CN111100903B - Method for quantitatively detecting iota-carrageenan by enzyme method - Google Patents
Method for quantitatively detecting iota-carrageenan by enzyme method Download PDFInfo
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Abstract
The invention relates to the technical field of biotechnology and biochemical detection, in particular to a method for quantitatively detecting iota-carrageenan by an enzyme method. Degrading iota-carrageenan into reducing sugar by utilizing the specificity of iota-carrageenan, inactivating enzyme, adding p-hydroxybenzoyl hydrazine solution for color reaction, measuring the light absorption value of the supernatant at 400-420nm after centrifugation, and comparing a standard curve to obtain the content of iota-carrageenan. The detection method has the advantages of good linear range and high accuracy, and can be popularized and used in the market.
Description
Technical Field
The invention relates to the technical field of biotechnology and biochemical detection, in particular to a method for quantitatively detecting iota-carrageenan by an enzyme method.
Background
Carrageenans (also known as carrageenans or carrageenans), which are natural anionic linear polysaccharides of sulfuric acid extracted from the cell walls of red algae such as carrageenans, eucheuma, gigartina, etc., are of various types depending on the number and attachment positions of sulfate groups on galactose and the content of 3, 6-anhydrogalactose in the structure. Iota-carrageenan is one of three common carrageenan types (kappa-, iota-and lambda-carrageenan), consists of alternate sulfated-alpha-D-3, 6-anhydrogalactose and sulfated-beta-D-galactose residues, is widely available and has various functions, so that the iota-carrageenan is applied to various industrial productions of food, medicine, cosmetics and the like and plays roles of a coagulant, a binder, a stabilizer, an emulsifier, a suspending agent, a thickener and the like.
Quantitative detection of iota-carrageenan is a basic link in iota-carrageenan gum quantity control, functional research and product development. At present, commonly used iota-carrageenan quantitative detection methods are a phenol-sulfuric acid method, a liquid chromatography-tandem mass spectrometry method and the like. The phenol-sulfuric acid method has high sensitivity, does not need expensive instruments, is easily influenced by other polysaccharides in the sample, and is time-consuming and high in sample and reagent consumption. The liquid chromatography-tandem mass spectrometry obtains the content of the iota-carrageenan by hydrolyzing, deriving and pretreating a sample and measuring the content of monosaccharides consisting of the iota-carrageenan.
P-hydroxybenzoyl hydrazine (pHBH) is an aromatic hydrazide compound which can generate a yellow product with a beta-diketone compound under a strong alkaline condition. Research shows that pHBH can react with reducing sugar such as glucose and the like to generate similar yellow substances under high-temperature and alkaline conditions, the color depth after reaction is in direct proportion to the concentration of the reducing sugar, and the sensitivity of the reaction is high. This provides feasibility for the quantitative detection of iota-carrageenan by an enzyme method.
In conclusion, the method for quantitatively detecting the iota-carrageenan has great significance, and is high in sensitivity, good in accuracy and simple and convenient to operate.
Disclosure of Invention
The invention aims to solve the technical problem that the commonly used iota-carrageenan quantitative detection methods are a phenol-sulfuric acid method and a liquid chromatography-tandem mass spectrometry method, but the two methods respectively have the defects of poor reproducibility, complex operation, time and labor waste, and can not realize the detection with high sensitivity, good accuracy and simple and convenient operation.
In order to solve the problems, the invention provides an enzymatic quantitative detection method for iota-carrageenan, which specifically degrades the iota-carrageenan into reducing sugar by utilizing specific iota-carrageenan enzyme, utilizes the color development reaction of p-hydroxybenzoyl hydrazine (pHBH), and measures the content of the iota-carrageenan by measuring the light absorption value at 400-420 nm.
In order to achieve the purpose, the invention is realized by the following technical scheme: an enzymatic quantitative detection method for iota-carrageenan is characterized by degrading iota-carrageenan into reducing sugar by utilizing iota-carrageenan enzyme specificity, inactivating enzyme, adding p-hydroxy-benzoyl hydrazine solution for color development reaction, measuring a light absorption value of a supernatant at 400-420nm after centrifugation, and comparing a standard curve to obtain iota-carrageenan content.
Further, the iota-carrageenase is iota-carrageenase Cgi1_ Wf, and the amino acid sequence of the iota-carrageenase is SEQ ID No.1. Because most of the enzymes in the research of the GH82 family iota-carrageenase at present have poor stability and quick inactivation, and have great limitation in practical application, the degradation iota-carrageenase is not completely controllable, and great experimental error can be caused.
SEQ ID NO.1：
NEIEQEITANNQQEFNLSREAKTGITSTGYNSTNYFQPPTNLPTKNFTGSTSTQLQTLIN NSSTGAIIKIPKKTYNWGEIKLKSKIQLEIESGTIIKPSNNNIKRIFSIGSSGNGTRVTDVSIIGV GGKFTIDLSATANLNQNMAVIKMGRVSNFKISNFIIKDRRTSLASILLNYIPSNSDNEPYPKN GVIEKINQTGISHTGYGLIQAYSASNVLFKNLYCKGGVTLRLETDDKTMKDAVKNGGKLFG LRNIYADMIKCTSGLCPIMFSPHFTENGKITARNITATGCAFAVRVEHGFIEVFDTNKTYALTS SGGNQFKNFIAGKISGTGNSSKFIGNQYKRANGTQWAIRLSDASINGSLDPYITNQIGYLKN GSFESTTIENVTTIYKPTNAKLKQSFLPFIPCNDWTSKIKNPTDTGMGNGFEYYGPSLGERF DNTNGTNSNGNYIINVNGTTTRFSTVRNILYNTPTACTSNAYGTIPTTSNSPGL
Furthermore, the optimum reaction temperature of the enzyme is 25 ℃, the activity of the enzyme can still be maintained at more than 80 percent at room temperature, the optimum reaction pH value is 8.0, the enzyme is basically kept stable in the pH value range of 5.0-9.0, the enzyme has good storage stability, can be stably stored for at least 30 days at 4 ℃, and can still maintain 80 percent of activity after being placed for 10 days at 25 ℃.
The enzyme has a sequence similarity of up to 41% with other known enzymes (the closest sequence is CgiA produced by Zobellia galactantivorans DsijT). The phylogenetic tree was constructed using MEGA6 with Cgi1_ Wf and all iota-carrageenan sequences annotated as the GH82 family, with the results shown in fig. 3: iota-carrageenase Cgi1_ Wf can be seen in the phylogenetic tree of the GH82 family iota-carrageenase. Thus Cgi1_ Wf is a new member of the iota-carrageenan GH82 family. Subjecting the amino acid sequence of iota-carrageenan Cgi1_ Wf to Blast analysis and multiple sequence alignment of Cgi1_ Wf with 2 iota-carrageenan sequences of GH82 family that have been characterized using ClustalX2, it can be seen from fig. 4 that Cgi1_ Wf shows a different degree of specificity at other sites except strictly conserved at key catalytic sites, and that Blast demonstrates that Cgi1_ Wf has only 41% of the highest similarity to the CgiA sequence produced by Zobellia galactanavora DsijT, indicating that Cgi1_ Wf is a novel iota-carrageenan in GH82 family.
The iota-carrageenase Cgi1_ Wf has high activity on iota-carrageenase, but has no degradation effect on other marine polysaccharides such as agar, algin, kappa-carrageenase and the like. The optimum reaction temperature is 25 ℃, the activity of more than 80 percent can be still kept after the mixture is placed in the environment at room temperature for 10 days, the optimum reaction pH value is 8.0, the mixture basically keeps stable in the pH value range of pH 5.0-9.0, the enzyme kinetic constant Km is 2.73mg/mL, and the Kcat is 560.75s -1 Km/Kcat of 501.72. Mu.M -1 s -1 . As described above, the iota-carrageenan Cgi1_ Wf of the present invention has superior enzymatic properties, good stability, and easy storage, and has strong specificity for substrate binding and a fast enzymatic hydrolysis rate, compared to other iota-carrageenases, and is an ideal enzyme for quantitative iota-carrageenan detection.
Furthermore, the nucleotide sequence of the gene for coding the iota-carrageenase is SEQ ID NO.2 or all genes capable of being translated into SEQ ID NO.1.
SEQ ID NO.2：
AACGAGATTGAACAAGAAATCACCGCAAACAATCAACAAGAATTTAACCTCTCTA GAGAAGCTAAAACCGGAATAACTTCAACAGGATATAATTCTACAAACTATTTTCAACCCC CAACCAACCTACCCACAAAAAATTTCACTGGAAGTACAAGTACACAACTTCAAACACTT ATTAATAATAGCAGCACTGGAGCCATTATTAAAATCCCTAAAAAAACGTATAACTGGGGA GAAATCAAATTAAAATCCAAAATACAATTAGAAATAGAAAGTGGAACTATTATTAAACCA TCTAACAATAATATTAAACGCATTTTCAGTATTGGTAGTTCTGGTAATGGTACTAGAGTTA CTGATGTTAGTATTATAGGTGTAGGCGGAAAATTTACAATAGACCTATCTGCCACCGCTA ACCTAAACCAAAACATGGCAGTGATAAAAATGGGAAGAGTATCTAATTTTAAAATCTCT AATTTTATTATTAAAGACAGAAGAACTTCACTAGCATCAATCTTACTAAATTACATCCCAT CAAACTCTGACAATGAACCTTATCCTAAAAATGGTGTCATCGAAAAAATTAACCAAACA GGAATTTCACACACAGGTTATGGGTTAATCCAAGCGTATTCTGCATCAAATGTATTATTTA AAAATCTATATTGTAAAGGTGGAGTAACTCTTAGACTAGAAACTGATGACAAAACCATG AAAGATGCTGTTAAAAATGGTGGTAAGTTATTTGGATTGAGAAATATTTACGCAGATATG ATAAAATGTACTAGTGGACTTTGCCCTATCATGTTTTCTCCTCACTTTACTGAAAACGGA AAAATTACAGCAAGAAATATTACCGCAACTGGATGTGCTTTTGCTGTAAGAGTTGAACA CGGGTTTATTGAAGTTTTTGACACTAATAAAACTTATGCTTTAACCTCATCTGGAGGAAA CCAATTTAAAAACTTTATTGCTGGTAAAATATCAGGAACTGGAAACTCTTCTAAATTCAT AGGCAACCAATACAAGAGAGCTAATGGCACACAGTGGGCTATTAGATTATCTGACGCTT CTATAAACGGCTCTCTAGATCCATACATCACTAATCAAATTGGATATTTAAAAAATGGTAG TTTTGAAAGCACAACCATAGAAAATGTAACAACAATTTACAAACCTACAAACGCCAAAT TAAAACAATCCTTTTTACCATTTATCCCTTGTAATGATTGGACTAGCAAGATTAAAAATCC TACTGACACAGGAATGGGGAATGGTTTTGAATACTATGGACCTTCTTTAGGAGAAAGAT TTGACAACACAAATGGTACCAACTCTAATGGAAACTACATCATCAATGTAAATGGAACA ACTACTAGGTTTTCAACTGTGAGAAACATTCTTTACAACACCCCAACCGCCTGTACAAG TAATGCATATGGTACAATTCCTACCACTTCTAATAGTCCTGGGTTATAA
Further, the method for quantitatively detecting iota-carrageenan by using the enzymatic method specifically comprises the following steps:
(1) Preparing an iota-carrageenan solution: weighing iota-carrageenan with chemical grade or higher purity, and dissolving the iota-carrageenan in a buffer solution to prepare iota-carrageenan standard solution with concentration gradient;
(2) preparation of a pHBH solution: pHBH is weighed and dissolved in HCl, and then NaOH is added to adjust the pH of the solution to be alkaline, so as to prepare 10-100mg/mL pHBH solution. This is because a concentrated pHBH reagent is easy to store in an acidic environment, but develops color in an alkaline environment, which is necessary for color development.
(3) Drawing a quantitative standard curve: mixing the iota-carrageenan solutions with different concentrations prepared in the step (1) with a proper amount of iota-carrageenan enzyme solution respectively to react; after the reaction, the mixture is placed in a metal bath at 100 ℃ for 5-10min to inactivate the enzyme; then adding pHBH solution, developing in 100 ℃ metal bath for 5-10min, quickly cooling to room temperature, centrifuging to obtain supernatant, measuring the light absorption value of the supernatant, and detecting the wavelength of 400-420nm (the reaction product of pHBH and reducing sugar has good linear relation in the wavelength range and high precision); mixing the iota-carrageenan solution with the same concentration gradient and the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the mixture as a contrast, and then calculating the light absorption value increment corresponding to the iota-carrageenan solution with different concentrations; taking the concentration of the iota-carrageenan standard solution as a horizontal coordinate, taking the light absorption value increment of iota-carrageenan with each concentration as a vertical coordinate, and obtaining a standard curve under a specific reaction condition through linear fitting;
(4) And (3) sample determination: adding a certain amount of iota-carrageenase into the sample to repeat the reaction in the step (3); substituting the light absorption value increment into a standard curve under the conditions of corresponding enzyme adding amount, reaction time, reaction temperature, reaction pH and the like, and calculating the iota-carrageenan concentration in the reaction system, thereby obtaining the iota-carrageenan content in the sample.
Further, the pH of the buffer solution in the step (1) is 7.0-9.0, and the enzyme has higher enzyme activity in the pH range.
Further, the adding amount of the enzyme in the step (2) is 1-1000U, the reaction time is 10-30min, and the reaction temperature is 20-50 ℃. Within the parameter range, the iota-carrageenase has higher enzymolysis activity, and can ensure the rapid proceeding of the enzymolysis reaction; the addition amount of the enzyme and the reaction time need to correspond to each other, and the reaction time is increased when the addition amount of the enzyme is small, so that the complete enzymolysis of the sample can be ensured.
Furthermore, reducing sugar in the sample is removed according to the method of national standard GB 5009.88-2014 before the measurement of the step (4).
The invention has the beneficial effects that:
(1) The invention provides a method for quantitatively detecting iota-carrageenan by an enzyme method, which utilizes iota-carrageenan enzyme to specifically degrade iota-carrageenan in a sample at a lower temperature to form reducing sugar, the reducing sugar can generate a color reaction with pHBH, the color depth of a solution is in direct proportion to the concentration of the reducing sugar, and the iota-carrageenan content in the detected sample can be obtained by detecting the increase of light absorption values before and after the reaction of a reaction solution. The detection method has the advantages of good linear range and high accuracy, and can be popularized and used in the market.
(2) The iota-carrageenan enzyme has the advantages of excellent enzymatic properties, good stability, easiness in storage, strong specificity on substrate combination and high enzymolysis rate, and is an ideal enzyme for quantitative detection of iota-carrageenan.
Drawings
FIG. 1: the objective gene Cgi1_ Wf of the iota-carrageenase is subjected to agarose gel electrophoresis;
FIG. 2 is a schematic diagram: an electrophoretogram of the purified iota-carrageenase Cgi1_ Wf;
FIG. 3: a phylogenetic tree constructed by Cgi1_ Wf and all iota-carrageenan enzymes annotated as GH82 family; wherein the black frame is iota-carrageenase Cgi1_ Wf;
FIG. 4 is a schematic view of: cgi1_ Wf multiple sequence alignment results; wherein, the black frame and white characters are conservative residues of iota-carrageenase.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: cloning, expressing and obtaining iota-carrageenase Cgi1_ Wf in escherichia coli
Cultivation of Wenyingzhuangia fuccanilytica CZ1127 in 2216E Medium T Until the end of logarithm, extracting whole genome DNA, designing upstream and downstream primers (5 '-GACACGATCCAAAGACAAGTAGCAGTAAATGATACTACA; 5' -GACACCTCGAGCTATTGATATACTCTTACATCTATTC) according to target genes, and carrying out PCR by using the whole genome as a template, wherein the PCR reaction conditions are as follows: carrying out 22 cycles of 95 ℃ and 3min,94 ℃ and 30s,58 ℃ and 30s,72 ℃ and 150s, and finally lasting for 5min at 72 ℃ to obtain the iota-carrageenase Cgi1_ Wf gene segment which is connected to a DH5 alpha vector to form a recombinant plasmid. The recombinant plasmid was introduced into BL21 (DE 3) competent cells to construct a recombinant strain. In an LB culture medium containing kanamycin, isopropyl thiogalactoside is used for induction expression, the induction temperature is 17 ℃, and the induction time is 12h. The cells were collected by centrifugation, and a predetermined amount of 20mM disodium hydrogenphosphate-sodium dihydrogenphosphate (Na) 2 HPO 4 -NaH 2 PO 4 ) Suspending the buffer solution, then carrying out ultrasonic disruption in an ice-water bath (power 400W, work for 2s, gap 6s, circulation for 99 times), centrifuging and collecting supernatant, namely the crude enzyme solution of the iota-carrageenan Cgi1_ Wf.
And (3) performing affinity chromatography purification on the target protein in the supernatant by using a HisTrpTM HP chromatographic column on the intracellular supernatant enzyme liquid after the ultrasonication, wherein the SDS-PAGE analysis result is shown in figure 2, and the purified enzyme protein is a single band, which shows that the purity is good. The activity of the purified enzyme was 88.25U/mg (1U activity was defined as the activity to generate 1. Mu. Mol of reducing sugar in 1 min).
Example 2: cloning, expressing and obtaining iota-carrageenase Cgi1_ Wf in bacillus subtilis
Cultivation of Wenyingzhuangia fuccanilytica CZ1127 in 2216E Medium T Until the end of logarithm, extracting whole genome DNA, designing upstream and downstream primers (5 '-GGCTAATGGGCAGCAGCCATCATCA;5' -AATGAATTATGTAAGAGTATCAATAATAG) according to target genes, carrying out PCR (polymerase chain reaction) by taking the whole genome as a template in example 1 to obtain an iota-carrageenase Cgi1_ Wf gene fragment, connecting the gene fragment to a pHT01 carrier to form recombinant plasmid, transforming the recombinant plasmid into a bacillus subtilis competent cell, screening positive clone, carrying out induction expression in LB culture solution by using isopropyl thiogalactoside, wherein the induction temperature is 38 ℃, the induction time is 14h, centrifugally collecting thalli, adding a certain amount of 20mM Na 2 HPO 4 -NaH 2 PO 4 Suspending the buffer solution, then carrying out ultrasonic disruption in an ice-water bath (power 400W, work time 2s, gap 6s, circulation 99 times), centrifuging and collecting supernatant, namely the crude enzyme solution of the iota-carrageenan Cgi1_ Wf. The purification procedure of example 1 is repeated to obtain pure enzyme solution, and the activity of the recombinase fermentation broth is detected to be 140.71U/mg.
Example 3: cloning expression and acquisition of iota-carrageenase Cgi1_ Wf in pichia pastoris
Cultivation of Wenyingzhuangia fuccanilytica CZ1127 in 2216E Medium T Until the end of logarithm, extracting whole genome DNA, designing upstream and downstream primers (5 '-TTATAAATGGGCAGCAGCCATCATCATCA; 5' -GCATGCAGATTATGTAAGAGTATCATAATAG) according to target genes, carrying out PCR (polymerase chain reaction) by taking the whole genome as a template according to the embodiment 1 to obtain an iota-carrageenase Cgi1_ Wf gene fragment, connecting the gene fragment to a pPIC9k carrier to form recombinant plasmids, and adding the recombinant plasmids into pichia pastoris GS115 competent cells to form recombinant cells; screening for Positive clones and splicingInoculating into YPD medium, culturing at 30 deg.C for 20h, inoculating into BMGY medium, shaking at 30 deg.C and 200rpm to OD600=2.0, centrifuging to collect thallus, discarding supernatant, resuspending precipitate with BMMY medium, and inducing with methanol at 29 deg.C and 200rpm for 72h. After the induction is finished, centrifuging and collecting supernatant fluid to obtain crude enzyme liquid. The activity of the recombinase fermentation liquor is detected to be 59.44U/mg.
Example 4: the method of the invention is verified in accuracy
The method and the phenol-sulfuric acid method are utilized to determine the iota-carrageenan content in the sample:
(1) Preparing an iota-carrageenan solution: weighing chemical grade iota-carrageenan, dissolving in 20mM Na with pH of 8.0 2 HPO 4 -NaH 2 PO 4 Buffer solution, preparing iota-carrageenan standard solutions with the concentrations of 1.00mg/mL, 3.00mg/mL, 5.00mg/mL, 7.00mg/mL and 9.00mg/mL respectively;
(2) preparation of a pHBH solution: weighing pHBH to be dissolved in 2mol/LHCl, preparing 200mg/mL pHBH mother liquor, and mixing the mother liquor with 2mol/LNaOH solution according to the weight ratio of 1:9, mixing to prepare 20mg/ml pHBH solution.
(3) Drawing a quantitative standard curve: 375 mu L of the iota-carrageenan solution prepared above with different concentrations and 10U Cgi1_Wf (buffer solution is filled to 375 mu L) react for 25min at 25 ℃. Standing in 100 deg.C metal bath for 10min after reaction to inactivate enzyme, adding 250 μ L pHBH solution, developing color in 100 deg.C metal bath for 8min, rapidly cooling to room temperature, centrifuging to obtain supernatant, and measuring light absorption value of the supernatant at 415 nm; simultaneously, mixing the inactivated enzyme solution with the same concentration gradient with the iota-carrageenan solution, repeating the operation, and measuring the light absorption value of the inactivated enzyme solution as a contrast so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the iota-carrageenan standard solution as an abscissa and the corresponding light absorption value increment as an ordinate, obtaining a standard curve y =1.3425x +0.0158 under specific reaction conditions by linear fitting, wherein R is 2 The value was 0.9991.
(4) And (3) sample determination: an appropriate amount of the sample was weighed, ground, washed with 85% ethanol solution to remove reducing sugars, and the ethanol solution was discarded for 3 consecutive times. After desugarization, the sample was dried overnight in an oven at 40 ℃ and the sample was dissolved in buffer after drying. And (4) taking 375 mu L of sample solution, repeating the operation in the third step, substituting the light absorption value increment into the standard curve y =1.3425x +0.0158, calculating the iota-carrageenan concentration in the reaction system, and further converting the iota-carrageenan content in the sample.
(5) The iota-carrageenan content of the samples was determined according to the phenol-sulfuric acid method in the national standard SN/T4260-2015.
Each method was performed in triplicate and the results are shown in the following table.
| The method of the invention | Phenol-sulfuric acid process | |
| Assay parallel 1 (mg/mL) | 10.24 | 9.62 |
| Assay parallel 2 (mg/mL) | 9.88 | 9.43 |
| Assay parallel 3 (mg/mL) | 9.50 | 10.20 |
| Determination of average value (mg/mL) | 9.87 | 9.75 |
From the above results, it can be seen that there is substantially no deviation between the measurement results of the method of the present invention and the measurement results of the phenol-sulfuric acid method, indicating that the method of the present invention has good accuracy.
Example 5: carrying out quantitative specificity verification on the method of the invention
The method of the invention is used for quantifying the iota-carrageenan in the three mixed solutions respectively.
Step one, preparing an iota-carrageenan solution: weighing chemical grade iota-carrageenan, dissolving in 20mM Na with pH of 7.0 2 HPO 4 -NaH 2 PO 4 A buffer solution, namely preparing an iota-carrageenan solution with the concentration of 4.00 mg/mL; simultaneously preparing agar, kappa-carrageenan and algin solutions with the concentration of 2.00 mg/mL; and respectively and uniformly mixing the agar solution, the kappa-carrageenan solution and the algin solution with the iota-carrageenan solution in equal volume to prepare a mixed solution, wherein the iota-carrageenan concentration of the mixed solution is 2.00mg/mL.
Step two, preparing a pHBH solution: weighing pHBH, dissolving the pHBH in 2mol/LHCl to prepare 200mg/mL pHBH mother liquor, and mixing the mother liquor with 2mol/LNaOH solution according to the weight ratio of 1:9, mixing to prepare 20mg/ml pHBH solution.
Step three, drawing a quantitative standard curve: 375 μ L of the prepared iota-carrageenan solutions with different concentrations were reacted with 50U Cgi1/wf (buffer make up to 375 μ L) at 25 deg.C for 25min. Standing in 100 deg.C metal bath for 8min after reaction to inactivate enzyme, adding 250 μ LpHBH solution, developing in 100 deg.C metal bath for 5min, rapidly cooling to room temperature, centrifuging to obtain supernatant, and measuring light absorption value of the supernatant at 400 nm; simultaneously, mixing the inactivated enzyme solution with the same concentration gradient with the iota-carrageenan solution, repeating the operation, and measuring the light absorption value of the inactivated enzyme solution as a contrast so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the iota-carrageenan standard solution as an abscissa and the corresponding light absorption value increment as an ordinate, obtaining a standard curve of y =1.3844x +0.0226 under specific reaction conditions by linear fitting, wherein R is 2 The value was 0.9990.
Step four, sample determination: and (4) respectively taking 375 mu L of mixed solution, repeating the operation in the third step, substituting the light absorption value increment into a standard curve y =1.3844x +0.0226, and calculating the iota-carrageenan concentration in the reaction system so as to convert the iota-carrageenan content in the mixed solution.
The measurement was carried out in triplicate, and the results were as follows.
| Iota-carrageenan and agar | Iota-carrageenan and kappa-carrageenan | Iota-carrageenan and algin | |
| Assay parallel 1 (mg/mL) | 2.05 | 2.08 | 1.98 |
| Assay parallel 2 (mg/mL) | 2.02 | 1.99 | 2.04 |
| Assay parallel 3 (mg/mL) | 2.04 | 1.95 | 2.04 |
| Determination of average value (mg/mL) | 2.04 | 2.01 | 2.02 |
| Relative error (%) | 2.0％ | 0.5％ | 1.0％ |
From the above results, it can be seen that the method of the present invention has good quantitative specificity.
Example 6: determining iota-carrageenan content in meat product
(1) Preparing an iota-carrageenan solution: weighing chemical grade iota-carrageenan, dissolving in 20mM Na with pH of 8.0 2 HPO 4 -NaH 2 PO 4 Buffer solution, preparing iota-carrageenan standard solution with the concentration of 0.20mg/mL, 0.40mg/mL, 0.60mg/mL, 0.80mg/mL and 1.00mg/mL respectively;
(2) preparation of a pHBH solution: weighing pHBH, dissolving the pHBH in 2mol/LHCl to prepare 200mg/mL pHBH mother liquor, and mixing the mother liquor with 2mol/LNaOH solution according to the weight ratio of 1:9, mixing to prepare 20mg/ml pHBH solution.
(3) Drawing a quantitative standard curve: 375 μ L of the prepared iota-carrageenan solutions with different concentrations are respectively reacted with 100U Cgi1_wf (buffer solution is filled to 375 μ L) at 35 ℃ for 15min. Standing at 100 deg.C for 5min after reaction to inactivate enzyme, adding 250 μ LpHBH solution, developing at 100 deg.C in metal bath for 5min, rapidly cooling to room temperature, centrifuging to obtain supernatant, and measuring light absorption value of the supernatant at 410 nm; simultaneously, mixing the inactivated enzyme solution with the same concentration gradient with the iota-carrageenan solution, repeating the operation, and measuring the light absorption value of the inactivated enzyme solution as a contrast so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the iota-carrageenan standard solution as an abscissa and the corresponding light absorption value increment as an ordinate, obtaining a standard curve y =1.4031x +0.0419 under specific reaction conditions by linear fitting, wherein R is 2 The value was 0.998.
(4) And (3) sample determination: an appropriate amount of the sample was weighed, ground, washed with 85% ethanol solution to remove reducing sugars, and the ethanol solution was discarded for 3 consecutive times. After desugarization, the sample was dried overnight in an oven at 40 ℃ and the sample was dissolved in buffer after drying. And (4) taking 375 mu L of sample solution, repeating the operation in the step (3), substituting the increase of the light absorption value into the standard curve y =1.4031x +0.0419, calculating the iota-carrageenan concentration in the reaction system, and calculating the iota-carrageenan content in the meat product to be 10.4mg/mL.
Example 7: determining content of iota-carrageenan in jelly
(1) Preparing an iota-carrageenan solution: weighing chemical grade iota-carrageenan, dissolving in 20mM Na with pH of 8.0 2 HPO 4 -NaH 2 PO 4 Buffer solution, preparing iota-carrageenan standard solutions with the concentrations of 0.30mg/mL, 0.60mg/mL, 0.90mg/mL, 1.20mg/mL and 1.50mg/mL respectively;
(2) preparation of pHBH solution: weighing pHBH, dissolving the pHBH in 2mol/LHCl to prepare 200mg/mL pHBH mother liquor, and mixing the mother liquor with 2mol/LNaOH solution according to the weight ratio of 1:9, mixing to prepare 20mg/ml pHBH solution.
(3) Drawing a quantitative standard curve: 375 mu L of the iota-carrageenan solution with different concentrations prepared above and 200U Cgi1_Wf (buffer solution is filled to 375 mu L) react for 20min at 30 ℃. Standing at 100 deg.C for 5min after reaction to inactivate enzyme, adding 250 μ LpHBH solution, developing color in 100 deg.C metal bath for 5min, rapidly cooling to room temperature, centrifuging to obtain supernatant, and measuring light absorption value of the supernatant at 415 nm; simultaneously, mixing the inactivated enzyme solution with the same concentration gradient with the iota-carrageenan solution, repeating the operation, and measuring the light absorption value of the inactivated enzyme solution as a contrast, thereby calculating the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the iota-carrageenan standard solution as an abscissa and the corresponding light absorption value increment as an ordinate, obtaining a standard curve of y =1.3726x +0.0429 under specific reaction conditions by linear fitting, wherein R is 2 The value was 0.9993.
(4) And (3) sample determination: an appropriate amount of the sample was weighed, ground, washed with 85% ethanol solution to remove reducing sugars, and the ethanol solution was discarded for 3 consecutive times. After desugarization, the sample was placed in an oven at 40 ℃ to dry overnight, after which the sample was dissolved in buffer. And (4) taking 375 mu L of sample solution, repeating the operation in the third step, substituting the light absorption value increment into the standard curve y =1.3726x +0.0429, calculating the concentration of the iota-carrageenan in the reaction system, and further calculating the content of the iota-carrageenan in the jelly to be 12.45mg/mL.
Finally, it should be noted that the above embodiments, although describing the specific embodiments of the present invention, do not limit the present invention; it will be understood by those skilled in the art that these are by way of example only and that the scope of the invention is defined by the appended claims. All changes, modifications and equivalents that come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Sequence listing
<110> China oceanic university
<120> method for quantitatively detecting iota-carrageenan by enzyme method
<130> China oceanic university
<140> 1
<141> 2019-12-10
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 492
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<213> Artificial Sequence (Artificial Sequence)
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Asn Glu Ile Glu Gln Glu Ile Thr Ala Asn Asn Gln Gln Glu Phe Asn
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Leu Ser Arg Glu Ala Lys Thr Gly Ile Thr Ser Thr Gly Tyr Asn Ser
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Thr Asn Tyr Phe Gln Pro Pro Thr Asn Leu Pro Thr Lys Asn Phe Thr
35 40 45
Gly Ser Thr Ser Thr Gln Leu Gln Thr Leu Ile Asn Asn Ser Ser Thr
50 55 60
Gly Ala Ile Ile Lys Ile Pro Lys Lys Thr Tyr Asn Trp Gly Glu Ile
65 70 75 80
Lys Leu Lys Ser Lys Ile Gln Leu Glu Ile Glu Ser Gly Thr Ile Ile
85 90 95
Lys Pro Ser Asn Asn Asn Ile Lys Arg Ile Phe Ser Ile Gly Ser Ser
100 105 110
Gly Asn Gly Thr Arg Val Thr Asp Val Ser Ile Ile Gly Val Gly Gly
115 120 125
Lys Phe Thr Ile Asp Leu Ser Ala Thr Ala Asn Leu Asn Gln Asn Met
130 135 140
Ala Val Ile Lys Met Gly Arg Val Ser Asn Phe Lys Ile Ser Asn Phe
145 150 155 160
Ile Ile Lys Asp Arg Arg Thr Ser Leu Ala Ser Ile Leu Leu Asn Tyr
165 170 175
Ile Pro Ser Asn Ser Asp Asn Glu Pro Tyr Pro Lys Asn Gly Val Ile
180 185 190
Glu Lys Ile Asn Gln Thr Gly Ile Ser His Thr Gly Tyr Gly Leu Ile
195 200 205
Gln Ala Tyr Ser Ala Ser Asn Val Leu Phe Lys Asn Leu Tyr Cys Lys
210 215 220
Gly Gly Val Thr Leu Arg Leu Glu Thr Asp Asp Lys Thr Met Lys Asp
225 230 235 240
Ala Val Lys Asn Gly Gly Lys Leu Phe Gly Leu Arg Asn Ile Tyr Ala
245 250 255
Asp Met Ile Lys Cys Thr Ser Gly Leu Cys Pro Ile Met Phe Ser Pro
260 265 270
His Phe Thr Glu Asn Gly Lys Ile Thr Ala Arg Asn Ile Thr Ala Thr
275 280 285
Gly Cys Ala Phe Ala Val Arg Val Glu His Gly Phe Ile Glu Val Phe
290 295 300
Asp Thr Asn Lys Thr Tyr Ala Leu Thr Ser Ser Gly Gly Asn Gln Phe
305 310 315 320
Lys Asn Phe Ile Ala Gly Lys Ile Ser Gly Thr Gly Asn Ser Ser Lys
325 330 335
Phe Ile Gly Asn Gln Tyr Lys Arg Ala Asn Gly Thr Gln Trp Ala Ile
340 345 350
Arg Leu Ser Asp Ala Ser Ile Asn Gly Ser Leu Asp Pro Tyr Ile Thr
355 360 365
Asn Gln Ile Gly Tyr Leu Lys Asn Gly Ser Phe Glu Ser Thr Thr Ile
370 375 380
Glu Asn Val Thr Thr Ile Tyr Lys Pro Thr Asn Ala Lys Leu Lys Gln
385 390 395 400
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Asn Pro Thr Asp Thr Gly Met Gly Asn Gly Phe Glu Tyr Tyr Gly Pro
420 425 430
Ser Leu Gly Glu Arg Phe Asp Asn Thr Asn Gly Thr Asn Ser Asn Gly
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Asn Tyr Ile Ile Asn Val Asn Gly Thr Thr Thr Arg Phe Ser Thr Val
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Arg Asn Ile Leu Tyr Asn Thr Pro Thr Ala Cys Thr Ser Asn Ala Tyr
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aacgagattg aacaagaaat caccgcaaac aatcaacaag aatttaacct ctctagagaa 60
gctaaaaccg gaataacttc aacaggatat aattctacaa actattttca acccccaacc 120
aacctaccca caaaaaattt cactggaagt acaagtacac aacttcaaac acttattaat 180
aatagcagca ctggagccat tattaaaatc cctaaaaaaa cgtataactg gggagaaatc 240
aaattaaaat ccaaaataca attagaaata gaaagtggaa ctattattaa accatctaac 300
aataatatta aacgcatttt cagtattggt agttctggta atggtactag agttactgat 360
gttagtatta taggtgtagg cggaaaattt acaatagacc tatctgccac cgctaaccta 420
aaccaaaaca tggcagtgat aaaaatggga agagtatcta attttaaaat ctctaatttt 480
attattaaag acagaagaac ttcactagca tcaatcttac taaattacat cccatcaaac 540
tctgacaatg aaccttatcc taaaaatggt gtcatcgaaa aaattaacca aacaggaatt 600
tcacacacag gttatgggtt aatccaagcg tattctgcat caaatgtatt atttaaaaat 660
ctatattgta aaggtggagt aactcttaga ctagaaactg atgacaaaac catgaaagat 720
gctgttaaaa atggtggtaa gttatttgga ttgagaaata tttacgcaga tatgataaaa 780
tgtactagtg gactttgccc tatcatgttt tctcctcact ttactgaaaa cggaaaaatt 840
acagcaagaa atattaccgc aactggatgt gcttttgctg taagagttga acacgggttt 900
attgaagttt ttgacactaa taaaacttat gctttaacct catctggagg aaaccaattt 960
aaaaacttta ttgctggtaa aatatcagga actggaaact cttctaaatt cataggcaac 1020
caatacaaga gagctaatgg cacacagtgg gctattagat tatctgacgc ttctataaac 1080
ggctctctag atccatacat cactaatcaa attggatatt taaaaaatgg tagttttgaa 1140
agcacaacca tagaaaatgt aacaacaatt tacaaaccta caaacgccaa attaaaacaa 1200
tcctttttac catttatccc ttgtaatgat tggactagca agattaaaaa tcctactgac 1260
acaggaatgg ggaatggttt tgaatactat ggaccttctt taggagaaag atttgacaac 1320
acaaatggta ccaactctaa tggaaactac atcatcaatg taaatggaac aactactagg 1380
ttttcaactg tgagaaacat tctttacaac accccaaccg cctgtacaag taatgcatat 1440
ggtacaattc ctaccacttc taatagtcct gggttataa 1479
Claims (7)
1. A method for quantitatively detecting iota-carrageenan by an enzymatic method is characterized by comprising the following steps: degrading iota-carrageenan into reducing sugar by utilizing the specificity of iota-carrageenan, inactivating enzyme, adding a p-hydroxybenzoyl hydrazine solution for color reaction, measuring the light absorption value of the supernatant at 400-420nm after centrifugation, and comparing with a standard curve to obtain iota-carrageenan content;
the iota-carrageenase is iota-carrageenase Cgi1_ Wf, and the amino acid sequence of the iota-carrageenase is SEQ ID No.1.
2. The enzymatic quantitative iota-carrageenan detection method according to claim 1, wherein the enzymatic quantitative iota-carrageenan detection method comprises the following steps: the iota-carrageenase has the optimum reaction temperature of 25 ℃, can still keep more than 80% of activity at room temperature, has the optimum reaction pH value of 8.0, basically keeps stable in the pH value range of 5.0-9.0, has good storage stability, can be stored stably for at least 30 days at 4 ℃, and can still keep 80% of activity after being placed for 10 days at 25 ℃.
3. The enzymatic quantitative iota-carrageenan detection method according to claim 1, wherein the enzymatic quantitative iota-carrageenan detection method comprises the following steps: the nucleotide sequence of the gene for coding the iota-carrageenase is SEQ ID NO.2 or all genes which can be translated into SEQ ID NO.1.
4. The enzymatic quantitative iota-carrageenan detection method according to claim 1, characterized by comprising the steps of:
(1) Preparing an iota-carrageenan solution: weighing iota-carrageenan with chemical grade or higher purity, dissolving the iota-carrageenan in a buffer solution, and preparing iota-carrageenan standard solution with concentration gradient;
(2) preparation of pHBH solution: weighing pHBH, dissolving the pHBH in HCl, adding NaOH to adjust the pH of the solution to be alkaline, and preparing into a pHBH solution of 10-100 mg/mL;
(3) Drawing a quantitative standard curve: mixing the iota-carrageenan solutions with different concentrations prepared in the step (1) with a proper amount of iota-carrageenan enzyme respectively to react; inactivating the enzyme after the reaction; adding pHBH solution, developing in 100 deg.C metal bath for 5-10min, cooling to room temperature rapidly, centrifuging to obtain supernatant, measuring light absorption value of the supernatant, and measuring the detection wavelength of 400-420nm; mixing the iota-carrageenan solution with the same concentration gradient and the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the mixture as a comparison, and calculating the light absorption value increment corresponding to the iota-carrageenan solution with different concentrations; taking the concentration of the iota-carrageenan standard solution as an abscissa and the light absorption value increment of the iota-carrageenan with each concentration as an ordinate, and obtaining a standard curve under a specific reaction condition through linear fitting;
(4) And (3) sample determination: adding a certain amount of iota-carrageenase into the sample to repeat the reaction in the step (3); and substituting the light absorption value increment into a standard curve under the conditions of corresponding enzyme addition amount, reaction time, reaction temperature, reaction pH and the like to calculate the iota-carrageenan concentration in the reaction system, thereby obtaining the iota-carrageenan content in the sample.
5. The enzymatic quantitative iota-carrageenan detection method according to claim 4, wherein the enzymatic quantitative iota-carrageenan detection method comprises the following steps: the pH value of the buffer solution in the step (1) is 7.0-9.0.
6. The enzymatic quantitative iota-carrageenan detection method according to claim 4, wherein the enzymatic quantitative iota-carrageenan detection method comprises the following steps: in the step (3), the addition amount of the enzyme is 1-1000U, the reaction time is 10-30min, and the reaction temperature is 20-50 ℃.
7. The enzymatic quantitative iota-carrageenan detection method according to claim 4, wherein the enzymatic quantitative iota-carrageenan detection method comprises the following steps: and (4) removing reducing sugar in the sample according to a national standard GB 5009.88-2014 method before measurement.
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| Title |
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| hypothetical protein [Wenyingzhuangia fucanilytica];无;《NCBI GenBank》;20160821;第1-2页 * |
| 基于pHBH法的岩藻聚糖硫酸酯酶酶活测定方法;张翠玉等;《中国食品学报》;20130731;第13卷(第7期);第3节 * |
| 来源于Wenyingzhuangia属海洋细菌的一种β-琼胶酶的克隆表达及性质研究;田雪健等;《食品与发酵工业》;20190218(第08期);摘要和第1.2.4节 * |
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