CN111100855B

CN111100855B - Porphyra polysaccharase and application thereof

Info

Publication number: CN111100855B
Application number: CN201911359527.2A
Authority: CN
Inventors: 常耀光; 张玉莹; 薛长湖; 申晶晶; 曹斯琦; 李兆杰; 徐杰
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2023-04-07
Anticipated expiration: 2039-12-25
Also published as: CN111100855A

Abstract

The invention relates to the technical field of biotechnology and biochemical detection, in particular to porphyra polysaccharase and application thereof. The porphyra polysaccharide enzyme Por16B _ Wf has a novel amino acid sequence and good enzymological properties, and the amino acid sequence is SEQ ID NO.1. Based on Por16B _ Wf, the invention establishes a quantitative detection method of porphyra polysaccharide, which comprises the following steps: mixing Porphyra polysaccharase Por16B _ Wf with a sample to be detected for reaction, hydrolyzing the Porphyra polysaccharose contained in the sample into reducing sugar, adding p-hydroxybenzoyl hydrazine (pHBH) to perform a color reaction with the reducing sugar, and substituting the color absorbance value into a standard curve to obtain the content of the Porphyra polysaccharose in the sample. The detection method has the advantages of rapidness, accuracy, strong specificity and the like.

Description

Porphyra polysaccharase and application thereof

Technical Field

The invention relates to the technical field of biotechnology and biochemical detection, in particular to porphyra polysaccharase and application thereof.

Background

The laver has edibility and high nutritive value, and is one of the most commercially valuable marine algae at present. Porphyra polysaccharide is the main water-soluble polysaccharide in laver, exists in cell wall and intercellular space, and is (1 → 4) -6-OSO ₃ A linear structure consisting of alternating between-alpha-L-galactopyranose and (1 → 3) -beta-D-galactopyranose. Research shows that the porphyra polysaccharide has various physiological activities of eliminating active oxygen free radicals, inhibiting the growth of cancer cells, reducing cholesterol and the like.

Quantitative detection of porphyra polysaccharide is a basic link in quality control, function research and product development of the porphyra polysaccharide. At present, the commonly used quantitative detection methods of the porphyra polysaccharide are a phenol-sulfuric acid method, a liquid chromatography-tandem mass spectrometry method and the like. The phenol-sulfuric acid method has high sensitivity and does not need expensive instruments, but the reproducibility is greatly influenced by test conditions, the time is consumed, and the consumption of samples and reagents is high. The liquid chromatography-tandem mass spectrometry obtains the content of the porphyra polysaccharide by hydrolyzing, deriving and pretreating a sample and measuring the content of monosaccharide consisting of the porphyra polysaccharide.

The porphyra polysaccharase can specifically degrade the porphyra polysaccharose. However, the research on the laver polysaccharase is less at present, five kinds of laver polysaccharase are reported at home and abroad so far, wherein the five kinds of laver polysaccharase comprise one kind of laver reported at home, and only one kind of laver enzyme at home has the research on biochemical properties at present, but the laver polysaccharase has poor stability and quick inactivation, has great limitation in practical application, and can cause great experimental error.

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.

Therefore, the porphyra polysaccharide enzyme with strong specificity, high activity and good stability is found, and has very important significance for quantitative detection of the porphyra polysaccharide.

Disclosure of Invention

The technical problem to be solved by the invention is that the reported porphyra polysaccharide enzyme is unstable and quick to inactivate, has great limitation in practical application, and limits the development of a technology for detecting the content of the porphyra polysaccharide by adopting an enzyme method.

In order to solve the above problems, the present invention provides a novel porphyran polysaccharase Por16B _ Wf having a sequence similarity of at most 79% to other known enzymes (the closest sequence is PorB produced by Zobellia galactanivorans). The optimum reaction temperature of the enzyme is 40 ℃, the optimum reaction pH value is 7.0, and the enzyme is basically kept stable in the pH value range of pH 5.0-10.0; the enzyme has good storage stability, can be stably stored for at least 3 months at 4 ℃, and can still maintain 90% of initial activity after being placed for 24 hours at the temperature of 25 ℃. The enzyme has good reaction specificity, has high activity on porphyra polysaccharide, and has no degradation effect on other marine polysaccharides such as agar, algin, carrageenan and the like. In conclusion, the porphyra polysaccharide enzyme Por16B _ Wf has good application potential, is particularly applied to detecting the content of the porphyra polysaccharide, and has the characteristics of simple and convenient operation, high accuracy and good stability.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a porphyra polysaccharase is porphyra polysaccharase Por16B _ Wf, and the amino acid sequence thereof is SEQ ID NO.1.

SEQ ID NO.1：

QQSPTFIDGEDPKPDNTKWKLVKNMSDEFNGTKVDEEKWQISGQGWIGRAPGLFLADNVKVTNGSLQITTTMLPKPIIKNNKEFTHGGGYVGSRNGMTYGYYECEMKANKTFMSSTFWLINEGKNIKGCDKRTTELDIQECVGQITNDAEWMKNFDQAMNSNTHSRNIPEGCNYIKGSEKSGATIGAKVYNDFHVYGVWWKSKDEILFFLDGKFQSKVKPPSDFDIEMYLRMVVETYDWNPVPADGGMAYSKEDRTTTYNWVRSWTLVNPKK

The enzyme has a sequence similarity of at most 79% to other known enzymes (the closest sequence is PorB produced by Zobellia galactani vorans). Phylogenetic trees were constructed from Por16B _ Wf using MEGA6 with GH16 family porphyra polysaccharase sequences in the CAZy database, and the results are shown in FIG. 3: it can be seen that the porphyran polysaccharase Por16B _ Wf is in the phylogenetic tree of the GH16 family of porphyran polysaccharases. Thus, por16B _ Wf is a new member of the porphyran polysaccharase GH16 family. The Porphyra polysaccharase Por16B _ Wf amino acid sequence was subjected to Blast analysis and multi-sequence alignment of Por16B _ Wf with 4 reported Porphyra polysaccharase sequences of GH16 family using ClustalX2, the results are shown in FIG. 4: the 4 porphyran polysaccharidases were BpGH16B (Genbank EDY 95423.1) from Bacteroides plebeius DSM 17135 and from Zobellia galactanavora Dsij, respectively ^T PorB (Genbank CAZ 95074.1) from Zobellia galacta vorans Dsij ^T PorA (Genbank CAZ 96750.1) and Czochytica CZ1127 from Wenyingzhuangia fuccanolytica ^T Por16A _ Wf (Genbank WP _ 068825731.1), as can be seen in FIG. 4, por16B _ Wf shows different degrees of specificity at other sites besides strictly conserved at key catalytic sites, indicating that Por16B _ Wf is a novel porphyran enzyme in the GH16 family.

The porphyra polysaccharase Por16B _ Wf has high activity on porphyra polysaccharide, but has no degradation effect on other marine polysaccharides such as agar, algin, carrageenan and the like. The optimum reaction temperature is 40 ℃, the optimum reaction pH value is 7.0, and the optimum reaction pH value is 5.The pH value range of 0-10.0 is basically kept stable; the enzyme has good storage stability, can be stably stored for at least 3 months at 4 ℃, and can still maintain 90% of initial activity after being placed for 24 hours at the temperature of 25 ℃; enzyme kinetic constant K _m Is 2.73mg/mL, K _cat Is 38.20s ^-1 ，K _m /K _cat Is 14 μ M ^-1 s ^-1 ，V _max It was 66.23U/mg. As described above, the porphyran polysaccharase Por16B _ Wf of the present invention has superior enzymatic properties, good stability, easy storage, strong specificity for substrate binding, and a fast enzymatic hydrolysis rate, compared to other porphyran polysaccharases, and is an ideal enzyme for quantitative detection of porphyra polysaccharides.

The nucleotide sequence of the gene for coding the porphyra polysaccharide enzyme Por16B _ Wf is SEQ ID NO.2 and all genes capable of being translated into SEQ ID NO.1.

SEQ ID NO.2：

CAACAGTCACCAACTTTTATTGATGGAGAAGACCCAAAACCAGACAATACAAAATGGAAATTGGTTAAAAATATGTCCGATGAGTTTAATGGTACAAAGGTAGATGAAGAAAAATGGCAAATATCTGGTCAAGGATGGATCGGTAGGGCGCCAGGATTATTTCTTGCTGATAATGTAAAAGTTACAAATGGAAGTTTGCAAATAACCACAACCATGTTGCCAAAACCAATAATAAAAAATAATAAGGAGTTTACGCATGGAGGTGGTTATGTTGGATCTAGAAACGGGATGACTTATGGTTATTATGAGTGTGAAATGAAGGCTAATAAAACATTTATGTCTTCTACTTTTTGGTTGATAAATGAAGGAAAAAACATAAAAGGCTGTGATAAAAGAACCACAGAATTAGACATACAAGAATGCGTTGGACAAATTACAAATGATGCTGAGTGGATGAAAAATTTTGACCAAGCCATGAATTCCAATACACATAGTCGAAATATTCCTGAAGGTTGTAATTATATTAAAGGTTCAGAAAAATCAGGAGCCACTATTGGAGCAAAGGTATATAACGATTTTCACGTGTATGGTGTTTGGTGGAAGTCTAAAGATGAAATACTTTTCTTTTTAGATGGTAAATTTCAATCGAAAGTAAAACCACCATCCGATTTTGATATTGAGATGTATTTAAGAATGGTTGTTGAAACTTATGATTGGAATCCAGTTCCAGCTGATGGTGGAATGGCCTACTCTAAAGAAGATAGAACCACCACTTATAATTGGGTTAGGTCTTGGACATTGGTAAATCCTAAAAAATAA

According to the discovery, the invention establishes a quantitative detection method of the porphyra polysaccharide based on the porphyra polysaccharide Por16B _ Wf, adopts the reaction of the Por16B _ Wf and a sample to determine the light absorption value at 400-420nm, and has the advantages of specificity, rapidness, simplicity and convenience and the like.

Further, p-hydroxybenzoyl hydrazine (pHBH) was used as a color developer in the measurement.

The method specifically comprises the following steps of:

(1) Preparing a laver polysaccharide solution: weighing porphyra polysaccharide with chemical grade or higher purity, and dissolving the porphyra polysaccharide in buffer solution to prepare a porphyra polysaccharide standard solution with concentration gradient;

(2) preparation of a pHBH solution: pHBH is weighed and dissolved in HCl to prepare pHBH mother liquor, then NaOH is added to adjust the pH of the solution to be alkaline, and 10-100mg/mL of pHBH solution is prepared. This is because pHBH develops color with the reducing end under alkaline conditions and is easy to store under acidic conditions;

(3) Drawing a quantitative standard curve: mixing the porphyra polysaccharide solutions with different concentrations prepared in the step (1) with a proper amount of Por16B _ Wf enzyme solution respectively for reaction, wherein the addition amount of the enzyme is 1-1000U, the reaction time is 1-30min, the reaction temperature is 20-60 ℃, and Por16B _ Wf has high enzymolysis activity within the parameter range, so that the rapid enzymolysis reaction is ensured; the addition amount of the enzyme and the reaction time need to correspond to each other, and if the addition amount of the enzyme is small, the reaction time is increased, so that the complete enzymolysis of the sample is ensured; the Por16B _ Wf has higher enzymolysis activity and can keep stable within the range of 20-60 ℃; after the reaction, the mixture is placed in a metal bath at 100 ℃ for 5-10min to inactivate the enzyme; adding pHBH solution, developing in 100 deg.C metal bath for 5-10min, rapidly cooling to room temperature, centrifuging to obtain supernatant, measuring light absorption value of the supernatant, and detecting wavelength of 400-420nm; mixing the porphyra polysaccharide solution with the same concentration gradient and the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the reaction as a control, and then calculating the light absorption value increment corresponding to the porphyra polysaccharide solution with different concentrations; taking the concentration of the laver polysaccharide standard solution as a horizontal coordinate, taking the light absorption value increment of the laver polysaccharide 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 Por16B _ Wf into the sample solution 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 adding amount, reaction time, reaction temperature, reaction pH and the like to calculate the concentration of the porphyra polysaccharide in the reaction system, thereby obtaining the content of the porphyra polysaccharide in the sample.

Further, the pH of the buffer solution in the step (1) is 7.0-9.0.Por16B _ Wf has higher enzymolysis activity and stability in the pH range of 6.0-9.0.

Furthermore, the reducing sugar in the sample is removed according to the method of national standard GB 5009.88-2014 before the determination of the step (4).

The invention has the beneficial effects that:

(1) The present invention provides a porphyra polysaccharase, the sequence similarity of the porphyra polysaccharase and other known enzymes is 79% at most. The porphyra polysaccharide enzyme has the advantages of excellent enzymological properties, good stability, easy storage, strong specificity for substrate combination and high enzymolysis rate, and is an ideal enzyme for detecting the quantitative detection of the porphyra polysaccharide.

(2) The porphyra polysaccharase has high activity on the porphyra polysaccharose, but has no degradation effect on other marine polysaccharides such as agar, algin, carrageenan and the like.

(3) The quantitative detection method of the porphyra polysaccharide utilizes the porphyra polysaccharide enzyme to degrade the porphyra polysaccharide in the sample to form reducing sugar, the reducing sugar can perform color reaction with pHBH, the solution color is in direct proportion to the concentration of the reducing sugar, and the content of the porphyra polysaccharide in the detection sample can be obtained by detecting the light absorption value increment before and after the reaction of the reaction solution. The detection method has the advantages of good linear range and high accuracy, and can be popularized and used in the market.

Drawings

FIG. 1: the target gene electrophoresis pattern of the porphyra polysaccharase Por16B _ Wf is disclosed;

FIG. 2: the invention relates to an electrophoretogram after purifying porphyra polysaccharase Por16B _ Wf;

FIG. 3: a phylogenetic tree constructed by Por16B _ Wf and all known GH16 family porphyra polysaccharase; wherein the asterisk is porphyra polysaccharase Por16B _ Wf;

FIG. 4 is a schematic view of: por16B _ Wf multiple sequence alignment results; among these, conserved residues of Por16B _ Wf are in black boxes.

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: clone expression and acquisition of Porphyra polysaccharidase Por16B _ Wf in Escherichia coli

Cultivation of Wenyingzhuangianducanthritica CZ1127 in 2216E Medium ^T Until the end of logarithm, extracting whole genome DNA, designing upstream and downstream primers (5 '-GACACGATCCAAAGAAAGCAGTAAATGATACTACA; 5' -GACACCTCGAGCTATTGATATACTCTTACATTATTTC) according to target genes, and carrying out PCR by taking the whole genome as a template, wherein the PCR reaction conditions are as follows: 95 ℃ for 3min,95 ℃ for 20s,42 ℃ for 22s,72 ℃ for 60s,22 cycles, and finally, the temperature of 72 ℃ lasts for 5min to obtain the porphyra polysaccharide enzyme Por16B _ Wf gene fragment, and the porphyra polysaccharide enzyme Por16B _ Wf gene fragment is connected to a pET-28a (+) vector to form a recombinant plasmid. The recombinant plasmid is 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. Centrifuging to collect thallus, adding 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate (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 porphyra polysaccharide enzyme Por16B _ Wf.

The crude enzyme solution obtained above was subjected to HisTrap ^TM HP purification and HiTrap ^TM Desalting desaltings to obtain purified enzyme solution. The activity of 1U is defined as the activity of generating 1 mu mol of reducing sugar within 1min, and the activity of recombinase fermentation liquor is 45.31U/mg.

Example 2: clone expression and acquisition of Porphyra polysaccharase Por16B _ 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 '-TGCCAAGTTATTATTCCAACAGTCAACTTTTATTG; 5' -GTTATGTTAGATCTC) according to target geneTTATTTTTTAGGATTTACCAATGT), PCR is carried out using the whole genome as a template as in example 1 to obtain a porphyrinase Por16B _ Wf gene fragment, which is ligated to pHT01 vector to construct a recombinant plasmid. Transforming the recombinant plasmid into bacillus subtilis competent cells, screening positive clones, and carrying out induced expression in LB culture solution by using isopropyl thiogalactoside, wherein the induction temperature is 37 ℃ and the induction time is 12h. Centrifuging to collect thallus, adding 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 for 2s, gap 6s, circulation for 99 times), centrifuging and collecting supernatant, namely the crude enzyme solution of the porphyra polysaccharide enzyme Por16B _ Wf. The purification operation in example 1 was repeated to obtain a pure enzyme solution, and the activity of the recombinase fermentation broth was detected to be 60.71U/mg.

Example 3: clone expression and acquisition of porphyra polysaccharidase Por16B _ Wf in pichia pastoris

Wenyingzhuangiantucantanica CZ1127 cultured in 2216E medium ^T Until the end of logarithm, extracting whole genome DNA, designing upstream and downstream primers (5 '-TAACAGTATTATTCCAACAGTCACAACTTTTATTG; 5' -TGGATGTTAGATCTTTTTTTTAGGATTTACAATGT) according to target genes, carrying out PCR (polymerase chain reaction) by taking the whole genome as a template in example 1 to obtain a porphyria polysaccharase Por16B _ Wf gene fragment, connecting the porphyria polysaccharase Por16B _ Wf gene fragment to a pPIC9k vector to form recombinant plasmids, and adding the recombinant plasmids into pichia pastoris GS115 competent cells to form recombinant cells; screening positive clones, inoculating to YPD medium, culturing at 30 deg.C for 20h, inoculating to BMGY medium, shake culturing at 30 deg.C and 200rpm until OD600=2.0, centrifuging to collect thallus, discarding supernatant, resuspending the precipitate in 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 broth is detected to be 82.84U/mg (1U activity is defined as the activity of generating 1 mu mol of reducing sugar within 1 min).

Example 4: the method of the invention is subjected to accuracy verification

The method and the phenol-sulfuric acid method are utilized to determine the content of the porphyra polysaccharide in the sample:

(1) Preparing a laver polysaccharide solution: weighing chemical grade violetThe vegetable polysaccharide is dissolved in 20mM Na with pH of 8.0 ₂ HPO ₄ -NaH ₂ PO ₄ Buffer solution, preparing standard solutions of porphyra polysaccharide with the concentrations of 2.00mg/mL, 3.00mg/mL, 4.00mg/mL, 5.00mg/mL and 6.00mg/mL respectively;

(2) preparation of a pHBH solution: weighing pHBH, dissolving the pHBH in 2mol/L HCl to prepare 200mg/mL pHBH mother liquor, and mixing the mother liquor with 2mol/L NaOH solution according to the ratio of 1:9, mixing to prepare 20mg/ml pHBH solution.

(3) Drawing a quantitative standard curve: 375 μ L of the prepared porphyra polysaccharide solution with different concentrations is respectively reacted with 10U of Por16B _wf (buffer solution is filled to 375 μ L) at 35 deg.C for 20min. Standing in 100 deg.C metal bath for 10min 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 laver polysaccharide solution, repeating the operation, and measuring the light absorption value of the inactivated enzyme solution as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the laver polysaccharide standard solution as an abscissa and the corresponding absorbance increment as an ordinate, obtaining a standard curve y =1.2194x +0.1035 under specific reaction conditions by linear fitting, wherein R is ² The value was 0.9976.

(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 (3) taking 375 mu L of sample solution, repeating the operation in the step (3), substituting the increment of the light absorption value into the standard curve y =1.2194x +0.1035, calculating the concentration of the porphyra polysaccharide in the reaction system, and converting the content of the porphyra polysaccharide in the sample.

(5) The content of porphyra polysaccharide in the sample is determined according to a phenol-sulfuric acid method in national standard SN/T4260-2015.

Three replicates of each procedure were performed and the results are shown in the table below.

	The method of the invention	Phenol-sulfuric acid process
			Assay parallel 1 (mg/mL)	4.96	5.06
Assay parallel 2 (mg/mL)	5.04	5.01
			Assay parallel 3 (mg/mL)	5.00	4.96
Determination of average value (mg/mL)	5.00	5.01

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: the specificity of the method of the invention is verified

The method of the invention is used for quantifying the porphyra polysaccharide in the three mixed solutions respectively.

Step one, preparation of a laver polysaccharide solution: weighing chemical grade porphyra polysaccharide, dissolving in 20mM Na with pH of 7.0 ₂ HPO ₄ -NaH ₂ PO ₄ A buffer solution, namely preparing a laver polysaccharide solution with the concentration of 8.00 mg/mL; simultaneous systemPreparing agar, carrageenan and algin solution with the concentration of 8.00 mg/mL; and uniformly mixing agar, carrageenan and alginate solution with the laver polysaccharide solution in equal volume respectively to prepare a mixed solution, wherein the laver polysaccharide concentration of the mixed solution is 4.00mg/mL.

Step two, preparation of a pHBH solution: weighing pHBH to be dissolved in 2mol/LHCl to prepare 200mg/mL pHBH mother liquor, and mixing the mother liquor with 2mol/L NaOH solution according to the weight ratio of 1:9 to prepare 20mg/mL pHBH solution.

Step three, drawing a quantitative standard curve: 375 μ L of the prepared porphyra polysaccharide solution with different concentrations is respectively reacted with 200U Por16B _wf (buffer solution is filled to 375 μ L) at 25 deg.C 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 400 nm; simultaneously, mixing the inactivated enzyme solution with the same concentration gradient with the laver polysaccharide solution, repeating the operation, and measuring the light absorption value of the inactivated enzyme solution as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the laver polysaccharide standard solution as an abscissa and the corresponding light absorption value increment as an ordinate, obtaining a standard curve of y =1.2444x +0.0226 under specific reaction conditions through linear fitting, wherein R is ² The value was 0.9990.

Step four, sample determination: and (3) respectively taking 375 mu L of mixed solution, repeating the operation in the third step, substituting the increment of the light absorption value into the standard curve y =1.2444x +0.0226, calculating the concentration of the porphyra polysaccharide in the reaction system, and converting the content of the porphyra polysaccharide in the mixed solution.

The measurement was carried out in triplicate, and the measurement results were as follows.

	Laver polysaccharide and agar	Thallus Porphyrae polysaccharide and carrageenan	Thallus Porphyrae polysaccharide and algin
				Assay parallel 1 (mg/mL)	4.02	4.08	3.98
Assay parallel 2 (mg/mL)	4.00	3.99	4.04
				Assay parallel 3 (mg/mL)	4.04	3.95	4.04
Determination of average value (mg/mL)	4.02	4.01	4.02
				Relative error (%)	0.5％	0.25％	0.5％

From the above results, it can be seen that the method of the present invention has good specificity.

Example 6: measuring the content of laver polysaccharide in laver

(1) Preparing a laver polysaccharide solution: weighing chemical-grade laver juiceSugar dissolved in 20mM Na pH8.0 ₂ HPO ₄ -NaH ₂ PO ₄ Buffer solution, preparing standard solution of laver polysaccharide with concentration of 0.10mg/mL, 0.20mg/mL, 0.30mg/mL, 0.40mg/mL and 0.50mg/mL respectively;

(3) Drawing a quantitative standard curve: 375 μ L of the above prepared porphyra polysaccharide solution with different concentrations is respectively reacted with 150U Por16B _wf (buffer solution is filled to 375 μ L) at 35 deg.C for 25min. Standing in 100 deg.C metal bath for 6min after reaction to inactivate enzyme, adding 250 μ L pHBH 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 410 nm; simultaneously, mixing the inactivated enzyme solution with the same concentration gradient with the laver polysaccharide solution, repeating the operation, and measuring the light absorption value of the inactivated enzyme solution as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the laver polysaccharide standard solution as an abscissa and the corresponding light absorption value increment as an ordinate, obtaining a standard curve of y =1.2247x +0.0147 under specific reaction conditions by linear fitting, wherein R is ² The value was 0.9990.

(4) And (3) sample determination: taking commercially available laver as an example, a proper amount of laver sample is weighed, ground, washed with 85% ethanol solution to remove reducing sugar, and the ethanol solution is discarded for 3 times. After desugarization, the sample was placed in an oven at 40 ℃ to dry overnight, after which the sample was dissolved in buffer. And (3) taking 375 mu L of sample solution, repeating the steps in the third step, substituting the increment of the light absorption value into the standard curve y =1.2247x +0.0147, calculating the concentration of the porphyra polysaccharide in the reaction system, and converting the content of the porphyra polysaccharide in the sample. The content of thallus Porphyrae polysaccharide in the thallus Porphyrae sample is 22.43 + -0.42 mg/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.

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Sequence listing

<110> China oceanic university

<120> porphyra polysaccharase and application thereof

<130> China oceanic university

<140> 1

<141> 2019-12-10

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 272

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gln Gln Ser Pro Thr Phe Ile Asp Gly Glu Asp Pro Lys Pro Asp Asn

1 5 10 15

Thr Lys Trp Lys Leu Val Lys Asn Met Ser Asp Glu Phe Asn Gly Thr

20 25 30

Lys Val Asp Glu Glu Lys Trp Gln Ile Ser Gly Gln Gly Trp Ile Gly

35 40 45

Arg Ala Pro Gly Leu Phe Leu Ala Asp Asn Val Lys Val Thr Asn Gly

50 55 60

Ser Leu Gln Ile Thr Thr Thr Met Leu Pro Lys Pro Ile Ile Lys Asn

65 70 75 80

Asn Lys Glu Phe Thr His Gly Gly Gly Tyr Val Gly Ser Arg Asn Gly

85 90 95

Met Thr Tyr Gly Tyr Tyr Glu Cys Glu Met Lys Ala Asn Lys Thr Phe

100 105 110

Met Ser Ser Thr Phe Trp Leu Ile Asn Glu Gly Lys Asn Ile Lys Gly

115 120 125

Cys Asp Lys Arg Thr Thr Glu Leu Asp Ile Gln Glu Cys Val Gly Gln

130 135 140

Ile Thr Asn Asp Ala Glu Trp Met Lys Asn Phe Asp Gln Ala Met Asn

145 150 155 160

Ser Asn Thr His Ser Arg Asn Ile Pro Glu Gly Cys Asn Tyr Ile Lys

165 170 175

Gly Ser Glu Lys Ser Gly Ala Thr Ile Gly Ala Lys Val Tyr Asn Asp

180 185 190

Phe His Val Tyr Gly Val Trp Trp Lys Ser Lys Asp Glu Ile Leu Phe

195 200 205

Phe Leu Asp Gly Lys Phe Gln Ser Lys Val Lys Pro Pro Ser Asp Phe

210 215 220

Asp Ile Glu Met Tyr Leu Arg Met Val Val Glu Thr Tyr Asp Trp Asn

225 230 235 240

Pro Val Pro Ala Asp Gly Gly Met Ala Tyr Ser Lys Glu Asp Arg Thr

245 250 255

Thr Thr Tyr Asn Trp Val Arg Ser Trp Thr Leu Val Asn Pro Lys Lys

260 265 270

<210> 2

<211> 819

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caacagtcac caacttttat tgatggagaa gacccaaaac cagacaatac aaaatggaaa 60

ttggttaaaa atatgtccga tgagtttaat ggtacaaagg tagatgaaga aaaatggcaa 120

atatctggtc aaggatggat cggtagggcg ccaggattat ttcttgctga taatgtaaaa 180

gttacaaatg gaagtttgca aataaccaca accatgttgc caaaaccaat aataaaaaat 240

aataaggagt ttacgcatgg aggtggttat gttggatcta gaaacgggat gacttatggt 300

tattatgagt gtgaaatgaa ggctaataaa acatttatgt cttctacttt ttggttgata 360

aatgaaggaa aaaacataaa aggctgtgat aaaagaacca cagaattaga catacaagaa 420

tgcgttggac aaattacaaa tgatgctgag tggatgaaaa attttgacca agccatgaat 480

tccaatacac atagtcgaaa tattcctgaa ggttgtaatt atattaaagg ttcagaaaaa 540

tcaggagcca ctattggagc aaaggtatat aacgattttc acgtgtatgg tgtttggtgg 600

aagtctaaag atgaaatact tttcttttta gatggtaaat ttcaatcgaa agtaaaacca 660

ccatccgatt ttgatattga gatgtattta agaatggttg ttgaaactta tgattggaat 720

ccagttccag ctgatggtgg aatggcctac tctaaagaag atagaaccac cacttataat 780

tgggttaggt cttggacatt ggtaaatcct aaaaaataa 819

Claims

1. An application of porphyra polysaccharase in quantitative detection of porphyra polysaccharide is characterized in that: is Porphyra polysaccharase Por16B _ Wf with amino acid sequence of SEQ ID NO.1.

2. A quantitative detection method of porphyra polysaccharide is characterized in that: the measurement of absorbance at 400-420nm using Por16B _ Wf, a porphyrase enzyme as defined in claim 1, reacted with a sample.

3. The quantitative determination method of porphyra polysaccharide as claimed in claim 2, wherein: p-hydroxybenzoyl hydrazine (pHBH) is used as a color developing agent during detection.

4. The quantitative measurement method of porphyra polysaccharide as claimed in claim 2, characterized by comprising the steps of:

(2) preparation of a pHBH solution: weighing pHBH, dissolving the pHBH in HCl to prepare pHBH mother liquor, adding NaOH to adjust the pH of the solution to be alkaline, and preparing 10-100mg/mL pHBH solution;

(3) Drawing a quantitative standard curve: mixing the porphyra polysaccharide solutions with different concentrations prepared in the step (1) with a proper amount of Por16B _ Wf enzyme solution respectively for reaction, wherein the adding amount of the enzyme is 5-20 mu L, the reaction time is 1-30min, and the reaction temperature is 20-60 ℃; after the reaction, the mixture is placed in a metal bath at 100 ℃ for 5-10min to inactivate the enzyme; adding pHBH solution, developing in 100 deg.C metal bath for 5-10min, rapidly cooling to room temperature, centrifuging to obtain supernatant, measuring light absorption value of the supernatant, and detecting wavelength of 400-420nm; mixing the porphyra polysaccharide solution with the same concentration gradient and the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the reaction as a control, and then calculating the light absorption value increment corresponding to the porphyra polysaccharide solution with different concentrations; taking the concentration of the laver polysaccharide standard solution as an abscissa and the light absorption value increment of the laver polysaccharide 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 Por16B _ Wf 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 adding amount, reaction time, reaction temperature, reaction pH and the like, and calculating the concentration of the porphyra polysaccharide in the reaction system so as to obtain the content of the porphyra polysaccharide in the sample.

5. The quantitative determination method of porphyra polysaccharide as claimed in claim 4, wherein: the pH value of the buffer solution in the step (1) is 7.0-9.0.

6. The quantitative determination method of porphyra polysaccharide as claimed in claim 4, wherein: and (4) performing proper pretreatment on the sample according to the sample property before the measurement.