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Measurement of the Galactanase Activity of the GanB Galactanase Protein from Bacillus subtilis
枯草芽孢杆菌中GanB半乳聚糖酶活性的测定   

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Abstract

The activity of the endo-β-1,4-galactanase GanB from B. subtilis on the high molecular weight β-1,4-galactan was determined quantitatively by the measurement of the increase of the reducing power or with the dyed substrate Azo-galactan. The generated degradation products were analyzed using thinlayer-chromatography (TLC) or high-performance anion-exchange chromatography (HPAEC).

Keywords: Galactanase-assay(半乳聚糖酶分析), Endo-β-galactanase(内切β-半乳聚糖酶), Galactan(半乳聚糖), AZCL-galactan(AZCL-半乳聚糖), GanB from Bacillus subtilis(来自枯草芽孢杆菌的GanB), Galacto-oligosaccharides(半乳寡聚糖)

Background

Bacillus subtilis possesses comprehensive systems for the utilization of plant cell wall polysaccharides including the gene cluster ganSPQAB which encodes galactan utilization elements (Watzlawick et al., 2016). Galactans are high molecular weight galacto-saccharides and are found as side chains of rhamnogalacturonan type I in pectin. Its degradation is carried out by endo-beta1,4 galactanases (EC 3.2.1.89). The utilization of galactan by B. subtilis involves the extracellular galactanase GanB, cleaving the high molecular galactan inside the chain and resulting short oligomers that enter the cell wall to get there further degraded. The ganB gene from B. subtilis was cloned and expressed in E.coli (Watzlawick et al., 2016) and the enzymatic properties of the purified His-tagged mature protein were characterized by galactanase assays.

Materials and Reagents

  1. Eppendorf tubes 1.5 ml
  2. TLC-plate: Silica Gel 60 F254 (Merck Millipore, Darmstadt, Germany)
  3. Sodium acetate trihydrate (Carl Roth, catalog number: 6779 )
  4. Galactan from lupin (Arabinofuranosidase treated lupin pectic galactan, Gal:Ara:Rha:Xyl: GalUA = 91:2:1.8:0.2:5.0) (Megazyme, catalog number: P-GALLU )
  5. β-1,4-galactobiose (Sigma-Aldrich, catalog number: G9662 )
  6. D-(+)-galactose (Sigma-Aldrich, catalog number: G5388 )
  7. 3,5-dinitrosalicylic acid (Sigma-Aldrich, catalog number: D0550 )
  8. Potassium-sodium-tartrate tetrahydrate (Carl Roth, catalog number: 8087 )
  9. Sodium sulfite (Carl Roth, catalog number: P033 )
  10. Sodium hydroxide 50% (Carl Roth, catalog number: 8655 )
  11. AZCL-galactan: Azurine-crosslinked-potato (AZCL) galactan (which has been purified from potato fibre and treated to remove most of the arabino- furanosyl residues) (Sigma-Aldrich, catalog number: 38127 )
    Note: This product has been discontinued.
  12. Ammonium heptamolybdate tetrahydrate (Carl Roth, catalog number: 7311 )
  13. Cerium sulfate tetrahydrate (Carl Roth, catalog number: P014 )
  14. Sulfuric acid (H2SO4)
  15. Potassium-phosphate dibasic (KH2PO4) (Carl Roth, catalog number: P018 )
  16. di-Potassium hydrogen phosphate (K2HPO4) (Carl Roth, catalog number: P749 )
    Note: Prepare a 0.1 M solution in pure H2O and use it for preparation of the potassium phosphate buffer described in Recipes section.
  17. Sodium borate decahydrate (Sigma-Aldrich, catalog number: G5388 )
  18. Sulfuric acid 95% (Carl Roth, catalog number: HN62 )
  19. Acetone (Carl Roth, catalog number: 9372 )
  20. Milli-Q pure H2O
  21. Purified recombinant His6-tagged GanB (see Recipes)
    Note: This protein was produced in E. coli JM109 harboring the plasmid pHWG1119 after induction with rhamnose as described by (Watzlawick et al., 2016). Crude extracts were purified by immobilized-metal affinity chromatography using TALON® Metal Affinity resins (Clontech Laboratories, USA) according to the supplier’s instruction. Fractions of the purified proteins were combined and the imidazole was removed with NAP10 columns (GE Healthcare, Munich, Germany) equilibrated with 0.1 M KPP.
  22. Lupin-galactan (see Recipes)
  23. DNS-reagent (see Recipes)
  24. AZCL-galactan (see Recipes)
  25. TLC developing reagent (see Recipes)
  26. Potassium phosphate buffer (KPP), 0.1 M (see Recipes)
  27. 0.4 M sodium borate solution (see Recipes)
  28. 0.3 M sodium acetate, pH 5.2 (see Recipes)
  29. TLC mobile phase solution (see Recipes)
  30. HPAEC isocratic mobile phase (see Recipes)

Equipment

  1. Pipette P2 , P20 , P200 , P1000 (Gilson Pipetman classic)
  2. Vortex Minishaker (Heidolph Instrument, model: Heidolph Reax 2000 )
  3. Centrifuge (Eppendorf, model: 5415 D )
  4. Thermomixer compact (Eppendorf)
  5. Blow-dryer
  6. HPLC apparatus: The HPLC apparatus consists of a pump (220, Bischoff, Leonberg, Germany) and an ESA Coulochem II electrochemical detector (Bischoff, Leonberg, Germany) with an analytical cell (5040, ESA Coulochem II, Bischoff)
  7. HPLC-column (Thermo Fisher Scientific, model: DionexTM CarboPacTM PA1 )
  8. TLC-chamber (size: 12 x 12 x 7 cm) (Desaga, Heidelberg, Germany)
  9. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: GENESYSTM 10 Vis )
  10. pH meter (Metteler Toledo, model: FE20 ATC FiveEasyTM )

Procedure

In order to measure the galactanase activity of His6-GanB two different assays were applied: one using the natural substrate galactan is described in Procedure A and another using a dyed galactan substrate is described in Procedure B. The reaction of the galactanase enzyme on galactan for producing galacto-oligosaccharide is described in Procedure C. Galacto-oligosaccharides generated after the degradation of galactan were identified by two different chromatographic methods. First Thinlayer-chromatography (TLC) was used to identify the products qualitatively and is described in Procedure D. Quantitative analysis of individual galacto-oligosaccharides was performed using high-performance anion-exchange chromatography (HPAEC) as described in Procedure E.

  1. Galactanase activity determination by measurement of the increase of the reducing power
    In order to calculate endo-β-galactanase activity in units, the reduced sugars released from the lupin galactan degradation were determined by an adapted 3,5-dinitrosalicylic acid (DNS) assay (Miller, 1959).
    1. Mix appropriate amounts of His6-GanB (maximal 0.5 µg) in 40 µl of 0.1 M potassium phosphate buffer (KPP) pH 6.5 with 40 µl of 2% lupin galactan in an Eppendorf tube.
    2. Incubate the Eppendorf tube in a thermomixer at 37 °C for 15 min with agitation at 300 rpm.
    3. Stop the reaction by adding of 300 µl of DNS-reagent.
    4. Heat the Eppendorf tube for 20 min at 95 °C.
    5. Add 1 ml H2O to the reaction mixture and keep the Eppendorf tube on ice for 20 min.
    6. Measure the absorbance of the developed orange color at 540 nm (see Figure1)
      As the blank the reaction mixture ingredients without enzyme is used.
      For unit calculation, the absorption of 1 µmol galactose is determined from a calibration curve applying 1 to 10 mM galactose in the reaction mixture of the assay (1 µmol galactose is equivalent to 0.034 A540 units). One unit of galactanase enzyme was defined as the release of 1 µmol of reducing sugar, here galactose, per minute determined by the DNS-assay. Calculate units per milliliter from the formula (see also Data analysis):




      Figure 1. DNS-assay for determination of the reducing power from the reaction of His6-GanB with galactan: Color development of different amounts of His6-GanB after step A5

  2. Colorimetric assay: Activity on the dyed substrate AZCL-galactan (Azurine-crosslinked-potato galactan) by measurement of the water soluble dyed fragments after degradation of the insoluble AZCL-galactan.
    1. Delude appropriate amounts of His6-GanB (e.g., 2 µl of 0.6 mg/ml; 2 µl of 0.12 mg/ml; 2 µl of 0.06 mg/ml) in 200 µl of 0.1 M KPP pH 6.5 and mix with 50 µl of a well mixed suspension of 1% AZCL-galactan in an Eppendorf tube.
    2. Incubate the Eppendorf tube in a thermomixer at 37 °C for 15 min with agitation at 300 rpm.
    3. Stop the reaction by adding of 250 µl of 0.4 M sodium borate.
    4. Centrifuge at 13,000 x g for 5 min.
    5. Take 100 µl of the blue colored supernatant and dilute with 900 µl of H2O.
    6. Measure the absorbance at 595 nm (for 1.2 µg His6-GanB an OD595 of 0.43 was determined).
      As the blank the reaction mixture ingredients without enzyme was used.
      Figure 2 shows the development of blue color in step B3 (before centrifugation) from the degradation of the unsoluble AZCL-galactan suspension with different amounts of GanB-enzyme. Tube 1 represents the blank.


      Figure 2. AZCL-galactan assay: Development of the blue color of different amounts of His6-GanB after step B3
      Note: The dyed substrate AZCL-galactan is used for semi-quantitatively activity determination as molar extinction coefficient ε595 of the blue dye was unknown.

  3. Enzymatic reaction for analysis of the generated galacto-oligosaccharides
    1. Mix appropriate amounts of His6-GanB (e.g., 1 µg) in 40 µl 0.1 M KPP pH 6.5 with 40 µl of 2% lupin-galactan in an Eppendorf tube and incubate the reaction at 37 °C with agitation at 300 rpm.
    2. Withdraw 10 µl of the reaction mixture at different times of reaction and put it in a new Eppendorf tube
    3. Stop the reaction by heating for 5 min at 95 °C.
    4. Centrifuge at 13,000 x g for 5 min.
    5. Use aliquots of the heat-denatured enzymatic reaction for analyzing the products by TLC (Procedure D step 1) or HPLC (Procedure E step 1).

  4. TLC-analysis
    1. Spot 4 µl of the heat-denatured enzymatic reaction, withdrawn at different reaction times from Procedure C, on a TLC-plate (Silica Gel 60 F254).
    2. As a control spot 2 µl of a solution of galactose (10 mg/ml) and 2 µl of 1% lupin-galactan on the plate.
    3. Separate the products with the mobile phase acetone-H2O (87:13) in a thin-layer-chamber till the migration front reach the top.
    4. Dry the TLC-plate with a blow-dryer approximately 5 min.
    5. Soak the TLC-plate in the developing reagent.
    6. Back the plates in an oven at 80 °C till blue spots are visible.
    7. Figure 3 shows the TLC-chromatogram after separation and visualization of the reaction products generated from lupin galactan degradation of GanB after different reaction times.


      Figure 3. TLC-Chromatogram of the degradation products of lupin-galactan by His6-GanB (60 ng) withdrawn at reaction times of 30 min, 1, 2, 4 and 8 h from Procedure C. The reaction products were separated with the mobile phase acetone-H2O (87:13) and visualized by the developing reagent.

  5. HPAEC-analysis
    1. Dilute samples of the enzymatic reaction from Procedure C 1:10 with H2O.
    2. Load 10 μl on the CarboPac PA1 column by injection in the HPLC apparatus and separate the products with an isocratic mobile phase of 0.22 N NaOH/0.02 M sodium acetate on a HPLC-System at a flow rate of 0.75 ml/min.
    3. Detect the eluted carbohydrate by pulsed amperometry with an analytical cell (5040, ESA Coulochem II, Bischoff).
    4. Individual sugars like galactose and β-1,4 galactobiose were identified by comparing retention times with those of standards.
    5. A typical HPLC-profile is given in Figure 4.


      Figure 4. HPLC-chromatogram of the degradation products of lupin-galactan by purified His6-GanB of the reaction of 1 µg His6-GanB in 40 µl 0.1 M KPP pH 6.5 and 40 µl 2% Galactan after incubation at 37 °C for 30 min. The identity of the individual sugars were galactose (Gal); β1,4-galactobiose (Gal2), Galactotriose (Gal3) and Galactotetraose (Gal4).

    Note: Galactanase activity in U/ml could be only calculated from Procedure A. Procedure B is used for semi-quantitative activity determination. The chromatograms of Procedure D is only for a qualitative view of the products and with Procedure E the produced galacto-oligosaccharide were separated but due to the lack of standards of Gal3 and Gal4 it was not quantified.

Data analysis

  1. During the enzymatic assays each value was determined in triplicates.
  2. The reducing power during the galactan degradation by GanB described in Procedure A was determined using the DNS assay and quantified according to a galactose standard curve given in Figure 5. 1 U of galactanase was defined as the release of 1 µmol galactose per minute and is calculated from the equation:




    Figure 5. Standard curve of galactose determined by DNS-assay. The A540 nm values were obtained by mixing of 80 µl of a solution of 5 to 20 mM galactose with 300 µl DNS-reagent and proceeded according to Procedure A step 4.

Recipes

  1. Purified recombinant His6-tagged GanB
    0.6 mg/ml in 0.1 M potassium phosphate buffer pH 6.5
    Note: The protein quantity was determined by the method of Bradford (Bradford, 1976) using bovine serum albumin as standard.
  2. Lupin-galactan
    Dissolve 2% lupin-galactan in Milli-Q pure H2O
  3. DNS-reagent
    Mix 1.76 g dinitrosalicylic acid, 50 g potassium-sodium-tartrate, 1.26 g sodium sulfite, 6 ml 50% NaOH with 183 ml Milli-Q pure H2O
  4. AZCL-galactan
    Make a suspension of 1% AZCL-galactan in Milli-Q pure H2O and mix thoroughly before using
  5. TLC developing reagent
    Mix 10.5 g ammonium heptamolybtate, 0.5 g cerium sulfate, 15 ml concentrated H2SO4 in 245 ml Milli-Q pure H2O
  6. Potassium phosphate buffer (KPP), 0.1 M
    Mix the solutions of 0.1 M KH2PO4 and 0.1 M K2HPO4 till the pH reached 6.5
  7. 0.4 M sodium borate solution
    Dissolve 152 g sodium borate in 1 L Milli-Q pure H2O and adjust the pH to 9.8
  8. 0.3 M sodium acetate, pH 5.2
    Dissolve 40.8 g sodium acetate in 1 L Milli-Q pure H2O and adjust the pH to 5.2 with acetic acid
  9. TLC mobile phase solution
    Mix 87 ml acetone with 13 ml Milli-Q pure H2O
  10. HPAEC isocratic mobile phase
    Mix 11 ml 50% sodium hydroxide, 63.7 ml 0.3 M sodium acetate pH 5.2 with 890.3 ml Milli-Q pure H2O

Acknowledgments

This protocol was first used in Watzlawick et al. (2016).

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3): 426-428.
  3. Watzlawick, H., Morabbi Heravi, K. and Altenbuchner, J. (2016). Role of the ganSPQAB operon in degradation of galactan by Bacillus subtilis. J Bacteriol 198(20): 2887-2896.

简介

来自B的内切-β-1,4-半乳糖苷酶GanB的活性。 通过测量还原力的增加或染色底物偶氮半乳聚糖来定量测定高分子量β-1,4-半乳聚糖上的枯草芽孢杆菌。 使用薄层色谱(TLC)或高效阴离子交换层析(HPAEC)分析产生的降解产物。

枯草芽孢杆菌具有用于植物细胞壁多糖的综合系统,包括编码半乳聚糖利用元素的基因簇ganSPQAB(Watzlawick等人,2016)。 半乳聚糖是高分子量半乳糖,并且在果胶中被认为是I型鼠李糖半乳糖醛酸的侧链。 其降解通过内切β1,4半乳聚糖酶(EC 3.2.1.89)进行。 半乳聚糖的利用。 枯草芽孢杆菌涉及细胞外半乳聚糖酶GanB,裂解链内的高分子半乳聚糖,并产生进入细胞壁的短的低聚物进一步降解。 来自的 ganB 基因。 枯草芽孢杆菌在大肠杆菌(Watzlawick等人,2016)中克隆并表达,纯化的His标记的成熟蛋白的酶学性质由 半乳聚糖酶测定。

关键字:半乳聚糖酶分析, 内切β-半乳聚糖酶, 半乳聚糖, AZCL-半乳聚糖, 来自枯草芽孢杆菌的GanB, 半乳寡聚糖

材料和试剂

  1. Eppendorf管1.5 ml
  2. TLC板:Silca Gel 60F 254(Merck Millipore,Darmstadt,Germany)
  3. 醋酸钠三水合物(Carl Roth,目录号:6779)
  4. 来自羽扇豆的半乳聚糖(Arabinofuranosidase treated lupin pectic galactan,Gal:Ara:Rha:Xyl:GalUA = 91:2:1.8:0.2:5.0)(Megazyme,目录号:P-GALLU)
  5. β-1,4-半乳糖苷(Sigma-Aldrich,目录号:G9662)
  6. D - (+) - 半乳糖(Sigma-Aldrich,目录号:G5388)
  7. 3,5-二硝基水杨酸(Sigma-Aldrich,目录号:D0550)
  8. 四水合钾盐(Carl Roth,目录号:8087)
  9. 亚硫酸钠(Carl Roth,目录号:P033)
  10. 氢氧化钠50%(Carl Roth,目录号:8655)
  11. AZCL-半乳聚糖:Azurine交联马铃薯(AZCL)半乳聚糖(已从马铃薯纤维中纯化并处理以除去大部分阿拉伯呋喃糖残基)(Sigma-Aldrich,目录号:38127)
    注意:本产品已停产。
  12. 七钼酸铵四水合物(Carl Roth,目录号:7311)
  13. 四水合硫酸铈(Carl Roth,目录号:P014)
  14. 硫酸(H 2 SO 3 SO 4)
  15. 磷酸氢二钾(KH 2 PO 4)(Carl Roth,目录号:P018)
  16. 磷酸氢二钾(K 2 HPO 4)(Carl Roth,目录号:P749)
    注意:在纯H O中制备0.1 M溶液,并用于制备食谱部分中描述的磷酸钾缓冲液
  17. 硼酸钠十水合物(Sigma-Aldrich,目录号:G5388)
  18. 硫酸95%(Carl Roth,目录号:HN62)
  19. 丙酮(Carl Roth,目录号:9372)
  20. Milli-Q纯H 2 O O
  21. 纯化的重组His <6>标签GanB(参见食谱)
    注意:如(Watzlawick等人,2016)所述,在用鼠李糖诱导后,在携带质粒pHWG1119的大肠杆菌JM109中产生该蛋白质。根据供应商的说明书,使用TALON金属亲和树脂(Clontech Laboratories,USA)通过固定化金属亲和层析纯化粗提取物。将纯化的蛋白质的级分合并,用用0.1M KPP平衡的NAP10柱(GE Healthcare,Munich,Germany)除去咪唑。
  22. 羽扇豆半乳糖(见食谱)
  23. DNS试剂(参见食谱)
  24. AZCL-半乳聚糖(参见食谱)
  25. TLC显影剂(参见食谱)
  26. 磷酸钾缓冲液(KPP),0.1M(参见食谱)
  27. 0.4 M硼酸钠溶液(见配方)
  28. 0.3 M醋酸钠,pH 5.2(参见食谱)
  29. TLC流动相溶液(参见食谱)
  30. HPAEC等静止流动相(参见食谱)

设备

  1. 移液器P2,P20,P200,P1000(Gilson Pipetman经典)
  2. 涡流迷你机(Heidolph Instrument,型号:Heidolph Reax 2000)
  3. 离心机(Eppendorf,型号:5415 D)
  4. Thermomixer compact(Eppendorf)
  5. 吹风机
  6. HPLC装置:HPLC装置由分析电池(5040,ESA Coulochem II,Bischoff)的泵(220,Bischoff,Leonberg,Germany)和ESA Coulochem II电化学检测器(Bischoff,Leonberg,Germany)组成。
  7. HPLC柱(Thermo Fisher Scientific,型号:Dionex CarboPac TM PA1)
  8. TLC室(尺寸:12×12×7厘米)(德沙加,海德堡,德国)
  9. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:GENESYSTM 10 Vis)
  10. pH计(Metteler Toledo,型号:FE20 ATC FiveEasy TM

程序

为了测量His 6 -GanB的半乳聚糖酶活性,应用了两种不同的测定方法:在方法A中描述了一种使用天然底物半乳聚糖,另一种使用染色的半乳聚糖底物在方法B中描述。半乳聚糖酶在半乳聚糖上产生半乳寡聚糖的反应在方法C中描述。半乳聚糖降解后产生的半乳寡糖通过两种不同的色谱方法鉴定。使用第一薄层色谱(TLC)来定性鉴定产物,并在方法D中描述。使用方法E中所述的高效阴离子交换层析(HPAEC)进行个体半乳寡糖的定量分析。

  1. 半乳聚糖酶活性测定通过测量降低功率的增加 为了以单位计算内切β-半乳糖苷酶活性,通过适应的3,5-二硝基水杨酸(DNS)测定(Miller,1959)测定从羽扇豆半乳聚糖降解释放的还原糖。
    1. 在40μl0.1M磷酸钾缓冲液(KPP)pH6.5中将适量的His 6 -GanB(最大0.5μg)与40μl2%羽扇豆半乳聚糖在Eppendorf管中混合。
    2. 在300rpm的搅拌下,将温热混合器中的Eppendorf管在37℃下孵育15分钟。
    3. 加入300μlDNS试剂停止反应。
    4. 在95°C下将Eppendorf管加热20分钟。
    5. 向反应混合物中加入1ml H 2 O,并将Eppendorf管保持在冰上20分钟。
    6. 测量540nm处开发的橙色的吸光度(见图1)
      作为空白,使用不含酶的反应混合物成分。
      对于单位计算,在测定的反应混合物(1μmol半乳糖相当于0.034A 540单位)中,从施用1至10mM半乳糖的校准曲线确定1μmol半乳糖的吸收。一个单位的半乳聚糖酶被定义为通过DNS测定法测定的每分钟1μmol还原糖(这里是半乳糖)的释放。从公式计算每毫升单位(另见数据分析):




      图1.用于测定His6-GanB与半乳聚糖的反应的还原能力的DNS测定:步骤A5后不同量的His6-GanB的显色发展
      />
  2. 比色测定:通过在不溶性AZCL-半乳聚糖降解后测量水溶性染色片段,在染色底物AZCL-半乳聚糖(Azurine交联的马铃薯半乳聚糖)上的活性。
    1. 将适量的His -GanB(例如,2μl0.6mg/ml;2μl0.12mg/ml;2μl0.06mg/ml)在200μl0.1M KPP pH6.5中,并与50μl1%AZCL-半乳聚糖的充分混合的悬浮液在Eppendorf管中混合。
    2. 在300rpm的搅拌下,将温热混合器中的Eppendorf管在37℃下孵育15分钟。
    3. 加入250μl0.4M硼酸钠停止反应
    4. 以13,000 x g离心5分钟。
    5. 取100μl蓝色上清液,用900μlH 2 O稀释。
    6. 测量595nm处的吸光度(对于1.2μgHis 6 -GanB,测定0.43的OD 595 )。
      作为空白,使用没有酶的反应混合物成分。
      图2显示了在不同量的GanB酶的不溶性AZCL-半乳聚糖悬浮液的降解中,步骤B3(离心前)蓝色的发展。管1代表空白。


      图2. AZCL-半乳聚糖测定:在步骤B3之后开发不同量的His6-GanB的蓝色
      注意:染色底物AZCL-半乳聚糖用于半定量活性测定,因为蓝色染料的摩尔消光系数ε595是未知的。

  3. 用于分析产生的低聚半乳糖的酶反应
    1. 将适量的His 6 -GanB(例如,1μg)在40μl0.1M KPP pH6.5中与40μl2%羽扇豆白蛋白在Eppendorf管中混合并在37℃下以300rpm的搅拌温育该反应。
    2. 在不同的反应时间取出10μl反应混合物,并将其放入新的Eppendorf管中
    3. 在95℃加热5分钟停止反应。
    4. 以13,000 x g离心5分钟。
    5. 使用热变性酶反应的等分试样通过TLC分析产物(方法D步骤1)或HPLC(方法E步骤1)。

  4. TLC分析
    1. 点样4μl热变性酶反应,在不同的反应时间从方法C,在TLC板(硅胶60 F254)上取出。
    2. 作为对照点,将2μl半乳糖(10mg/ml)和2μl1%羽扇豆半乳聚糖的溶液置于板上。
    3. 将产品与流动相丙酮-H 2 O(87:13)在薄层室中分离,直到迁移前端到达顶部。
    4. 用吹风机干燥TLC板大约5分钟。
    5. 将TLC板浸泡在显影试剂中
    6. 将板材放在80°C的烘箱中,直到看到蓝色斑点。
    7. 图3显示了在不同反应时间后甘蓝半乳聚糖降解产生的反应产物的分离和可视化后的TLC-色谱图。


      图3.通过His 6 -GanB(60ng)在30分钟,1,2,4和8小时的反应时间取出的羽扇豆半乳聚糖的降解产物的TLC-色谱图方法C。将反应产物用流动相丙酮-H 2 O(87:13)分离,并用显影试剂显色。

  5. HPAEC分析
    1. 用H 2 O从步骤C 1:10稀释酶反应样品。
    2. 在HPLC装置中注入CarboPac PA1柱上10μl,并以0.75ml/min的流速将产物与0.22N NaOH/0.02M乙酸钠的等度流动相在HPLC系统上分离。 >
    3. 通过脉冲电流分析法(5040,ESA Coulochem II,Bischoff)检测洗脱的碳水化合物。
    4. 通过将保留时间与标准物质的保留时间进行比较,确定了半乳糖和β-1,4半乳糖二糖等单糖。
    5. 典型的HPLC-profile在图4中给出

      图4.通过纯化的His 6 -GanB的1μgHis 6 -GanB在40μl的反应中的羽扇豆半乳聚糖的降解产物的HPLC-色谱图0.1M KPP pH6.5和40μl2%半乳聚糖,37℃温育30分钟。单个糖的身份是半乳糖(Gal); β1,4-半乳糖二糖(Gal2),半乳糖三糖(Gal3)和半乳糖四糖(Gal4)。

    注意:U/ml中的半乳聚糖酶活性可以仅从方法A计算。程序B用于半定量活性测定。方法D的色谱图仅用于产品的定性观察,并且在方法E中分离所产生的低聚半乳糖,但是由于缺少Gal 3和Gal 4的标准物,未被量化。

数据分析

  1. 在酶测试期间,每个值一式三份确定。
  2. 在方法A中描述的GanB半乳聚糖降解期间的还原能力使用DNS测定法确定,并根据图5给出的半乳糖标准曲线进行定量。将1U半乳聚糖酶定义为每分钟1μmol半乳糖的释放,并计算从等式:




    图5.通过DNS测定法测定的半乳糖的标准曲线 A540nm值是通过将80μl5至20mM半乳糖的溶液与300μlDNS-试剂混合而获得的,并根据步骤4.步骤4.

食谱

  1. 纯化的重组His 标记GanB
    0.6mg/ml的0.1M磷酸钾缓冲液pH6.5
    注意:使用牛血清白蛋白作为标准,通过Bradford(Bradford,1976)的方法测定蛋白质量。
  2. 羽扇豆半乳聚糖
    在Milli-Q纯H 2 O溶液中溶解2%羽扇豆半乳聚糖
  3. DNS试剂
    混合1.76g二硝基水杨酸,50g硫酸钾钠盐,1.26g亚硫酸钠,6ml 50%NaOH与183ml Milli-Q纯H 2 O /
  4. AZCL-半乳聚糖
    在Milli-Q纯H 2 O 2中悬浮1%AZCL-半乳聚糖,并在使用之前彻底混合,
  5. TLC显影试剂
    将10.5g七聚山梨酸铵,0.5g硫酸铈硫酸盐,15ml浓缩的H 2 SO 4 SO 4在245ml Milli-Q纯H 2 O中, br />
  6. 磷酸钾缓冲液(KPP),0.1M
    将0.1M KH 2 PO 4和0.1MH 2 HPO 4的溶液混合直到pH达到6.5, br />
  7. 0.4M硼酸钠溶液
    将152g硼酸钠溶于1L Milli-Q纯H 2 O中,并将pH调节至9.8°C /
  8. 0.3M乙酸钠,pH5.2
    将40.8g乙酸钠溶于1L Milli-Q纯H 2 O中,用乙酸调节pH至5.2
  9. TLC流动相溶液
    混合87 ml丙酮与13 ml Milli-Q纯H 2 O
  10. HPAEC等静止流动相
    混合11ml 50%氢氧化钠,63.7ml 0.3M乙酸钠pH 5.2,加入890.3ml Milli-Q纯H 2 O /

致谢

该协议首先用于Watzlawick等人。 (2016)。

参考文献

  1. Bradford,MM(1976)。  快速敏感的方法用于利用蛋白质 - 染料结合原理定量微量的蛋白质。
    Anal Biochem 72:248-254。
  2. Miller,GL(1959)。  使用二硝基水杨酸试剂用于测定还原糖。 31(3):426-428。
  3. Watzlawick,H.,Morabbi Heravi,K.和Altenbuchner,J.(2016)。 ganSPQAB操纵子在枯草芽孢杆菌降解半乳聚糖中的作用。 J Bacteriol 198(20):2887-2896。 br />
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引用:Watzlawick, H. (2017). Measurement of the Galactanase Activity of the GanB Galactanase Protein from Bacillus subtilis. Bio-protocol 7(7): e2206. DOI: 10.21769/BioProtoc.2206.
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