集胞藻细胞中转化酶酶活的测定方法

Xiaoming  Tan; Kuo  Song; Xuefeng  Lu

doi:10.21769/BioProtoc.2856

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Peer-reviewed

Enzymatic Activity Assay for Invertase in Synechocystis Cells

集胞藻细胞中转化酶酶活的测定方法

XT Xiaoming Tan

KS Kuo Song

XL Xuefeng Lu email

发布: 2018年05月20日第8卷第10期 DOI: 10.21769/BioProtoc.2856 浏览次数: 7237

评审: Anonymous reviewer(s)

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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

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

Equipment

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

Software

Chromeleon^TM software (Thermo Fisher Scientific, Thermo Scientific^TM, version 6.80; catalog number: CHROMELEON6)

Procedure

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如何引用

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.