲一区二区| 久久6这里有精品| 老女人水多毛片| 欧美一区二区精品小视频在线| 偷拍熟女少妇极品色| 亚洲图色成人| 亚洲精品亚洲一区二区| 18禁在线无遮挡免费观看视频| 中文字幕av在线有码专区| www.色视频.com| 青春草国产在线视频 | 高清日韩中文字幕在线| 久久久久性生活片| 亚洲中文字幕一区二区三区有码在线看| 我要搜黄色片| 丝袜喷水一区| 精华霜和精华液先用哪个| 日韩高清综合在线| 在线播放无遮挡| 亚洲自偷自拍三级| 国产91av在线免费观看| 全区人妻精品视频| 99久久中文字幕三级久久日本| 日本黄色片子视频| 国产白丝娇喘喷水9色精品| 又粗又硬又长又爽又黄的视频 | 伦精品一区二区三区| 国产男人的电影天堂91| 免费在线观看成人毛片| 免费av不卡在线播放| 搡老妇女老女人老熟妇| 久久精品夜色国产| 欧美区成人在线视频| 一级毛片电影观看 | 国产国拍精品亚洲av在线观看| 内地一区二区视频在线| 国产精品偷伦视频观看了| 亚洲精品久久午夜乱码| 久久精品国产a三级三级三级| 久久热精品热| 久久久久久久精品精品| 日韩精品免费视频一区二区三区 | 亚洲精品日本国产第一区| 少妇人妻精品综合一区二区| 99国产综合亚洲精品| 校园人妻丝袜中文字幕| 成人综合一区亚洲| av福利片在线| 丝袜美足系列| 午夜免费男女啪啪视频观看| 欧美精品人与动牲交sv欧美| 国产国语露脸激情在线看| 国产高清有码在线观看视频| 精品视频人人做人人爽| 欧美人与性动交α欧美精品济南到 | 免费日韩欧美在线观看| 日韩视频在线欧美| 亚洲熟女精品中文字幕| a 毛片基地| 看免费成人av毛片| 亚洲av.av天堂| 国产69精品久久久久777片| 日韩,欧美,国产一区二区三区| 日本vs欧美在线观看视频| 久久久国产一区二区| 久久久久国产精品人妻一区二区| 国产在线免费精品| 日本av手机在线免费观看| 日韩一区二区视频免费看| 十分钟在线观看高清视频www| 中文字幕av电影在线播放| 久久久久久人妻| 日本av免费视频播放| 欧美日韩av久久| 精品国产一区二区三区久久久樱花| 久久久久久久精品精品| 国产成人午夜福利电影在线观看| 午夜激情久久久久久久| 精品亚洲乱码少妇综合久久| 日本色播在线视频| 美女大奶头黄色视频| 老司机亚洲免费影院| 你懂的网址亚洲精品在线观看| 简卡轻食公司| 2022亚洲国产成人精品| 99热这里只有精品一区| 精品一区二区免费观看| h视频一区二区三区| 中文字幕最新亚洲高清| 女人久久www免费人成看片| 精品国产国语对白av| 日本-黄色视频高清免费观看| 水蜜桃什么品种好| 91国产中文字幕| 建设人人有责人人尽责人人享有的| 午夜av观看不卡| 久久久久久人妻| 日韩中文字幕视频在线看片| 另类亚洲欧美激情| 国产免费视频播放在线视频| 国产毛片在线视频| 午夜91福利影院| 我的老师免费观看完整版| 亚洲欧美日韩另类电影网站| 免费播放大片免费观看视频在线观看| 丝袜喷水一区| 亚洲av电影在线观看一区二区三区| 免费看光身美女| 国语对白做爰xxxⅹ性视频网站| 国产精品 国内视频| 久热久热在线精品观看| 亚州av有码| 热99久久久久精品小说推荐| 久久韩国三级中文字幕| 制服丝袜香蕉在线| 欧美丝袜亚洲另类| 色吧在线观看| 少妇被粗大的猛进出69影院 | 在线观看免费高清a一片| 制服丝袜香蕉在线| 免费看av在线观看网站| 七月丁香在线播放| kizo精华| 丰满少妇做爰视频| 亚洲av中文av极速乱| 成年av动漫网址| 久久久久视频综合| 午夜福利,免费看| 丰满少妇做爰视频| 久久久午夜欧美精品| 欧美成人午夜免费资源| 日韩av免费高清视频| 伊人久久精品亚洲午夜| 亚洲四区av| 国产午夜精品久久久久久一区二区三区| av在线app专区| 多毛熟女@视频| 国产探花极品一区二区| 最近手机中文字幕大全| 只有这里有精品99| 在线观看人妻少妇| 中文字幕最新亚洲高清| 国产男女超爽视频在线观看| 国产成人aa在线观看| 亚洲精品,欧美精品| 999精品在线视频| 美女主播在线视频| 国产精品久久久久久精品古装| 精品人妻偷拍中文字幕| 国产精品久久久久久精品电影小说| 夜夜看夜夜爽夜夜摸| 国产在视频线精品| 成年美女黄网站色视频大全免费 | 插逼视频在线观看| 大香蕉97超碰在线| 高清黄色对白视频在线免费看| 国产老妇伦熟女老妇高清| 欧美xxⅹ黑人| 曰老女人黄片| 亚洲av欧美aⅴ国产| 少妇 在线观看| 只有这里有精品99| 婷婷色av中文字幕| 欧美bdsm另类| 国产在线一区二区三区精| 成人国语在线视频| 日韩视频在线欧美| 黑人猛操日本美女一级片| 熟女av电影| 久久99热这里只频精品6学生| 国产成人精品福利久久| 国产免费一区二区三区四区乱码| 满18在线观看网站| 中文字幕制服av| 午夜久久久在线观看| 黄色一级大片看看| av女优亚洲男人天堂| 婷婷色综合大香蕉| 午夜免费观看性视频| 丝袜美足系列| av在线观看视频网站免费| 久久久a久久爽久久v久久| 观看美女的网站| 高清黄色对白视频在线免费看| 女的被弄到高潮叫床怎么办| 亚洲国产欧美在线一区| 久久精品国产亚洲av涩爱| 大香蕉97超碰在线| 精品久久久久久久久亚洲| 中文字幕久久专区| 91精品国产国语对白视频| 亚洲精品乱码久久久久久按摩| 97在线人人人人妻| 人人澡人人妻人| 永久免费av网站大全| 自线自在国产av| 激情五月婷婷亚洲| 全区人妻精品视频| 91精品国产九色| 亚洲欧洲日产国产| 99精国产麻豆久久婷婷| 妹子高潮喷水视频| 亚洲内射少妇av| 夫妻性生交免费视频一级片| 青春草国产在线视频| 大码成人一级视频| 伦理电影大哥的女人| 最近2019中文字幕mv第一页| 久久久久久伊人网av| 久久精品国产亚洲av涩爱| a 毛片基地| 久久久久久久久大av| 麻豆成人av视频| 免费日韩欧美在线观看| 人人妻人人澡人人看| 免费看不卡的av| 欧美 亚洲 国产 日韩一|