从小球藻Chlorella sorokiniana提取O-乙酰丝氨酸 (硫醇) 裂解酶 (OASTL) 并测定其活性

Giovanna Salbitani; Simona Carfagna

doi:10.21769/BioProtoc.2342

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Peer-reviewed

Extraction and Activity of O-acetylserine(thiol)lyase (OASTL) from Microalga Chlorella sorokiniana

从小球藻Chlorella sorokiniana提取O-乙酰丝氨酸 (硫醇) 裂解酶 (OASTL) 并测定其活性

Giovanna Salbitani

Simona Carfagna email

发布: 2017年06月20日第7卷第12期 DOI: 10.21769/BioProtoc.2342 浏览次数: 11247

评审: Dennis NürnbergAnonymous reviewer(s)

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Abstract

O-acetylserine(thiol)lyase (OASTL) is an enzyme catalysing the reaction of inorganic sulphide with O-acetylserine to form the S-containing amino acid L-cysteine. Here we describe an improved protocol to evaluate the activity of this enzyme from the microalga Chlorella sorokiniana. It is a colorimetric assay based on the reaction between cysteine, the product of OASTL activity, and ninhydrin reagent, which forms a thiazolidine (Thz).

Keywords: Chlorella sorokiniana (Chlorella sorokiniana)

Colorimetric assay (比色法)

Cysteine (半胱氨酸)

Microalgae (微藻类)

Ninhydrin (茚三酮)

O-acetylserine(thiol)lyase (O-乙酰（硫醇）裂解酶)

Sulphur (硫)

Background

In archaea, bacteria, microalgae and plants, the synthesis of cysteine (Cys) represents a decisive stage of assimilatory sulphate reduction (Hell and Wirtz, 2008). Cys biosynthesis is the last step of sulphur assimilation and proceeds by two interconnected reactions catalysed by serine acetyltransferase (SAT, EC 2.3.1.30) and O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) (Salbitani et al., 2014; Carfagna et al., 2015).
OASTLs catalyse the reaction between O-acetilserine (OAS) and sulphide to form Cys and acetate (Figure 1).

Figure 1. Schematic mechanism of cysteine biosynthesis catalyzed by O-acetylserine(thiol)lyase

In vascular plants, OASTLs are localized in chloroplasts, mitochondria and the cytosol with different functions for Cys synthesis (Jost et al., 2000; Birke et al., 2013). In microalgae, OASTLs seem to be mainly localized essentially in the chloroplasts (Merchant et al., 2007; Bromke, 2013). However, in Chlorella sorokiniana two isoforms, chloroplastic and cytosolic OASTL, were found under S-deprivation conditions (Carfagna et al., 2011).

Many researchers have developed and modified protocols to determine OASTLs activity in plants and bacteria (Gaitonde, 1967; Burnell and Whatley, 1977; Lèon et al., 1987; Rolland et al., 1992). Here we describe a protocol for the determination of OASTL activity, optimized for the green microalga Chlorella sorokiniana 211-8K (Figure 2). This OASTL assay is a spectrophotometric analysis based on the colorimetric reaction of the formed L-cysteine with ninhydrin reagent to form a thiazolidine (Thz) (Prota and Posiglione, 1973).

Figure 2. Optical microscope image of Chlorella sorokiniana cells

Materials and Reagents

Eppendorf tubes (1.5-2.0 ml)
Cuvettes 1.5 ml (BRAND, catalog number: 759115 )
CO₂ tank
Chlorella sorokininana Shihira & Krauss, strain 211/8K (CCAP, Cambridge University) (Figure 2)
Liquid nitrogen
Milli-Q water
O-Acetyl-L-serine (OAS) (Sigma-Aldrich, catalog number: CDS020792 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
Sodium sulfide nonahydrate (Na₂S·9H₂O) (Sigma-Aldrich, catalog number: S2006 )
Trichloracetic acid (TCA) (Sigma-Aldrich, catalog number: 91228 )
Acetic acid (CH₃COOH) (Avantor Performance Materials, J.T. Baker^®, catalog number: 401424 )
Ethanol (EtOH) (Avantor Performance Materials, J.T. Baker^®, catalog number: 8007 )
Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5655 )
Potassium phosphate dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: 04248 )
Note: This product has been discontinued.
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M2643 )
Ethylenediaminetetraacetic acid ferric sodium salt (Fe-EDTA) (Sigma-Aldrich, catalog number: E6760 )
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C5670 )
Potassium nitrate (KNO₃) (Sigma-Aldrich, catalog number: P8291 )
Copper(II) sulfate (CuSO₄) (Sigma-Aldrich, catalog number: 451657 )
Ammonium molybdate tetrahydrate, (NH₄)₆Mo₇O₂₄·4H₂O (Sigma-Aldrich, catalog number: M1019 )
Manganese(II) chloride (MnCl₂) (Sigma-Aldrich, catalog number: 13217 )
Note: This product has been discontinued.
Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Sigma-Aldrich, catalog number: Z0251 )
Boric acid (H₃BO₃) (Sigma-Aldrich, catalog number: B6768 )
Pyridoxal-phosphate (PLP) (Sigma-Aldrich, catalog number: P3657 )
Ninhydrin (Sigma-Aldrich, catalog number: N4876 )
Hydrochloric acid 37% (Avantor Performance Materials, J.T. Baker^®, catalog number: 6012 )
HEPES (Sigma-Aldrich, catalog number: H4034 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7030 )
Basal medium (see Recipes)
Phosphate buffer (see Recipes)
Extraction buffer (see Recipes)
Ninhydrin solution (see Recipes)
1 M HEPES solution (see Recipes)

Equipment

Culture flask (WHEATON, catalog number: 356954 )
Fluorescent lamps (Philips Lighting, model: TL-D 30W/55 )
Bench centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: IEC CL30 )
French pressure cell press (AMINCO RESOURCES, model: FA-078 )
Superspeed centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: Sorvall RC-5C Plus )
Vortex mixer (Troemner, catalog number: 945302 )
Eppendorf ThermoMixer^® Comfort (Eppendorf, model: 5355 )
Eppendorf MiniSpin^® (Eppendorf, model: 5453000011 )
Thermo bath (Labortechnik medingen, model: MWB 5 )
Spectrophotometer (Cole-Parmer, JENWAY, model: 7315 )
Optical microscope (Esselte, Leitz, model: Leitz Laborlux K )

Software

SigmaPlot^® 12 software

Procedure

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Salbitani, G. and Carfagna, S. (2017). Extraction and Activity of O-acetylserine(thiol)lyase (OASTL) from Microalga Chlorella sorokiniana. Bio-protocol 7(12): e2342. DOI: 10.21769/BioProtoc.2342.