Method for detecting l-serine based on <i>Escherichia coli </i>cysteine desulfurase

Abstract

The present disclosure provides a method for detecting L-serine based on Escherichia coli cysteine desulfurase, and belongs to the technical field of amino acid detection. The method includes the steps of: reacting an unknown sample containing L-serine with E. coli cysteine desulfurase in vitro to produce a red substance, and qualitatively or quantitatively determining L-serine content in the unknown sample by observing a color of the red substance or determining content thereof. The method provided by the present disclosure is simple and feasible in technical operation, few in reaction steps, and capable of directly qualitatively detecting by naked eyes and quantitatively detecting the L-serine content.

Claims

1. A method for detecting L-serine in an unknown sample with Escherichia coli cysteine desulfurase, comprising the steps of: reacting the unknown sample with Escherichia coli cysteine desulfurase in vitro; observing a color of a resulting reaction solution to determine whether a stable red substance is produced after the reaction; observing a color depth of the red substance with naked eyes; and qualitatively determining the presence or absence of the L-serine in the unknown sample and an L-serine content of the unknown sample; wherein if the red substance is present, the L-serine content in the unknown sample is quantitatively determined by a process comprising: measuring absorbances of L-serine standard solutions after reacting the solutions with Escherichia coli cysteine desulfurase to produce the red substance; constructing a standard curve based on the absorbances of the L-serine standard solutions; measuring an absorbance of the unknown sample; and substituting the absorbance of the unknown sample into an equation of the standard curve to quantitatively obtain the L-serine content in the unknown sample; wherein the reacting of the L-serine standard solutions and the unknown sample with Escherichia coli cysteine desulfurase occurs for a reaction time of 60-180 min; wherein the absorbances of the L-serine standard solutions and the unknown sample are measured at a wavelength of 528 nm; and wherein the Escherichia coli cysteine desulfurase has the amino acid sequence of SEQ ID NO: 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to more clearly illustrate the examples of the present disclosure or the technical solution in the prior art, the accompanying drawings required in the examples will be briefly introduced below. Obviously, the drawings in the following description are only some examples of the present disclosure. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

(2) FIG. 1 is an illustrative diagram of qualitative and semi-quantitative determination of L-serine and a standard colorimetric card;

(3) FIG. 2 illustrates optimization of incubation time and establishment of a standard curve; A: effects of incubation time and wavelength on enzymatic reaction; B: screening of optimal incubation time; C: standard curve.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(4) The technical solution of the present disclosure will now be specifically described by way of examples, which should not be construed as limiting the present disclosure, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present disclosure.

(5) The test methods used in the following examples are conventional, unless otherwise specified; the materials and reagents used are commercially available reagents and materials, unless otherwise specified.

(6) E. coli cysteine desulfurase used in the following examples has an amino acid sequence shown in SEQ ID NO: 1:

(7) TABLE-US-00001 MELPIYLDYSATTPVDPRVAEKMMQFMTMDGTFGNPASRSHRFGWQAEEA VDIARNQIADLVGADPREIVFTSGATESDNLAIKGAANFYQKKGKHIITS KTEHKAVLDTCRQLEREGFEVTYLAPQRNGIIDLKELEAAMRDDTILVSI MHVNNEIGVVQDIAAIGEMCRARGIIYHVDATQSVGKLPIDLSQLKVDLM SFSGHKIYGPKGIGALYVRRKSRVRIEAQMHGGGHERGMRSGTLPVHQIV GMGEAYRIAKEEMATEMERLRGLRNRLWNGIKDIEEVYLNGDLEHGAPNI LNVSFNYVEGESLIMALKDLAVSSGSACTSASLEPSYVLRALGLNDELAH SSIRFSLGRFTTEEEIDYTIELVRKSIGRLRDLSPLWEMYKQGVDLNSIE WAHHHHHH

Example 1 Method for Qualitative/Semi-Quantitative Detection of L-Serine Based on E. coli Cysteine Desulfurase

(8) 1. Reagents

(9) A. 1 M L-serine standard solution;

(10) B. Reaction buffer: 40 mM Tris-HCl buffer (pH 9.0), 100 ?M E. coli cysteine desulfurase, and 0.4 mM L-cysteine;

(11) 2. The L-serine standard solution was diluted to five concentrations, respectively: A: 0.00 mM, B: 0.25 mM, C: 0.50 mM, D: 1.00 mM, E: 2.00 mM. Each serial dilution of the standard solution and the unknown sample (100 ?L) were each mixed with 100 ?L of reaction buffer, incubated at room temperature (25? C.) for 2 h, observed with the naked eye, and photographed, and a standard colorimetric card was established.

(12) As shown in FIG. 1, in the concentration range of 0 to 2 mM, the color after enzymatic reaction of the L-serine standard solution with the reaction buffer changed from light yellow to light pink and gradually darkened to red. By observing with the naked eye, this color change has good discrimination, and different colors and their depths can reflect the presence or absence of L-serine well, as well as the level of concentration. It can be seen that the experimental method for qualitative/semi-quantitative detection of L-serine of the present disclosure is feasible, convenient and efficient.

Example 2 Method for Quantitatively Detecting L-Serine Based on E. coli Cysteine Desulfurase

(13) 1. Reagents

(14) A. 1 M L-serine standard solution;

(15) B. Reaction buffer: 40 mM Tris-HCl buffer (pH 9.0), 100 ?M E. coli cysteine desulfurase, and 0.4 mM L-cysteine.

(16) 2. Determination of an Optimal Reaction Time and Linear Range

(17) A. Determination of a Reaction Time

(18) L-serine to be tested was mixed with the reaction buffer in equal volumes, and incubated at 25? ? C. for different times; the absorbance was measured at a wavelength of 528 nm; a curve was plotted with the time as the horizontal axis and the absorbance value at 528 nm as the longitudinal axis.

(19) B. Determination of the Optimal Linear Range

(20) The L-serine standard solution was diluted to three concentrations, respectively: 0.50 mM, 1.00 mM, and 2.00 mM. Each serial dilution of the standard solution and the unknown sample (100 ?L) were each mixed with 100 ?L of reaction buffer, incubated at room temperature (25? C.) for 2 h, and the absorbance at 528 nm was measured for sample and standard tubes, respectively.

(21) 3. Plotting of a Standard Curve

(22) A standard curve was plotted with the concentration of the standard solution as the abscissa and the absorbance measured at a wavelength of 528 nm as the ordinate, and the curve was fitted to obtain a curve equation and an R.sup.2 value.

(23) 4. Determination of L-Serine Content in Samples

(24) The L-serine concentration was obtained according to the standard curve equation obtained above and the absorbance value of the sample tube.

(25) As shown in FIG. 2, with the prolongation of the incubation time between the L-serine standard and the reaction buffer, the absorption peak at 528 nm, which represents red, appears from nothing and gradually increases (FIG. 2A). The absorption peak reaches the highest at 2 h, and plateaus within 1 h (FIG. 2B). It can be seen that the optimal incubation time is 2 h, and as determined according to the optimized incubation time, the plotted standard curve has a good linear range in the range of 0.5 to 2.0 mM (FIG. 2C). It can be seen that the experimental method for quantitatively detecting L-serine provided by the present disclosure is feasible.

Example 3 Determination of the Ability to Resist Other L-Amino Acids

(26) 500 ?L each of L-serine and other L-amino acids (4 mM) to be tested were mixed with 500 ?L of E. coli cysteine desulfurase, respectively, and incubated on a shaker at 37? C. and 250 rpm for 2 h, and the color depth was observed with the naked eye and photographed.

(27) The results show that only L-serine and E. coli cysteine desulfurase appear red after incubation, and other amino acids and the control without amino acid appear light yellow, indicating that the method provided by the present disclosure can directly observe whether the red color is produced or not and determine whether L-serine is contained, and it is easy to know that this method has better resistance to the interference of other L-amino acids and better specificity. In addition, the inventors have further confirmed through experiments that the detection method has good anti-interference ability for D-serine, cycloserine and serine derivatives. The above results show the method provided by the present disclosure is feasible to qualitatively detect L-serine, and the operation is simple and feasible, with excellent specificity.

Example 4 Determination of L-Serine Content in L-Serine Accumulating Strain IscA.SUP.?./SufA.SUP.? Double Knockout Strain

(28) 1. Culture of IscA/SufA Double Knockout Strain

(29) IscA.sup.?/sufA.sup.? double mutant strain (for the construction method, refer to the master's thesis Functions of IscA and SufA in the Biogenesis of Iron-Sulfur Clusters in Escherichia coli from TONG Zhenzhen) and control wild-type E. coli MC4100 were cultured in LB broth at 250 rpm (round per minute) and 37? C. overnight, and the bacterial suspension cultured overnight was diluted 1:50 into Erlenmeyer flasks containing 500 mL of freshly prepared LB broth, respectively; the culture was continued under the same conditions until the OD.sub.600 nm reached 0.6 (logarithmic growth phase), and the bacteria were harvested by centrifugation.

(30) 2. Metabolite Extraction

(31) The above E. coli cells were sampled, 50 mL of pre-cooled methanol/acetonitrile/water (2:2:1, v/v/v) was added to each sample, vortexed to mix, sonicated in an ice bath for 20 min, and incubated at ?20? C. for 1 h to precipitate the protein; the protein was centrifuged at 14,000 rpm and 4? C. for 20 min, and the supernatant was collected and dried in vacuum. During detection, 100 ?L of buffer (40 mM Tris-HCl, pH 9.0) was added to reconstitute, centrifuged at 14,000 rcf and 4? C. for 20 min, and the supernatant was collected.

(32) 3. Determination of Sample Concentration

(33) The supernatant in step 2 and L-serine standard were taken and incubated with the reaction buffer for 2 h, respectively; pictures were taken and the absorbance at 528 nm was measured; a standard curve was plotted, and the sample concentration was calculated.

(34) Macroscopically, compared with the control without L-serine (light yellow), the extract of wild-type MC4100 turned pale pink after reacting with the reaction buffer (compared with the colorimetric card, it was judged that the L-serine concentration was between 0.00 and 0.25 mM), and the extract of the iscA.sup.?/sufA.sup.? double mutant strain turned dark pink after reacting with the reaction buffer (compared with the colorimetric card, it was judged that the L-serine concentration was between 0.50 and 1 mM); as calculated by quantitative determination, the L-serine content in the extract of the wild-type MC4100 was 0.21 mM. The L-serine content of the extract of the iscA.sup.?/sufA.sup.? double mutant strain was 0.53 mM. It can be seen that the results of the two methods are consistent.

(35) The above examples are only intended to describe the preferred implementation of the present disclosure and not intended to limit the scope of the present disclosure. Various alterations and improvements made by those of ordinary skill in the art based on the technical solution of the present disclosure without departing from the design spirit of the present disclosure shall fall within the scope of the appended claims of the present disclosure.

(36) TABLE-US-00002 SequenceListingInformation: DTDVersion:V1_3 FileName:SEQUENCE LISTING.xml SoftwareName:WIPOSequence SoftwareVersion:2.0.0 ProductionDate:2022-07-04 GeneralInformation: Currentapplication/Applicantfilereference:HLP20220302223 Earliestpriorityapplication/IPOffice:CN Earliestpriorityapplication/Applicationnumber:202111282894.4 Earliestpriorityapplication/Filingdate:2021-11-01 Applicantname:WenzhouMedicalUniversity Applicantname/Language:en Inventiontitle:METHODFORDETECTINGL-SERINEBASEDONESCHERICHIA COLICYSTEINEDESULFURASE(en) SequenceTotalQuantity:1 Sequences: SequenceNumber(ID):1 Length:408 MoleculeType:AA FeaturesLocation/Qualifiers: -source,1..408 >mol_type,protein >note,aminoacidsequenceofE.colicysteinedesulfurase >organism,syntheticconstruct Residues: MELPIYLDYSATTPVDPRVAEKMMQFMTMDGTFGNPASRSHRFGWQAEEAVDIARNQIAD60 LVGADPREIVFTSGATESDNLAIKGAANFYQKKGKHIITSKTEHKAVLDTCRQLEREGFE120 VTYLAPQRNGIIDLKELEAAMRDDTILVSIMHVNNEIGVVQDIAAIGEMCRARGIIYHVD180 ATQSVGKLPIDLSQLKVDLMSFSGHKIYGPKGIGALYVRRKSRVRIEAQMHGGGHERGMR240 SGTLPVHQIVGMGEAYRIAKEEMATEMERLRGLRNRLWNGIKDIEEVYLNGDLEHGAPNI300 LNVSFNYVEGESLIMALKDLAVSSGSACTSASLEPSYVLRALGLNDELAHSSIRFSLGRF360 TTEEEIDYTIELVRKSIGRLRDLSPLWEMYKQGVDLNSIEWAHHHHHH408 END