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Enzymatic Activity Assay for Invertase in Synechocystis Cells
集胞藻细胞中转化酶酶活的测定方法   

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Frontiers in Microbiology
Jun 2017

Abstract

Invertase can catalyze the hydrolysis of sucrose, and is widely distributed in cells of cyanobacteria and plants. Being responsible for the first step for sucrose metabolism, invertase plays important physiological roles and its enzymatic activity is frequently needed to be determined. All the methods for determination of the invertase activity are dependent on detection of the glucose product generated by the invertase. Here we describe an ion chromatography based protocol of our laboratory for determination of cyanobacterial intracellular invertase activity.

Keywords: Cyanobacteria (蓝藻), Synechocystis (集胞藻), Invertase enzymatic activity (转化酶的酶活性), Sucrose (蔗糖), Ion chromatography (离子色谱法)

Background

Invertase and sucrose play important physiological roles in cyanobacteria (Curatti, et al., 2008; Kolman et al., 2015) and higher plants (Vargas et al., 2003; Vargas et al., 2010). Invertase (EC 3.2.1.26) can catalyze the sucrose degradation into glucose and fructose. Due to this characteristic of the invertase, any methods which could be used for the determination of glucose or fructose would be theoretically used for the invertase enzymatic activity assay. In fact, most of the invertase enzymatic activity assays are based on the detection of the generated glucose product.

Some companies have developed several kits for the invertase activity assay, for instances, ab197005 from abcam (USA), KA1629 from Novus Biologicals (USA), MAK118 from Sigma-Aldrich (USA). By using these kits, the glucose product generated from the invertase reactions would be oxidized and determined by a colorimetric (570 nm) or fluorimetric method (λem/ex = 585/530 nm). In other methods, the amount of reducing sugar liberated by invertase was measured by coupling hexokinase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase, while the resulting NADPH was further spectrophotometrically determined at 340 nm (Vargas et al., 2003). Ion chromatography could directly detect various sugars including glucose, fructose, sucrose (Du et al., 2013) and glucosylglycerol (Tan et al., 2015). Compared with the spectrophotometer-based methods for sugar determinations, ion chromatography (IC) could be more beneficial for analyzing the enzymatic mixtures containing multiple compounds, especially for the enzymatic assay of cell crude extracts. By using ion chromatography, the sucrose consumption and the glucose production could be shown at the same time in the case of the invertase enzymatic assay, which would provide complete information for the enzymatic assays.

Here, we report our recent IC-based protocol for determination of the invertase activity in cyanobacterial cells.

Materials and Reagents

  1. Pipette tips
  2. 2 ml tubes (Cypress, China)
  3. 10 ml conical tubes (Kangjian, China)
  4. 1 ml syringe (Jianshi, China)
  5. Syringe membrane filters, 0.22 μm (Jinteng, China)
  6. Synechocystis sp. PCC 6803 (Tan et al., 2011)
  7. Milli-Q water (Millipore, Germany)
  8. Glass beads (Sigma-Aldrich, catalog number: G9018-250G )
  9. Fructose standard (Sinopharm Chemical Reagent, catalog number: 63003034 )
  10. Glucose standard (Sinopharm Chemical Reagent, catalog number: 10010518 )
  11. Liquid nitrogen
  12. 200 mM NaOH (prepared with Milli-Q water)
  13. Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
  14. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 10013018 )
  15. Calcium chloride dihydrate (CaCl2·2H2O) (Sinopharm Chemical Reagent, catalog number: 20011160 )
  16. Critic acid monohydrate (C6H8O7·H2O) (Sinopharm Chemical Reagent, catalog number: 10007118 )
  17. Ferric ammonium citrate ((NH4)3FeC12H10O14/C6H8O7·xFe3·yNH3) (Sinopharm Chemical Reagent, catalog number: 30011428 )
  18. EDTA·2Na·2H2O (Sinopharm Chemical Reagent, catalog number: 10009717 )
  19. Sodium carbonate (Na2CO3) (Sinopharm Chemical Reagent, catalog number: 10019260 )
  20. Boric acid (H3BO3) (Sinopharm Chemical Reagent, catalog number: 10004818 )
  21. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sinopharm Chemical Reagent, catalog number: 20026118 )
  22. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 10024018 )
  23. Sodium molybdate dihydrate (Na2MoO4·2H2O) (Sinopharm Chemical Reagent, catalog number: 10019818 )
  24. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sinopharm Chemical Reagent, catalog number: 10008218 )
  25. Cobalt(II) chloride hexahydrate (CoCl2·6H2O) (Sinopharm Chemical Reagent, catalog number: 10007216 )
  26. Sodium nitrate (NaNO3) (Sinopharm Chemical Reagent, catalog number: 10019918 )
  27. Potassium dihydrogen phosphate (KH2PO4) (Sinopharm Chemical Reagent, catalog number: 10017618 )
  28. Dipotassium hydrogen phosphate trihydrate (K2HPO4∙3H2O) (Sinopharm Chemical Reagent, catalog number: 10017518 )
  29. Phenylmethanesulfonyl fluoride (Sigma-Aldrich, catalog number: P7626 )
  30. Isopropanol (Sinopharm Chemical Reagent, catalog number: 40064360 )
  31. BG11 medium (see Recipes)
  32. 100 mM potassium phosphate buffer (pH 7.0) (see Recipes)
  33. 100 mM PMSF (see Recipes)

Equipment

  1. 50 ml flasks
  2. Pipettes (Eppendorf, Germany)
  3. Shaker (Taicang Huamei, model: THZ-701B )
  4. Water bath (Shanghai Yarong, model: B-260 )
  5. Centrifuge (Beckman Coulter, model: Microfuge® 22R )
  6. -20 °C freezer (Haier, model: BCD-219D )
  7. Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 )
  8. Thermal Cycler for PCR (Bio-Rad Laboratories, model: T-100 )
  9. Ion chromatography (Thermo Fisher Scientific, Thermo ScientificTM, model: DionexTM ICS-5000+ )
  10. DionexTM CarboPacTM PA10 analytical column (4 x 250 mm, Thermo Fisher Scientific, model: DionexTM CarboPacTM PA10 )

Software

  1. ChromeleonTM software (Thermo Fisher Scientific, Thermo ScientificTM, version 6.80; catalog number: CHROMELEON6)

Procedure

  1. Cultivation of Synechocystis sp. PCC 6803 (Tan et al., 2011)
    Note: Monitor the growth of cyanobacterial cells by measuring the optical density (OD) at 730 nm with a spectrophotometer. Culture volume should be less than 30 ml in 50 ml flasks.
    1. Grow Synechocystis cells in 20 ml liquid BG-11 media (see Recipe 1) in 50 ml flasks with an initial OD730 of 0.05.
    2. Cultivate the Synechocystis cultures at 30 °C with shaking (150 rpm) and constant white light illumination (50 μE/m2/sec) for ~4 days to reach the exponential growth phase.
  2. Preparation of cell crude extracts from Synechocystis cells
    1. Collect 8 ml of the exponential phase culture (OD730 ≈ 2) of Synechocystis by centrifugation at 8,000 x g for 10 min at room temperature.
    2. Resuspend Synechocystis cells in 0.5 ml of 100 mM potassium phosphate buffer (pH 7.0) in a 2 ml tube.
    3. Add 1 g of glass beads (150-212 μm; Sigma-Aldrich) into the 2 ml tube containing cell suspensions and 100 mM Phenylmethanesulfonyl fluoride (PMSF) to reach a final concentration of 1 mM. Then, vortex the tube at the highest speed for 1 min. For each 10 sec vortexing, cool the tube on ice for 5 sec.
    4. After centrifugation at 12,000 x g, 4 °C for 30 min, transfer the supernatant into a new tube and store on ice before use.
      Note: The cell crude extracts should be used immediately.
  3. Detection of the in vitro invertase activity
    1. For 50 µl in vitro invertase reactions, mix potassium phosphate buffer (pH 7.0), sucrose and crude extracts together (The final concentrations of potassium phosphate and sucrose are 100 mM and 2 g/L respectively. Whereas, the protein concentrations of crude extracts are 2.5~4.0 g/L.) Use the reaction mixture without the cell crude extract as a negative control.
    2. Incubate the mixtures at 30 °C in a thermal cycler (T100, Bio-Rad, USA) for 1.5 h. Then, stop the reaction by three freeze-thaw cycles. In each freeze-thaw cycle, freeze the reaction mixtures in liquid nitrogen and next thaw by incubation in a 65 °C water bath. Measure the amount of the resulting glucose and fructose as well as the rest of the sucrose substrate using ion chromatography (Figure 1).
    3. Spin down the reaction mixtures at 12,000 x g for 10 sec. Then, subject 25 μl of the diluted mixtures to ICS-5000+ ion-exchange chromatography system equipped with an electrochemical detector and a DionexTM CarboPacTM PA10 analytical column (4 x 250 mm, ThermoFisher, Waltham, MA, USA). Elute the column with 200 mM NaOH at a flow rate of 1.0 ml/min.
      Note: The reaction mixtures should be diluted to at least 50 folds before analyzed by ion chromatography.
    4. Prepare a series of glucose standard solutions (0.01, 0.05, 0.1, 0.5, 1, 2 mg/L), establish the standard curve by using ion chromatography (see below).
  4. Quantification of glucose resulted from the sucrose degradation catalyzed by invertase
    1. Retention times of glucose and fructose using the method described above are 3.5 and 3.8 min respectively (Figure 1).
    2. Establish the glucose standard curve by using the peak area calculated by the Chromeleon software (Figure 2).


      Figure 1. Ion chromatography profiles of standards and the in vitro invertase reaction mixtures. A. Chromatogram of the glucose-fructose standard mixture. 100 mg/L of the glucose and fructose standards were mixed together and analyzed by ion chromatography. B. Chromatogram of the in vitro invertase reaction mixture without the cell crude extract (negative control). C. Chromatogram of the in vitro invertase reaction mixture. Glu, Glucose; Fru, Fructose; Suc, Sucrose.

    3. For quantification of glucose in reaction mixtures, calculate the areas of the target peak obtained by ion chromatography by the Chromeleon software, and then determine the glucose concentration using the standard curve of the glucose standards (Figure 2).


      Figure 2. The glucose standard curve

Data analysis

Calculate the in vitro invertase enzymatic activities according to equations:

Invertase activities [nmol glucose/min/mg protein] = Cglucose x D/MWglucose/RT/Cprotein

where, Cglucose, the glucose resulted from the invertase reactions, unit: nmol/L;
D, dilution ratio for the cell crude extract, namely 50 in this protocol;
MWgluose, molecular weight of glucose, namely 180;
RT, reaction time, namely 90 min in this protocol;
Cprotein, the protein concentration of the cell crude extract, unit: mg/L.

Recipes

  1. BG11 medium (Rippka et al., 1979)
    1. 8 kinds of 10x stocks were prepared as follows:
      Stock 1 (40 g/L K2HPO4·3H2O)
      Stock 2 (75 g/L MgSO4·7H2O)
      Stock 3 (36 g/L CaCl2·2H2O)
      Stock 4 (6 g/L Critic acid)
      Stock 5 (6 g/L Ferric ammonium citrate)
      Stock 6 (1 g/L EDTA·2Na·2H2O)
      Stock 7 (20 g/L Na2CO3)
      Stock A5 (2.86 g/L H3BO3, 1.81 g/L MnCl2·4H2O, 0.22 g/L ZnSO4·7H2O, 0.39 g/L NaMoO4·2H2O, 0.08 mg/L CuSO4·5H2O, 0.01 g/L CoCl2·6H2O)
      Autoclave Stocks 1 and 5 at 121 °C for 20 min. Store the autoclaved stocks and other stocks at 4 °C before use
    2. Dissovle 1.5 g NaNO3 in 992 ml of ddH2O, and add 1 ml of Stock 2, 3, 4, 6, 7 and A5 into the medium. Autoclave the medium at 121 °C for 20 min, and supplement with 1 ml of Stocks 1 and 5
  2. 100 mM potassium phosphate buffer (pH 7.0)
    1. Prepare 0.2 M K2HPO4 solution by dissolving 45.6 g K2HPO4∙3H2O in ddH2O and adjusting the volume to 1 L
    2. Prepare 0.2 M KH2PO4 solution by dissolving 27.2 g KH2PO4 in ddH2O and adjusting the volume to 1 L
    3. Prepare 100 mM potassium phosphate buffer (pH 7.0) by mixing 38 ml of 0.2 M KH2PO4 solution, 62 ml of 0.2 M KH2PO4 solution and 100 ml of ddH2O
  3. 100 mM PMSF
    Dissolve 0.174 g PMSF into 10 ml of isopropanol, and store at -20 °C before use

Acknowledgments

This work was supported by the National Science Fund for Distinguished Young Scholars of China (31525002 to X. Lu), Shandong Key basic Research project (ZR2017ZB0211), the Joint Sino-German Research Project (grant GZ 984 to X. Lu), the Shandong Taishan Scholarship (X. Lu), the National Science Foundation of China (31301018 to X. Tan), the Key Research Program of the Chinese Academy of Sciences (ZDRW-ZS-2016-3 to X. Tan) and Qingdao Innovative Leading Talent (15-10-3-15-(31)-zch).

References

  1. Curatti, L., Giarrocco, L. E., Cumino, A. C. and Salerno, G. L. (2008). Sucrose synthase is involved in the conversion of sucrose to polysaccharides in filamentous nitrogen-fixing cyanobacteria. Planta 228(4): 617-625.
  2. Du, W., Liang, F., Duan, Y., Tan, X. and Lu, X. (2013). Exploring the photosynthetic production capacity of sucrose by cyanobacteria. Metab Eng 19: 17-25.
  3. Kolman, M. A., Nishi, C. N., Perez-Cenci, M. and Salerno, G. L. (2015). Sucrose in cyanobacteria: from a salt-response molecule to play a key role in nitrogen fixation. Life (Basel) 5(1): 102-126.
  4. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111(1):1-61.
  5. Tan, X., Yao, L., Gao, Q., Wang, W., Qi, F. and Lu, X. (2011). Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria. Metab Eng 13(2): 169-176.
  6. Tan, X., Du, W. and Lu, X. (2015). Photosynthetic and extracellular production of glucosylglycerol by genetically engineered and gel-encapsulated cyanobacteria. Appl Microbiol Biotechnol 99(5): 2147-2154.
  7. Vargas, W., Cumino, A. and Salerno, G. L. (2003). Cyanobacterial alkaline/neutral invertases. Origin of sucrose hydrolysis in the plant cytosol? Planta 216(6): 951-960.
  8. Vargas, W. A. and Salerno, G. L. (2010). The Cinderella story of sucrose hydrolysis: Alkaline/neutral invertases, from cyanobacteria to unforeseen roles in plant cytosol and organelles. Plant Sci 178(1):1-8.

简介

转化酶可催化蔗糖的水解,广泛分布于蓝细菌和植物细胞中。 负责蔗糖代谢的第一步,转化酶起着重要的生理作用,其酶活性经常需要确定。 所有测定转化酶活性的方法都依赖于转化酶产生的葡萄糖产物的检测。 在这里我们描述了我们的实验室用于测定蓝细菌细胞内转化酶活性的基于离子色谱的方案。

【背景】转化酶和蔗糖在蓝细菌中发挥重要的生理作用(Curatti,et al。,2008; Kolman等人,2015)和高等植物(Vargas等人, ,2003; Vargas et。,2010)。转化酶(EC 3.2.1.26)可以催化蔗糖降解成葡萄糖和果糖。由于转化酶的这种特性,任何可用于测定葡萄糖或果糖的方法理论上都将用于转化酶酶活性测定。实际上,大多数转化酶酶活性测定基于检测产生的葡萄糖产物。

一些公司已经开发了用于转化酶活性测定的几种试剂盒,例如来自abcam(USA)的ab197005,来自Novus Biologicals(USA)的KA1629,来自Sigma-Aldrich(USA)的MAK118。通过使用这些试剂盒,由转化酶反应产生的葡萄糖产物将被氧化并通过比色(570nm)或荧光法(λem/ ex = 585 / 530nm)测定。在其他方法中,转化酶释放的还原糖的量通过将己糖激酶,磷酸葡萄糖异构酶和葡萄糖-6-磷酸脱氢酶偶联来测量,而所产生的NADPH在340nm处进一步通过分光光度法测定(Vargas等人,2003)。离子色谱可以直接检测各种糖,包括葡萄糖,果糖,蔗糖(Du等人,2013)和葡糖基甘油(Tan等人,2015)。与用于糖测定的基于分光光度计的方法相比,离子色谱法(IC)可以更有利于分析含有多种化合物的酶混合物,特别是用于细胞粗提取物的酶促测定。通过使用离子色谱法,蔗糖消耗量和葡萄糖产量可以在转化酶酶促测定的情况下同时显示,这将为酶测定提供完整的信息。

在这里,我们报告了我们最近基于IC的方案来测定蓝细菌细胞中的转化酶活性。

关键字:蓝藻, 集胞藻, 转化酶的酶活性, 蔗糖, 离子色谱法

材料和试剂

  1. 移液器吸头
  2. 2毫升试管(赛普拉斯,中国)
  3. 10毫升锥形管(康健,中国)
  4. 1毫升注射器(中国建始)

  5. 注射器膜过滤器,0.22微米(金腾,中国)
  6. Synechocystis sp。 PCC 6803(Tan等人,2011)
  7. Milli-Q水(德国Millipore)
  8. 玻璃珠(Sigma-Aldrich,目录号:G9018-250G)
  9. 果糖标准品(国药集团化学试剂,目录号:63003034)
  10. 葡萄糖标准品(国药集团化学试剂,目录号:10010518)
  11. 液氮
  12. 200mM NaOH(用Milli-Q水制备)
  13. 蔗糖(国药集团化学试剂,目录号:10021418)
  14. 硫酸镁七水合物(MgSO 4·7H 2 O)(国药集团化学试剂,目录号:10013018)
  15. 氯化钙二水合物(CaCl 2·2H 2 O)(国药集团化学试剂有限公司,产品目录号:20011160)
  16. 批号酸一水合物(C 6 H 8 O 7·H 2 O)(国药集团化学试剂,目录号:10007118)
  17. 柠檬酸铁铵((NH 4)3 FeCl 2·2H 2 O·14 O 14, / C 6 H 8 O 7·xFe 3·yNH 3)((1)国药集团化学试剂,目录号:30011428)
  18. EDTA·2Na·2H 2 O(国药集团化学试剂,目录号:10009717)
  19. 碳酸钠(Na 2 CO 3)(国药集团化学试剂,目录号:10019260)
  20. 硼酸(H 3 BO 3)(Sinopharm Chemical Reagent,目录号:10004818)
  21. 氯化锰(II)四水合物(MnCl 2·4H 2 O)(国药集团化学试剂,目录号:20026118)
  22. 硫酸锌七水合物(ZnSO 4·7H 2 O)(国药集团化学试剂,目录号:10024018)
  23. 钼酸钠二水合物(Na 2 MoO 4·2H 2 O)(国药集团化学试剂,目录号:10019818)
  24. 五水合硫酸铜(II)(CuSO 4·5H 2 O)(国药集团化学试剂,产品目录号:10008218)
  25. 氯化钴(II)六水合物(CoCl 2·6H 2 O)(国药集团化学试剂,产品目录号:10007216)
  26. 硝酸钠(NaNO 3)(国药集团化学试剂,目录号:10019918)
  27. 磷酸二氢钾(KH 2 PO 4)(国药集团化学试剂,目录号:10017618)
  28. 磷酸氢二钾三水合物(KH 2 HPO 4·3H 2 O)(国药集团化学试剂,目录号:10017518)
  29. 苯基甲磺酰氟(Sigma-Aldrich,目录号:P7626)
  30. 异丙醇(国药集团化学试剂,目录号:40064360)
  31. BG11中(见食谱)
  32. 100 mM磷酸钾缓冲液(pH 7.0)(见食谱)
  33. 100毫米PMSF(见食谱)

设备


  1. 50毫升烧瓶
  2. 移液器(Eppendorf,德国)
  3. 摇床(太仓华美,型号:THZ-701B)
  4. 水浴(上海亚龙,型号:B-260)
  5. 离心机(Beckman Coulter,型号:Microfuge®22R)
  6. -20°C冰箱(海尔,型号:BCD-219D)
  7. Vortex-Genie 2(Scientific Industries,型号:Vortex-Genie 2)
  8. 用于PCR的热循环仪(Bio-Rad Laboratories,型号:T-100)
  9. 离子色谱法(Thermo Fisher Scientific,Thermo Scientific TM,型号:Dionex TM ICS-5000 +)
  10. Dionex TM CarboPac TM PA10分析柱(4×250mm,Thermo Fisher Scientific,型号:Dionex TM TM CarboPac TM TM > PA10)

软件

  1. Chromeleon TM软件(Thermo Fisher Scientific,Thermo Scientific TM TM 6.80;产品目录号:CHROMELEON6)

程序

  1. Synechocystis sp。的培养PCC 6803(Tan等人,2011)
    注意:通过用分光光度计测量730nm处的光密度(OD)来监测蓝细菌细胞的生长。在50ml烧瓶中培养体积应小于30ml。
    1. 在20ml液体BG-11培养基(参见配方1)中于50ml烧瓶中培养集胞藻(Synechocystis)细胞,其初始OD 730值为0.05。
    2. 在30℃摇动(150rpm)和恒定的白光照度(50μE/ m 2 /秒)培养集胞藻培养物约4天达到指数成长阶段。
  2. 从集胞蓝细胞制备细胞粗提物
    1. 通过在室温下以8,000×gg离心10分钟收集8ml的集胞蓝细菌的对数期培养物(OD 730≤2)。
    2. 在2ml管中的0.5ml 100mM磷酸钾缓冲液(pH7.0)中重悬集胞藻细胞。
    3. 加入1克玻璃珠(150-212微米;西格玛奥德里奇公司)到2毫升含有细胞悬液和100毫米苯甲磺酰氟(PMSF)的试管中,使终浓度达到1毫米。然后,以最高速度涡旋管1分钟。每10秒涡旋一次,将试管在冰上冷却5秒。
    4. 在12,000×gg,4℃离心30分钟后,将上清液转移到新管中并在使用前储存在冰上。
      注意:应立即使用细胞粗提取物。
  3. 检测体外转化酶活性
    1. 对于50μl体外转化酶反应,将磷酸钾缓冲液(pH7.0),蔗糖和粗提物混合在一起(磷酸钾和蔗糖的最终浓度分别为100mM和2g / L,粗提物的蛋白质浓度为2.5〜4.0 g / L。)使用不含细胞粗提物的反应混合物作为阴性对照。
    2. 在热循环仪(T100,Bio-Rad,USA)中于30℃孵育混合物1.5小时。然后,停止反应三次冻融循环。在每个冻融循环中,将反应混合物在液氮中冷冻,然后通过在65℃水浴中孵育解冻。使用离子色谱法测量所得葡萄糖和果糖以及其余蔗糖底物的量(图1)。
    3. 将反应混合物在12,000×gg下旋转10秒。然后,将25μl稀释的混合物置于配备有电化学检测器和Dionex TM CarboPac TM的ICS-5000 +离子交换色谱系统中, > PA10分析柱(4×250mm,ThermoFisher,Waltham,MA,USA)。
      用200 mM NaOH以1.0 ml / min的流速洗脱色谱柱 注意:反应混合物应稀释至少50倍,然后用离子色谱分析。
    4. 准备一系列葡萄糖标准溶液(0.01,0.05,0.1,0.5,1,2 mg / L),使用离子色谱法建立标准曲线(见下文)。
  4. 定量葡萄糖由蔗糖降解转化酶催化

    1. 使用上述方法保留葡萄糖和果糖的时间分别为3.5和3.8分钟(图1)

    2. 使用Chromeleon软件计算的峰面积建立葡萄糖标准曲线(图2)。


      图1.标准品和体外转化酶反应混合物的离子色谱图。A.葡萄糖 - 果糖标准混合物的色谱图。将100mg / L的葡萄糖和果糖标准混合在一起并通过离子色谱分析。 B.不含细胞粗提物的体外转化酶反应混合物的色谱图(阴性对照)。 C.体外转化酶反应混合物的色谱图。 Glu,葡萄糖; Fru,果糖; Suc,蔗糖。

    3. 为了定量反应混合物中的葡萄糖,计算Chromeleon软件通过离子色谱法得到的目标峰面积,然后使用葡萄糖标准品的标准曲线确定葡萄糖浓度(图2)。


      图2.葡萄糖标准曲线

数据分析

根据等式计算体外转化酶活性:

转化酶活性[nmol葡萄糖/分钟/ mg蛋白质] = C葡萄糖×D / MW葡萄糖/ RT / C蛋白质

其中C葡萄糖,由转化酶反应产生的葡萄糖,单位:nmol / L;
D,细胞粗提物的稀释比例,即在该方案中为50;
MW gluose ,葡萄糖的分子量,即180;
RT,反应时间,即本协议中的90分钟;
蛋白质浓度,细胞粗提物的蛋白质浓度,单位:毫克/升。

食谱

  1. BG11培养基(Rippka等人,1979)
    1. 如下准备了8种10x股票:
      库存1(40克/升K 2 HPO 4·3H 2 O)
      库存2(75g / L MgSO 4·7H 2 O)
      库存3(36克/升CaCl 2·2H 2 O)
      库存4(6克/升批发酸)
      库存5(6克/升柠檬酸铁铵)
      储备液6(1g / L EDTA·2Na·2H2O)
      库存7(20g / L Na 2 CO 3)
      库存A5(2.86g / LH 3 BO 3,1.81g / L MnCl 2·4H 2 O, 0.22g / L ZnSO 4·7H 2 O,0.39g / L NaMoO 4·2H 2 O, 0.08mg / L CuSO 4·5H 2 O,0.01g / L CoCl 2·6H 2 O)
      高压灭菌器1和5在121°C下保持20分钟。

      使用前将储存在4℃的高压灭菌的股票和其他股票存储
    2. 在992ml ddH 2 O中溶解1.5g NaNO 3,向培养基中加入1ml储备液2,3,4,6,7和A5。将培养基在121°C高压灭菌20分钟,并补充1毫升股票1和5
  2. 100mM磷酸钾缓冲液(pH7.0)
    1. 通过将45.6g K 2 HPO 4·3H 2 O溶解于水中,制备0.2MK 2 HPO 4溶液。在ddH <2> O中调整音量并将音量调整为1L
    2. 通过在ddH 2中溶解27.2g KH 2 PO 4来制备0.2M KH 2 PO 4溶液将音量调节至1L
    3. 通过混合38ml的0.2M KH 2 PO 4溶液,62ml的0.2M KH 2 O 2溶液来制备100mM磷酸钾缓冲液(pH 7.0) > PO 4溶液和100ml ddH 2 O
  3. 100毫摩尔PMSF
    将0.174克PMSF溶解于10毫升异丙醇中,并在使用前储存于-20°C

致谢

这项工作得到了中国杰出青年科学基金(31525002至X.鲁),山东省重点基础研究项目(ZR2017ZB0211),中德联合研究项目(授予GZ 984至X. Lu),中国科学院国家科学基金(31301018),中国科学院重点研究计划项目(ZDRW-ZS-2016-3至X.Tan),青岛创新领先企业山东泰山奖学金天赋(15-10-3-15-(31)-zch)。

参考

  1. Curatti,L.,Giarrocco,L.E。,Cumino,A.C。和Salerno,G.L。(2008)。 蔗糖合成酶参与蔗糖转化为丝状固氮蓝藻中的多糖。 Planta 228(4):617-625。
  2. Du,W.,Liang,F.,Duan,Y.,Tan,X.和Lu,X.(2013)。 探索蓝藻对蔗糖的光合生产能力 Metab Eng 19:17-25。
  3. Kolman,M.A.,Nishi,C.N.,Perez-Cenci,M.and Salerno,G.L。(2015)。 蓝藻中的蔗糖:来自盐分应答分子,在氮固定中发挥关键作用。 a> Life(Basel) 5(1):102-126。
  4. Rippka,R.,Deruelles,J.,Waterbury,J.B.,Herdman,M.,Stanier,R.Y。(1979)。 蓝藻纯培养物的一般分配,菌株历史和特性。
  5. Tan,X.,Yao,L.,Gao,Q.,Wang,W.,Qi,F。和Lu,X.(2011)。 光合作用驱动的二氧化碳转化为蓝细菌中的脂肪醇和碳氢化合物 Metab Eng 13(2):169-176。
  6. Tan,X.,Du,W.和Lu,X.(2015)。 通过基因工程和凝胶包裹的蓝细菌产生葡萄糖基甘油的光合和细胞外生产 Appl Microbiol Biotechnol 99(5):2147-2154。
  7. Vargas,W.,Cumino,A.和Salerno,G.L。(2003)。 蓝藻碱性/中性转化酶。植物细胞质中蔗糖水解的起源?植物细胞216(6):951-960。
  8. Vargas,W.A。和Salerno,G.L.(2010)。 灰姑娘的蔗糖水解故事:碱性/中性转化酶,从蓝藻到植物细胞质中不可预见的作用和细胞器。 植物科学 178(1):1-8。
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引用:Tan, X., Song, K. and Lu, X. (2018). Enzymatic Activity Assay for Invertase in Synechocystis Cells. Bio-protocol 8(10): e2856. DOI: 10.21769/BioProtoc.2856.
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