CN111100854B - Cold-adapted kelp amylase and application thereof - Google Patents

Cold-adapted kelp amylase and application thereof Download PDF

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CN111100854B
CN111100854B CN201911359486.7A CN201911359486A CN111100854B CN 111100854 B CN111100854 B CN 111100854B CN 201911359486 A CN201911359486 A CN 201911359486A CN 111100854 B CN111100854 B CN 111100854B
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常耀光
李嘉靖
薛长湖
申晶晶
张玉莹
刘艳艳
徐杰
张恬恬
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Ocean University of China
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Abstract

The invention relates to the technical field of biotechnology and biochemical detection, in particular to cold-adapted kelp amylase and application thereof. The Laminaria japonica amylase Lam16A _ Wa has a novel amino acid sequence and good enzymological properties, and the amino acid sequence is SEQ ID NO. 1. Based on Lam16A _ Wa, the invention establishes a quantitative detection method of kelp starch: mixing Laminarin amylase Lam16A _ Wa with a sample to be detected for reaction, hydrolyzing Laminarin starch in the sample into reducing sugar, adding p-hydroxybenzoyl hydrazine (pHBH) to react with the reducing sugar for color development, and substituting the color-developing light absorption value into a standard curve to obtain the Laminarin starch content in the sample. The detection method has the advantages of rapidness, accuracy, strong specificity and the like.

Description

Cold-adapted kelp amylase and application thereof
Technical Field
The invention relates to the technical field of biotechnology and biochemical detection, in particular to cold-adapted kelp amylase and application thereof.
Background
The kelp starch is an important storage polysaccharide of marine brown algae and diatom, the main structure is a linear beta-1, 3-glucan main chain and a branched chain connected with the beta-1, 6-bond on the linear beta-1, 3-glucan main chain, and the kelp starch is reported to have biological activities of resisting pathogens, regulating immunity, resisting oxidation and the like. The kelp starch oligosaccharide obtained by degrading kelp starch is proved to have the activities of resisting apoptosis, activating monocyte, resisting tumor, inducing plant to generate disease resistance and the like.
The quantitative detection of the kelp starch is a basic link in the quality control, the function research and the product development of the kelp starch. At present, the commonly used quantitative detection methods of the kelp starch are a phenol-sulfuric acid method, a high performance liquid chromatography and the like. The phenol-sulfuric acid method has high sensitivity, does not need expensive instruments, but has higher accuracy and repeatability under the influence of test conditions, consumes more time and consumes more samples and reagents. The high performance liquid chromatography is used for hydrolyzing, derivatizing and pretreating a sample, and measuring the content of monosaccharide consisting of the kelp starch to obtain the content of the kelp starch.
The kelp amylase can specifically degrade beta-1, 3-bonds in kelp starch. In the study of GH16 family kelp amylases, extensive studies have been made on the properties, crystal structures, thermal stabilities and their influencing factors of kelp amylases derived from hyperthermophiles and mesophiles. In contrast, the properties of the kelp amylase with chilling property are less studied, and the chilling property is a good property for practical application. Most of the kelp amylase with properties researched at present has higher optimal temperature (above 50 ℃), the reaction needs to consume heat energy, and most of the enzymes are unstable at high temperature and are inactivated quickly, so that the kelp amylase has great limitation in practical application.
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, finding the kelp amylase with the psychrotrophy has very important significance for the quantitative detection of the kelp starch.
Disclosure of Invention
The technical problems to be solved by the invention are that most of kelp amylase which is researched by nature has higher optimal temperature (more than 50 ℃), the reaction needs to consume heat energy, and most of the enzymes are unstable at high temperature and are inactivated quickly, so that the kelp amylase has great limitation in practical application.
In order to solve the problems, the invention provides a cold-adapted kelp amylase Lam16A _ Wa with novel sequence, good enzymatic property and high activity, and provides a method for detecting the content of kelp starch in a sample based on the enzyme, wherein the method has the advantages of specificity, rapidness, simplicity and convenience.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a cold-adapted sea tangle amylase is sea tangle amylase Lam16A _ Wa, and its amino acid sequence is SEQ ID NO. 1.
SEQ ID NO.1：
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSKDKVAVNDTTEPTVDCPGKITNAVPKGNNNVLVWSDEFNTAGSPCTENWTYDIGNSGWGNNELQYYTRDNAVVENGILKITAKKETYRGAPYTSARMKTQDRFSFTYGKVEVRAKLPYGLGTWPAIWMLGDNISTVGWPTCGEIDIMERAGTSSANLKITTSAIHNSSGYGNTPYVGENEPKEDLSTEFHLYSINWTADKIEFMVDNVVHYTYQPATKDAANWPFDKNQFVILNVAMGGTLGGNVDPDFTESAMEIDYVRVYQ
The degree of sequence similarity of the enzyme with other known enzymes is only 55% at the most. The MEGA6 is used for constructing a phylogenetic tree by utilizing Lam16A _ Wa and a sequence of studied kelp amylase of GH16 family, and the result is shown in figure 3: laminarin amylase Lam16A _ Wa can be seen in the phylogenetic tree of GH16 family Laminarin amylases. Thus, Lam16A _ Wa is a new member of the GH16 family of kelp amylases. The Laminaria amylase Lam16A _ Wa amino acid sequence was subjected to Blast analysis and Laminaria 16A _ Wa was subjected to multiple sequence alignment with 7 Laminaria amylase sequences having a crystal structure known from GH16 family using ClustalX2, and the results are shown in FIG. 4: these 7 kelp amylases are BglF (Genbank BAE54302.1) derived from Nocardiaopsis sp.F96, BglII (Genbank AAC38290.1) derived from Cellulosimicrobium cellulans LL-G109/DSM10297/DK-1, ZgLamC (Genbank CAZ95067.1) derived from Zobellia galactanicvorans DsijT, LamR (Genbank AAC69707.1) derived from Rhodothermus marina ITI278, TmLam (Genbank D35118.1) derived from Thermotoga maritima MSB8, TpLam (Genbank ABQ46917.1) derived from Thermota petrophila RKU-1 and ZgLaaLak (Genbank 96583.1 mA) derived from Zoberia phytans DjT, respectively. As can be seen from FIG. 4, Lam16A _ Wa shows different degrees of specificity at other sites except for strict conservation at key catalytic sites, and Blast confirmed that Lam16A _ Wa has only 55% of the highest similarity with Orf00540 sequence produced by Formosa algae KMM 3553, indicating that Lam16A _ Wa is a novel kelp amylase in GH16 family.
The kelp amylase Lam16A _ Wa has high activity on kelp starch, but has no degradation effect on other marine polysaccharides such as agar, algin, carrageenan and the like. The optimum reaction temperature is 35 ℃, which is far lower than other kelp amylase subjected to property research, the activity of the kelp amylase can be still kept more than 80% at room temperature, the kelp amylase has good cold adaptability, the optimum reaction pH value is 6.0, the kelp amylase is basically kept stable (the enzyme activity residual rate is more than 80%) in the pH value range of pH 3.0-11.0, the enzyme kinetic constant Km is 1.29mg/mL, and the Kcat is 111.89s-1Vmax is 203.01U/mg. As described above, compared with other kelp amylases, the kelp amylase Lam16A _ Wa of the present invention has excellent enzymatic properties, good stability, easy storage, and particularly excellent cold-adapted properties, and is also strong in substrate binding specificity and rapid in enzymatic hydrolysis rate, and therefore, is an ideal enzyme for quantitative detection of kelp starch.
The nucleotide sequence of the gene for coding the kelp amylase Lam16A _ Wa is SEQ ID NO.2 and all genes capable of being translated into SEQ ID NO. 1.
SEQ ID NO.2：
ATGGGCAGCAGCCATCATCATCATCATCATAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCAAAGACAAAGTAGCAGTAAATGATACTACAGAACCTACAGTAGATTGTCCTGGAAAAATAACCAATGCAGTTCCAAAGGGAAATAATAACGTATTGGTTTGGTCTGATGAGTTTAATACAGCAGGATCTCCTTGTACAGAAAACTGGACTTACGACATAGGAAACAGTGGTTGGGGAAATAATGAACTTCAATATTACACAAGAGATAATGCAGTGGTTGAGAACGGAATTTTAAAAATTACGGCAAAAAAAGAAACCTATAGAGGAGCTCCATATACATCTGCAAGAATGAAAACTCAAGACAGATTTAGTTTTACTTACGGTAAAGTAGAAGTACGTGCAAAATTACCTTATGGTTTAGGTACTTGGCCTGCTATATGGATGTTGGGAGATAATATTAGTACAGTAGGGTGGCCAACCTGTGGAGAAATAGATATTATGGAACGTGCCGGAACTAGTTCAGCTAATTTAAAAATTACCACAAGTGCTATTCATAATTCTTCTGGATATGGAAACACACCTTACGTAGGAGAAAATGAACCTAAAGAAGATTTGTCTACAGAATTTCATTTGTATTCCATTAACTGGACAGCAGATAAGATTGAATTTATGGTAGATAATGTAGTGCATTATACCTATCAGCCTGCTACAAAAGATGCAGCTAATTGGCCTTTTGATAAAAATCAATTTGTCATTTTAAATGTAGCTATGGGTGGAACTTTAGGAGGTAATGTTGATCCTGATTTTACAGAATCGGCAATGGAAATAGATTATGTAAGAGTATATCAATAG
According to the findings, the invention establishes a quantitative detection method of the kelp starch based on Lam16A _ Wa, adopts the reaction of the enzyme Lam16A _ Wa and a sample to determine the light absorption value at 400-420nm, and has the advantages of specificity, rapidness, simplicity 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 kelp starch solution: weighing kelp starch with chemical grade or higher purity, and dissolving the kelp starch in buffer solution to prepare a kelp starch 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.
(3) Drawing a quantitative standard curve: mixing the kelp starch solutions with different concentrations prepared in the step (1) with a proper amount of Lam16A _ Wa enzyme solution respectively for reaction, wherein the addition amount of the enzyme is 1-1000U, the reaction time is 10-30min, and the reaction temperature is 20-50 ℃; after the reaction, the mixture is placed in a metal bath at 100 ℃ for 5-10min to inactivate the enzyme; then adding a pHBH solution, developing in a metal bath at 100 ℃ for 5-10min, quickly cooling to room temperature, centrifuging to obtain a 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 kelp starch solution with the same concentration gradient and the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the kelp starch solution as a control, and then calculating the light absorption value increment corresponding to the kelp starch solution with different concentrations; taking the concentration of the standard solution of the kelp starch as a horizontal coordinate, taking the light absorption value increment of the kelp starch with each concentration as a vertical coordinate, and obtaining a standard curve under a specific reaction condition through linear fitting;
the addition amount of the enzyme is 1-1000U: ensuring that the enzyme is in excess and does not form a waste of resources. Too much will be wasted, and too little will result in insufficient enzymatic hydrolysis, thereby affecting the quantitative results.
The reaction time is 10-30 min: less than 10min may result in insufficient enzymolysis and poor linear relationship of detection, while more than 30min wastes time. The addition amount of enzyme and the reaction time need to correspond to each other, and the reaction time is increased when the addition amount of enzyme is small, so that the complete enzymolysis of the sample can be ensured.
The reaction temperature is 20-50 ℃: the enzyme has high enzyme activity at the optimum temperature of 35 ℃ and 20-50 ℃, and is favorable for full enzymolysis.
After the reaction, the mixture is placed in a metal bath at 100 ℃ for 5-10min to inactivate the enzyme: the temperature of 100 ℃ is the common temperature for enzyme deactivation, less than 5min may cause incomplete enzyme deactivation, and more than 10min wastes time.
(4) And (3) sample determination: adding a certain amount of Lam16A _ Wa into the sample solution after carrying out proper pretreatment on the sample, and repeating 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 concentration of the kelp starch in the reaction system, thereby obtaining the content of the kelp starch in the sample.
Further, the pH of the buffer solution in the step (1) is 7.0-11.0. The enzyme has a higher enzymatic activity in this pH range.
Further, the reducing sugars in the sample are removed before the determination in step (4) according to the method of national standard GB 5009.88-2014.
The invention has the beneficial effects that:
(1) the invention provides a cold-adapted kelp amylase, and the sequence similarity of the kelp amylase and other known enzymes is only 55% at most. The enzyme has the advantages of excellent enzymological property, good stability, easy storage, particularly excellent cold-adapted property, strong specificity for substrate combination and high enzymolysis rate, and is an ideal enzyme for detecting the quantitative detection of the kelp starch.
(2) The cold-adapted kelp amylase disclosed by the invention has high activity on kelp starch, but has no degradation effect on other marine polysaccharides such as agar, algin, carrageenan and the like.
(3) The quantitative detection method of the kelp starch provided by the invention utilizes the cold-adapted kelp amylase to degrade the kelp starch in the sample at a lower temperature 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 kelp starch in the detected sample can be obtained by detecting the light absorption value increment before and after the reaction of the reaction solution. The detection method is simple to operate, good in linear range and high in accuracy, and can be popularized and used in the market.
Drawings
FIG. 1: agarose gel electrophoresis picture of the target gene;
FIG. 2: an electrophoretogram of the purified kelp amylase Lam16A _ Wa;
FIG. 3: a phylogenetic tree constructed by Lam16A _ Wa and all known GH16 family kelp amylases; wherein the arrow points to kelp amylase Lam16A _ Wa;
FIG. 4: lam16A _ Wa multiple sequence alignment; among them, the black box and white character is the conserved residue of Lam16A _ Wa.
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 kelp amylase Lam16A _ Wa in escherichia coli
Cultivation OF Wenyingzhuangia aestuarii OF219 in 2216E MediumUntil the end of logarithm, extracting the whole genome DNA, designing upstream and downstream primers (5 '-GACACGGATCCAAAGACAAAGTAGCAGTAAATGATACTACA; 5' -GACACCTCGAGCTATTGATATACTCTTACATAATCTATTTC) according to the target gene, and carrying out PCR by taking the whole genome as a template, wherein the PCR reaction conditions are as follows: 3min at 95 ℃, 20s at 95 ℃, 22s at 42 ℃, 60s at 72 ℃, 22 cycles, and finally 5min at 72 ℃ to obtain the Laminaria amylase Lam16A _ Wa gene fragment, and the fragment is connected to a pET-28a (+) vector to form a recombinant plasmid. The recombinant plasmid was introduced into BL21(DE3) 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 12 h. Centrifuging to collect thallus, adding 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate (Na)2HPO4-NaH2PO4) 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 kelp amylase Lam16A _ Wa.
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 77.14U/mg (1U activity was defined as the activity to generate 1. mu. mol of reducing sugar in 1 min).
Example 2: clone expression and acquisition of kelp amylase Lam16A _ Wa in bacillus subtilis
Culturing Wenyingzhuangiaa aestuarii OF219 in 2216E culture medium until the end OF logarithm, extracting whole genome DNA, designing upstream and downstream primers (5 '-GGCTAATGGGCAGCAGCCATCATCA; 5' -AATGAATTATGTAAGAGTATATCAATAG) according to target genes, performing PCR (polymerase chain reaction) by using the whole genome as a template in example 1 to obtain a kelp amylase Lam16A _ Wa gene fragment, connecting the kelp amylase fragment to a pHT01 vector to form a recombinant plasmid, transforming the recombinant plasmid into a bacillus subtilis competent cell, screening positive clones, performing induction expression in LB culture solution by using isopropylthiogalactoside, wherein the induction temperature is 37 ℃, the induction time is 12h, centrifugally collecting thalli, adding a certain amount OF 20mM Na2HPO4-NaH2PO4Suspending 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, thus obtaining crude enzyme solution of alginate lyase Aly 7A. 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 140.71U/mg.
Example 3: clone expression and acquisition of kelp amylase Lam16A _ Wa in pichia pastoris
Culturing Wenyingzhuangia aestuarii OF219 in 2216E culture medium until the end OF logarithm, extracting whole genome DNA, designing upstream and downstream primers (5 '-TTATAAATGGGCAGCAGCCATCATCA; 5' -GCATGCAGATTATGTAAGAGTATATCAATAG) according to a target gene, carrying out PCR (polymerase chain reaction) by taking the whole genome as a template according to example 1 to obtain a kelp amylase Lam16A _ Wa gene fragment, connecting the kelp amylase Lam16 _ Wa gene fragment to a pPIC9k vector to form a recombinant plasmid, and adding the recombinant plasmid into a pichia pastoris 115 competent cell to form a recombinant GS cell; 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 is 2.0, centrifuging, collecting thallus, discarding supernatant, resuspending precipitate with BMMY medium, and inducing with methanol at 29 deg.C and 200rpm for 72 h. After the induction is finished, centrifuging and collecting supernatant fluid to obtain crude enzyme liquid. Detecting the activity of the recombinase fermentation liquor to be 105.86U/mg.
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 kelp starch in a certain sample:
(1) preparing a kelp starch solution: weighing chemical grade herba Zosterae Marinae starch, and dissolving in 20mM Na with pH of 8.02HPO4-NaH2PO4Buffer solution, preparing standard solutions of the kelp starch 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/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: collecting 375 μ L of the above prepared herba Zosterae Marinae with different concentrationsThe starch solutions were reacted with 100U of Lam16A _ Wa (buffer made up to 375. mu.L) for 25min at 25 ℃. Standing in 100 deg.C metal bath for 10min after reaction to inactivate enzyme, adding 250 μ LpHBH solution, developing 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 kelp starch solution, repeating the operation, and measuring the light absorption value of the mixture as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the standard solution of the kelp starch as an abscissa and the corresponding light absorption value increment as an ordinate, and obtaining a standard curve of y-1.3426 x +0.0158 under specific reaction conditions by linear fitting, wherein R is2The value was 0.9994.
(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 third step, substituting the increase of the light absorption value into a standard curve y which is 1.3426x +0.0158, calculating the concentration of the kelp starch in the reaction system, and converting the kelp starch content in the sample.
(5) The content of the kelp starch in the sample is 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) 8.12 7.98
Assay parallel 2(mg/mL) 8.02 8.11
Assay parallel 3(mg/mL) 7.98 8.06
Determination of average value (mg/mL) 8.04 8.05
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 kelp starch in the three mixed solutions respectively.
Step one, preparing a kelp starch solution: weighing chemical grade herba Zosterae Marinae starch, and dissolving in 20mM Na with pH of 7.02HPO4-NaH2PO4Buffer solution, preparing kelp starch solution with concentration of 2.00 mg/mL; simultaneously preparing agar, carrageenan and algin solution with the concentration of 2.00 mg/mL; and uniformly mixing the agar, the carrageenan and the algin solution with the kelp starch solution in equal volume respectively to prepare a mixed solution, wherein the concentration of the kelp starch in the mixed solution is 1.00 mg/mL.
Step two, 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.
Step three, drawing a quantitative standard curve: taking 375 mu L of the above mixtureThe prepared kelp starch solutions with different concentrations are respectively reacted with 50U of Lam16A _ Wa (buffer solution is filled to 375 mu L) at 25 ℃ for 25 min. 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 kelp starch solution, repeating the operation, and measuring the light absorption value of the mixture as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the standard solution of the kelp starch as an abscissa and the corresponding light absorption value increment as an ordinate, and obtaining a standard curve of y-1.3844 x +0.0226 under specific reaction conditions by linear fitting, wherein R is2The value was 0.9990.
Step four, sample determination: and (3) respectively taking 375 mu L of the mixed solution, repeating the operation in the third step, substituting the increase of the light absorption value into a standard curve y which is 1.3844x +0.0226, calculating the concentration of the kelp starch in the reaction system, and converting the content of the kelp starch in the mixed solution.
The measurement was carried out in triplicate, and the results were as follows.
Kelp starch and agar Kelp starch and carrageenin Kelp starch and algin
Assay parallel 1(mg/mL) 1.05 1.08 0.98
Assay parallel 2(mg/mL) 1.02 0.99 1.04
Assay parallel 3(mg/mL) 1.04 0.95 1.04
Determination of average value (mg/mL) 1.04 1.01 1.02
Relative error (%) 4％ 1％ 2％
From the above results, it can be seen that the method of the present invention has good specificity.
Example 6: determination of kelp starch content in kelp raw material
(1) Preparing a kelp starch solution: weighing chemical grade herba Zosterae Marinae starch, and dissolving in 20mM Na with pH of 8.02HPO4-NaH2PO4Buffer solution, preparing standard solutions of kelp starch with the concentrations of 0.10mg/mL, 0.20mg/mL, 0.30mg/mL, 0.40mg/mL and 0.50mg/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: respectively taking 375 μ L of the prepared herba Zosterae Marinae starch solutions with different concentrationsReacted with 20U of Lam16A _ Wa (buffer made up to 375. mu.L) for 10min at 35 ℃. 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 kelp starch solution, repeating the operation, and measuring the light absorption value of the mixture as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations; taking the concentration of the standard solution of the kelp starch as an abscissa and the corresponding light absorption value increment as an ordinate, and obtaining a standard curve under specific reaction conditions by linear fitting, wherein the standard curve is that y is 1.3828x +0.0357, and R is2The value is 0.9992.
(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 steps in the third step, substituting the increase of the light absorption value into a standard curve y which is 1.3828x +0.0357, calculating the concentration of the kelp starch in the reaction system, and converting the kelp starch content in the sample. The content of kelp starch in kelp is 48.55 + -0.48 mg/mL (53.19%).
Finally, it should be noted that the above embodiments describe specific embodiments of the present invention, but 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.
Figure BDA0002336803040000091
Figure BDA0002336803040000101
Figure BDA0002336803040000111
Figure BDA0002336803040000121
Figure BDA0002336803040000131
Sequence listing
<110> China oceanic university
<120> cold-adapted kelp amylase and application thereof
<130> China oceanic university
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<141> 2019-11-20
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Glu Asn Trp Thr Tyr Asp Ile Gly Asn Ser Gly Trp Gly Asn Asn Glu
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Ala Arg Met Lys Thr Gln Asp Arg Phe Ser Phe Thr Tyr Gly Lys Val
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Glu Val Arg Ala Lys Leu Pro Tyr Gly Leu Gly Thr Trp Pro Ala Ile
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Glu Ile Asp Ile Met Glu Arg Ala Gly Thr Ser Ser Ala Asn Leu Lys
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Ile Thr Thr Ser Ala Ile His Asn Ser Ser Gly Tyr Gly Asn Thr Pro
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atggctagca tgactggtgg acagcaaatg ggtcgcggat ccaaagacaa agtagcagta 120
aatgatacta cagaacctac agtagattgt cctggaaaaa taaccaatgc agttccaaag 180
ggaaataata acgtattggt ttggtctgat gagtttaata cagcaggatc tccttgtaca 240
gaaaactgga cttacgacat aggaaacagt ggttggggaa ataatgaact tcaatattac 300
acaagagata atgcagtggt tgagaacgga attttaaaaa ttacggcaaa aaaagaaacc 360
tatagaggag ctccatatac atctgcaaga atgaaaactc aagacagatt tagttttact 420
tacggtaaag tagaagtacg tgcaaaatta ccttatggtt taggtacttg gcctgctata 480
tggatgttgg gagataatat tagtacagta gggtggccaa cctgtggaga aatagatatt 540
atggaacgtg ccggaactag ttcagctaat ttaaaaatta ccacaagtgc tattcataat 600
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acagaatttc atttgtattc cattaactgg acagcagata agattgaatt tatggtagat 720
aatgtagtgc attataccta tcagcctgct acaaaagatg cagctaattg gccttttgat 780
aaaaatcaat ttgtcatttt aaatgtagct atgggtggaa ctttaggagg taatgttgat 840
cctgatttta cagaatcggc aatggaaata gattatgtaa gagtatatca atag 894

Claims (6)

1. The application of the cold-adapted kelp amylase is characterized in that: laminarin amylase Lam16A _ Wa, the amino acid sequence of which is SEQ ID number 1; the optimal reaction temperature of the kelp amylase Lam16A _ Wa is 35 ℃, and the kelp amylase can still keep more than 80% of activity at room temperature; the optimum reaction pH value is 6.0, the reaction is basically kept stable in the pH value range of pH 3.0-11.0, the enzyme kinetic constant Km is 1.29mg/mL, the Kcat is 111.89 s-1, and the Vmax is 203.01U/mg; the method is applied to quantitative detection of the kelp starch.
2. A quantitative detection method of kelp starch is characterized in that: the kelp amylase Lam16A _ Wa as defined in claim 1 was reacted with the sample to determine the absorbance at 400-420 nm.
3. The method for quantitatively detecting kelp starch according to claim 2, characterized in that: p-hydroxybenzoyl hydrazine (pHBH) is used as a color developing agent in detection.
4. The method for quantitatively detecting kelp starch according to claim 3, characterized by comprising the steps of:
(1) preparing a kelp starch solution: weighing kelp starch with chemical grade or higher purity, and dissolving the kelp starch in buffer solution to prepare a kelp starch 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 a pHBH solution.
(3) Drawing a quantitative standard curve: mixing the kelp starch solutions with different concentrations prepared in the step (1) with a proper amount of Lam16A _ Wa enzyme solution respectively for reaction, wherein the addition amount of the enzyme is 5-20 mu L, the reaction time is 10-30min, and the reaction temperature is 20-50 ℃; 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, cooling to room temperature rapidly, centrifuging to obtain supernatant, measuring the light absorption value of the supernatant, and detecting wavelength of 400-420 nm; mixing the kelp starch solution with the same concentration gradient and the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the kelp starch solution as a control, and then calculating the light absorption value increment corresponding to the kelp starch solution with different concentrations; taking the concentration of the standard solution of the kelp starch as a horizontal coordinate, taking the light absorption value increment of the kelp starch 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 Lam16A _ Wa to 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 concentration of the kelp starch in the reaction system, thereby obtaining the content of the kelp starch in the sample.
5. The method for quantitatively detecting kelp starch according to claim 4, wherein: the pH value of the buffer solution in the step (1) is 7.0-11.0.
6. The method for quantitatively detecting kelp starch according to claim 4, wherein: 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
MK522485.1.Wenyingzhuangia sp. OF219 laminarinase (Lam16A) mRNA，partial cds.《GenBank》.2019,第1页. *
Wenyingzhuangia sp. OF219 laminarinase (Lam16A) mRNA，partial cds;MK522485.1;《GenBank》;20190729;第1页 *
糖激酶活性检测方法进展;关婉怡等;《河北师范大学学报 自然科学版》;20151130;第39卷(第6期);第534页2.2部分 *

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