Patent Publication Number: US-2023145499-A1

Title: Method for detecting l-serine based on escherichia coli cysteine desulfurase

Description:
CROSS REFERENCE TO RELATED APPLICATION(S) 
     This patent application claims the benefit and priority of Chinese Patent Application No. 202111282894.4, filed on Nov. 01, 2021, the disclosure of which is incorporated by reference herein in its entirety as part of the present application. 
     REFERENCE TO AN ELECTRONIC SEQUENCE LISTING 
     The contents of the electronic sequence listing (sequencelisting.xml; Size: 2,363 bytes; and Date of Creation: Jul. 7, 2022) is herein incorporated by reference in its entirety. 
     TECHNICAL FIELD 
     The present disclosure relates to the technical field of amino acid detection, in particular to a method for detecting L-serine based on  Escherichia   coli  cysteine desulfurase. 
     BACKGROUND ART 
     L-serine is a non-essential polar amino acid in the human body. It is mainly involved in the biosynthesis of proteins in the body. It is also an important precursor of purines, pyrimidines, and phospholipids. It plays an important role in immune regulation, tumor metabolism and other processes, and is expected to serve as a tumor marker of some cancers. In terms of medicinal use, L-serine is widely used in amino acid infusions and nutritional additives, and its derivatives also have excellent medicinal value and biological activity. In terms of cosmetic industry, L-serine is extensively added to high-grade cosmetics due to its wettability and moisture retention. With the in-depth research and understanding of L-serine and the continuous development of medical and health care, the demand for L-serine is also increasing. Therefore, providing a quick and easy detection method for determining the content of L-serine in mixed amino acids, especially a detection technology capable of eliminating the interference of D-serine, cycloserine, serine analogs and serine derivatives, is extremely important for the development and utilization of L-serine. 
     At present, methods for determining amino acid content inside and outside of China are mainly divided into instrumental methods, chromogenic methods, chemiluminescence methods, and enzymatic methods. Instrumental methods usually include HPLC, GC-MS, LC-MS, NMR, and automatic amino acid analyzer. The chromogenic methods include chromotropic acid-spectrophotometry, paper chromatography-spectrophotometry, and ninhydrin method. Chemiluminescence method is mainly capillary electrophoresis coupling with electrochemiluminescence. Enzymatic methods include serine aminotransferase method and cystathionine lyase method. 
     Among them, instrumental methods such as liquid chromatography, gas chromatography, and chromatography-mass spectrometry have good sensitivity and high accuracy, but require expensive equipment, specialized laboratories, professionally trained laboratory technicians, high maintenance costs, and relatively high laboratory consumables. As the most basic and traditional detection method, ninhydrin method is easy to operate and quick to react, but it has high requirements on reaction conditions, and requires precise control of reaction temperature, pH, and time. Moreover, the method has different sensitivities to different types of amino acids and is not suitable for analysis of samples that require high precision. Fluorescence quenching method can avoid the interference of most amino acids, but the sample needs to undergo complex phosphorylation pretreatment, and the detection result has a large error. The paper chromatography-spectrophotometry is easy to operate, but it is not suitable for the analysis and detection of large quantities of samples due to its poor stability. Chromotropic acid-spectrophotometry has fast reaction speed, simple operation, and high accuracy, but poor anti-interference ability. 
     Enzymatic methods have excellent specificity and accuracy, but the currently used enzymatic methods have many reaction steps and many factors that affect the determination, and the detection signal often cannot be directly observed or directly measured. Therefore, it is very necessary to develop an enzymatic method for determining L-serine which is simple and can be directly observed with the naked eye after the reaction. 
     SUMMARY 
     An objective of the present disclosure is to provide a method for detecting L-serine based on  E .  coli  cysteine desulfurase. The method 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. 
     To achieve the above objective, the present disclosure provides the following solution: 
     The present disclosure provides a method for detecting L-serine based on  E .  coli  cysteine desulfurase, including 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. 
     Preferably, a method for qualitatively detecting the L-serine content in the unknown sample may be implemented by: observing whether a stable red substance is produced after the reaction, and a color depth of the red substance with the naked eye, and qualitatively determining the presence or absence of the L-serine in the unknown sample and the content thereof. 
     Preferably, a method for quantitatively detecting the L-serine content in the unknown sample may include: 
     measuring an absorbance of an L-serine standard solution, and constructing a standard curve; and   measuring an absorbance of a reacted red substance, and substituting the absorbance into an equation of the standard curve to quantitatively obtain the L-serine content in the unknown sample.   

     Preferably, both the absorbance of the L-serine standard solution and the absorbance of the red substance may be measured at a wavelength of 528 nm. 
     Preferably, the  E .  coli  cysteine desulfurase may have an amino acid sequence shown in SEQ ID NO: 1. 
     Preferably, the unknown sample and the  E .  coli  cysteine desulfurase may be mixed and reacted in equal volumes. 
     Preferably, a reaction time may be 60-180 min. 
     The present disclosure provides the following technical effects: 
     According to the method for detecting L-serine based on  E .  coli  cysteine desulfurase provided by the present disclosure, because the  E .  coli  cysteine desulfurase takes L-cysteine as a substrate to catalyze the generation of L-alanine and elemental sulfur, and the purified cysteine desulfurase binds to the prosthetic group pyridoxal phosphate, it appears yellow with the naked eye, and there is a characteristic absorption peak at 395 nm when scanned by a UV-Vis spectrophotometer. According to the inventor’s previous research, it is easy to know that the enzyme is expressed in an IscA - /SufA -  double-deficient bacterium. The enzyme turns red when observed with the naked eye, and there is a stable characteristic absorption peak at 528 nm when scanned by the UV-Vis spectrophotometer. After a large number of experiments, a stable red substance may also be generated by in vitro reaction of IscA - /SufA -  double-deficient  E .  coli -derived cysteine desulfurase with the L-serine, and specificity thereof depends on the L-serine. More importantly, the depth of the red color, the level of the absorption peak at 528 nm, and the L-serine added in the reaction mixture are dose-dependent, thus realizing the qualitative and quantitative determination of the L-serine in an unknown sample. The detection method provided by the present disclosure features convenient technical operation and few reaction steps. Not only can the detection method achieve intuitive qualitative detection and precise quantification, but also can effectively prevent the interference of D-serine, cycloserine, serine analogs and other amino acids, providing a scientific method for the rapid detection of L-serine in different fields. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       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. 
         FIG.  1    is an illustrative diagram of qualitative and semi-quantitative determination of L-serine and a standard colorimetric card; 
         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 
     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. 
     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. 
       E .  coli  cysteine desulfurase used in the following examples has an amino acid sequence shown in SEQ ID NO: 1:  
     
       
         
           
               
            
               
                  MELPIYLDYSATTPVDPRVAEKMMQFMTMDGTFGNPASRSHRFGWQAEEAVDI 
               
               
                 ARNQIADLVGADPREIVFTSGATESDNLAIKGAANFYQKKGKHIITSKTEHKAVLDTCRQ 
               
               
                 LEREGFEVTYLAPQRNGIIDLKELEAAMRDDTILVSIMHVNNEIGVVQDIAAIGEMCRAR 
               
               
                 GIIYHVDATQSVGKLPIDLSQLKVDLMSFSGHKIYGPKGIGALYVRRKSRVRIEAQMHGG 
               
               
                 GHERGMRSGTLPVHQIVGMGEAYRIAKEEMATEMERLRGLRNRLWNGIKDIEEVYLNG 
               
               
                 DLEHGAPNILNVSFNYVEGESLIMALKDLAVSSGSACTSASLEPSYVLRALGLNDELAHS 
               
               
                 SIRFSLGRFTTEEEIDYTIELVRKSIGRLRDLSPLWEMYKQGVDLNSIEWAHHHHHH 
               
            
           
         
       
     
     Example 1 Method for Qualitative/Semi-Quantitative Detection of L-Serine Based on  E   Coli  Cysteine Desulfurase 
     1. Reagents 
     A. 1 M L-serine standard solution; 
     B. Reaction buffer: 40 mM Tris-HCl buffer (pH 9.0), 100 µM  E .  coli  cysteine desulfurase, and 0.4 mM L-cysteine; 
     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 µL) were each mixed with 100 µL 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. 
     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 
     1. Reagents 
     A. 1 M L-serine standard solution; 
     B. Reaction buffer: 40 mM Tris-HCl buffer (pH 9.0), 100 µM  E .  coli  cysteine desulfurase, and 0.4 mM L-cysteine. 
     2. Determination of an Optimal Reaction Time and Linear Range 
     A. Determination of a Reaction Time 
     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. 
     B. Determination of the Optimal Linear Range 
     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 µL) were each mixed with 100 µL 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. 
     3. Plotting of a Standard Curve 
     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 2  value. 
     4. Determination of L-Serine Content in Samples 
     The L-serine concentration was obtained according to the standard curve equation obtained above and the absorbance value of the sample tube. 
     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.  2 A ). The absorption peak reaches the highest at 2 h, and plateaus within 1 h ( FIG.  2 B ). 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.  2 C ). 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 
     500 µL each of L-serine and other L-amino acids (4 mM) to be tested were mixed with 500 µL 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. 
     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 - /SufA -  Double Knockout Strain 
     1. Culture of IscA/SufA Double Knockout Strain 
     IscA - /sufA -  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 600   nm  reached 0.6 (logarithmic growth phase), and the bacteria were harvested by centrifugation. 
     2. Metabolite Extraction 
     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 µL 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. 
     3. Determination of Sample Concentration 
     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. 
     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 - /sufA -  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 - /sufA -  double mutant strain was 0.53 mM. It can be seen that the results of the two methods are consistent. 
     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. 
     Sequence Listing Information: 
     DTD Version: V1­_3   File Name: SEQUENCE LISTING.xml   Software Name: WIPO Sequence   Software Version: 2.0.0   Production Date: 2022-07-04   

     General Information: 
     Current application / Applicant file reference: HLP20220302223   Earliest priority application / IP Office: CN   Earliest priority application / Application number: 202111282894.4   Earliest priority application / Filing date: 2021-11-01   Applicant name: Wenzhou Medical University   Applicant name / Language: en   Invention title: METHOD FOR DETECTING L-SERINE BASED ON ESCHERICHIA COLI CYSTEINE DESULFURASE ( en )   Sequence Total Quantity: 1   

     Sequences: 
     Sequence Number (ID): 1   Length: 408   Molecule Type: AA   Features Location/Qualifiers: 
   source, 1..408 
   mo1_type, protein   note, amino acid sequence of  E .  coli  cysteine desulfurase   organism, synthetic construct   
   
   

     Residues: 
     MELPIYLDYS ATTPVDPRVA EKMMQFMTMD GTFGNPASRS HRFGWQAEEA VDIARNQIAD 60   LVGADPREIV FTSGATESDN LAIKGAANFY QKKGKHIITS KTEHKAVLDT CRQLEREGFE 120   VTYLAPQRNG IIDLKELEAA MRDDTILVSI MHVNNEIGVV QDIAAIGEMC RARGIIYHVD 180   ATQSVGKLPI DLSQLKVDLM SFSGHKIYGP KGIGALYVRR KSRVRIEAQM HGGGHERGMR 240   SGTLPVHQIV GMGEAYRIAK EEMATEMERL RGLRNRLWNG IKDIEEVYLN GDLEHGAPNI 300   LNVSFNYVEG ESLIMALKDL AVSSGSACTS ASLEPSYVLR ALGLNDELAH SSIRFSLGRF 360   TTEEEIDYTI ELVRKSIGRL RDLSPLWEMY KQGVDLNSIE WAHHHHHH 408   
END