CN109371001A - A kind of enzymolysis preparation of functionality kappa-carrageenin oligose - Google Patents

A kind of enzymolysis preparation of functionality kappa-carrageenin oligose Download PDF

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CN109371001A
CN109371001A CN201811408665.0A CN201811408665A CN109371001A CN 109371001 A CN109371001 A CN 109371001A CN 201811408665 A CN201811408665 A CN 201811408665A CN 109371001 A CN109371001 A CN 109371001A
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kappa
carrageenan
carragheen
enzyme
carrageenin oligose
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林娟
张娅娇
许鑫琦
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Fuzhou University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01083—Kappa-carrageenase (3.2.1.83)

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Abstract

The present invention provides a kind of enzymolysis preparation of functional kappa-carrageenin oligose, belongs to bioengineering field.Carragheen is extracted using high temperature water extraction using Eucheuma as raw material, prepares kappa-carrageenin oligose using the kappa-carrageenan lyases degraded carrageenan that fermentation prepares.The kappa-carrageenan lyases that the present invention passes through the biodegradable OK a karaoke club glue polysaccharide of exploitation, recycle enzyme engineering technology that traditional chemical degradation method is replaced to prepare kappa-carrageenin oligose, the high viscosity polysaccharide utilized that makes to be not easy to be absorbed by the body is degraded into the carrageenan oligosaccharide rich in physiological activity, and some small-molecule active substances, realize the higher value application of red algae resource.

Description

A kind of enzymolysis preparation of functionality kappa-carrageenin oligose
Technical field
The present invention relates to a kind of enzymolysis preparations of functional kappa-carrageenin oligose, belong to bioengineering field.
Technical background
Seaweed is a kind of important marine resources, many kinds of, widely distributed, according to statistics, the seaweed of whole world discovery In kind, Rhodophyta shares 6150 kinds, and totally 4335 kinds of Chlorophyta, brown alga is 1765 kinds.Currently, seaweed is mainly used in food work Industry, in addition to this, feed, drug and in terms of also utility value with higher.
Carragheen (Carrageenan) is a kind of aqueous colloidal extracted from red algae, is primarily present in Rhodophyceae The cell wall of algae such as Eucheuma, Kappa Trentepohlia, Chondrus in, be three big seaweed glue (agar-agar, algin, card of the world One of draw glue) industrial products, mainly as food additives application.Carragheen also has special curative effect, energy in field of medicaments The activity (such as myxovirus, HIV, herpesviral, rhabdovirus) for enough inhibiting many important virus causing diseases has immune system There is duration, is effective anticoagulation, antiulcer, antipepsin work, anti-bolt substance.But carrageenan molecule amount Greatly, it is not easy to be absorbed by the body, utilization rate is low, limits its development and application.The carrageenan oligosaccharide of low polymerization degree is in addition to that can keep Outside the bioactivity of macromolecule carragheen, also has and the advantages such as be easy to absorb, have no toxic side effect.Compare carragheen, molecular weight Lower carrageenan oligosaccharide is more stronger than the antioxidant activity of the OK a karaoke club glue polysaccharide of high molecular weight, it may be possible to because, low molecular weight Oligosaccharides is easier to enter cell interior, can more effectively provide proton, plays oxidation resistant active function.Compared to ι-carragheen And lambda-carrageenan, kappa-carrageenan show higher antioxidant activity.
Degraded carrageenan is prepared there are mainly three types of the methods of carrageenan oligosaccharide at present: chemical method, physical method and enzyme process.Enzyme process Degradation is to be degraded using carragheen lyases to carragheen, enzymic degradation is high with specificity, reaction condition is easily controllable, The advantages that free from environmental pollution, and the sulfate groups structure of substrate is not destroyed when degradation, obtained molecular weight of product distribution model It encloses narrow, is the promising approach for preparing carrageenan oligosaccharide.But carrageenase higher cost become restrict enzyme process preparation it is main because Element.
Carragheen lyases (Carrageenase) is a kind of polysaccharide hydrolase, can with the β -1 of selective degradation carragheen, 4- glycosidic bond generates carraoligose.Carragheen lyases has substrate specificity, not according to degradable substrate type Together, carragheen lyases can be divided into lambda-carrageenan lyases, kappa-carrageenan lyases, ι-carragheen lyases etc..
The present invention uses the kappa-carrageenan lyases of the biodegradable OK a karaoke club glue polysaccharide of Modern microbiological engineering development, Recycle enzyme engineering technology that traditional chemical degradation method is replaced to prepare kappa-carrageenin oligose, make to be not easy to be absorbed by the body the height utilized Viscosity polysaccharide is degraded into carrageenan oligosaccharide and some small-molecule active substances rich in physiological activity, realizes red algae resource Higher value application.
Summary of the invention
The purpose of the present invention is to provide a kind of enzymolysis preparations of functional kappa-carrageenin oligose.It is original with Eucheuma Material extracts carragheen using high temperature water extraction, is prepared using the kappa-carrageenan lyases degraded carrageenan of development in laboratory preparation Kappa-carrageenin oligose, and its composition and bioactivity are analyzed, establish the enzymatic hydrolysis preparation process of functional kappa-carrageenin oligose.
Following technical scheme is used to achieve the above object:
(1) extraction of carragheen.Carragheen is extracted by raw material of Eucheuma, and solid-liquid ratio, water temperature raising degree, pH, time are carried out Optimization of orthogonal test determines optimum process condition are as follows: solid-liquid ratio 1:60w/v, 100 DEG C of temperature, pH7, water mention 3 h of time, herein Under the conditions of OK a karaoke club glue yield be 44.8%.
(2) exploitation and fermentation preparation of kappa-carrageenan lyases.With bacterial strainZobelliaSp. genomic DNA is template, Clone kappa-carrageenan enzymeκ-car16 full length gene, 1638 bp, sequence is as shown in SEQ ID NO.1, construction recombination plasmid pPICZα-car-16；By recombinant plasmid pPICZ alpha-car-16Linearized, electrotransformation is transferred in Pichia pastoris X33 bacterial strain, warp Zeocin resistance screening obtains transformant, is seeded in BMGY fluid nutrient medium and cultivates 48 h, then is forwarded in BMMY culture medium； Add methanol induction, additive amount 1%v/v, every 12 h addition is primary, and every 24 h takes 1 mL fermentation liquid, fermentation liquid in 4 DEG C, It is centrifuged 5 min under the conditions of 12000 rpm, takes supernatant to detect kappa-carrageenan enzyme activity, reaches highest in 144 h enzyme activities, be 11.27 U/mL；Collecting supernatant is the crude enzyme liquid that ferments.
(3) the enzymatic hydrolysis preparation of kappa-carrageenin oligose.Carragheen is made into certain density bottom with the PBS buffer solution of corresponding pH Object is uniformly dissolved, and the enzyme solution that step (2) obtain is added, oscillating reactions certain time under the conditions of certain temperature, boiling water goes out immediately Enzyme 5 min, 13000 rpm are centrifuged 5 min, above reset and add ethanol solution to final concentration of 70%, 4 DEG C of overnight, 13000 rpm centrifugation 5 Min goes to precipitate, filtering, and 45 DEG C of supernatant revolvings are close dry, and distilled water is settled to certain volume, phenol --- and sulfuric acid process surveys sugared content.
Using response surface experiments design optimization enzymatic hydrolysis condition, optimum process condition is obtained are as follows: pH6.0, temperature 45 C, substrate Concentration 0.7%(g/mL), 1 h of enzymolysis time, carragheen degree of hydrolysis is 49.80%.Confirmatory experiment is carried out under above-mentioned optimal conditions, Obtaining degree of hydrolysis is 51%.
(4) the HPLC analysis of carragheen enzymolysis product.Catabolite is kappa-carrageenan disaccharides, tetrose and six sugar, wherein four Sugar is primary product.With the extension of degradation time, six sugar amounts are gradually decreased, and the amount of tetrose and disaccharides gradually increases.
(5) the antioxidant activity analysis of kappa-carrageenin oligose.Kappa-carrageenin oligose has hydroxy radical, ABTS and super oxygen yin Ion radical Scavenging activity, Hydroxyl radical-scavenging ability are suitable with Vc.
The present invention has the advantages that
The present invention extracts carragheen using Eucheuma as raw material, using high temperature water extraction, has yield height, without consuming chemistry examination Agent, it is environmentally protective, at low cost the advantages that；Traditional chemical method is replaced to prepare kappa-carrageenan widow using carragheen lyases enzymatic hydrolysis Sugar, the high viscosity polysaccharide utilized that makes to be not easy to be absorbed by the body are degraded into the carrageenan oligosaccharide rich in physiological activity, realize red algae money The higher value application in source.The carragheen lyases of this laboratory ferment production, activity is high, facilitates the life for reducing carrageenan oligosaccharide Produce cost.
Detailed description of the invention
Fig. 1 is Pichia yeast engineering growth and producing enzyme curve (5 L fermentor).
Fig. 2 is that the HPLC of kappa-carrageenin oligose ingredient is analyzed: (A) 24 h(B) 12 h(C) 1 h.
Fig. 3 is the comparison of kappa-carrageenin oligose and carragheen to Hydroxyl radical-scavenging ability.
Fig. 4 is the comparison of kappa-carrageenin oligose and carragheen to ABTS free radical scavenging ability.
Fig. 5 is the comparison of kappa-carrageenin oligose and carragheen to ultra-oxygen anion free radical Scavenging activity.
Specific embodiment
The process optimization of 1 carrageenan extraction by eucheuma of embodiment
Using Eucheuma as raw material, carragheen is extracted using high temperature water extraction.On the basis of experiment of single factor, using orthogonal test Carry out the optimization for extracting condition of carragheen.By L9(34) orthogonal arrage carry out solid-liquid ratio (1:40,1:50,1:60), temperature (80 DEG C, 90 DEG C, 100 DEG C), pH(6,7,8), the time, (3 h, 4 h, 5 h) orthogonal test, experimental result were as shown in table 1.
The orthogonal test of 1 carrageenan extraction by eucheuma of table
Eucheuma extraction prepares the Optimal technique process of carragheen are as follows: solid-liquid ratio 1:60,100 DEG C of temperature, pH 7, water mention the time 3 H, OK a karaoke club glue yield is up to 44.8% with this condition.
The exploitation and fermentation preparation of 2 kappa-carrageenan lyases of embodiment
With bacterial strainZobelliaSp. genomic DNA is template, and clone obtains kappa-carrageenan enzyme geneκ-car16。κ-car16 sequences 1638 bp of column overall length, sequence encode the albumen and a termination codon of 545 amino acid composition as shown in SEQ ID NO.1 Son.Predicted, which is free of signal peptide, and the theoretical molecular weight of maturation protein is 61.93 kDa, isoelectric point 8.74, The albumen belongs to the 16th family of glycoside hydrolase.
κ-carExpression quantity of 16 genes in Escherichia coli is relatively low, is not suitable for large-scale production and application；Finish red ferment Mother can efficiently expressing exogenous gene and be widely used in industrial production, the present invention utilize X33 pairs of Pichia pastorisκ-car16 Gene carries out heterogenous expression.
With reference to pPICZ α A, B, and C (Invitrogen) operation manual, select expression vector pPICZ α-C upper intrinsic Signal peptide α-factor construction of expression vector pPICZ α-car-16.Design hasACC65I HeNotκ-card of I restriction enzyme site It draws glue enzyme primer (table 2), goes out the aim sequence containing restriction enzyme site using pMD18-T plasmid cloning.By to plasmid and purpose Segment double digestion simultaneously uses T4Ligase connects, and the multiple cloning sites area (MCS) on expression vector pPICZ α-C is replaced Forκ-car16 gene orders；Sequencing result be include c-myc antigenic determinant and 6 from α-factor signal peptide to target gene A histidine tag forms complete reading frame, and there is no frameshift mutation or deletion mutations.Recombinant plasmid pPICZ alpha-car-16Structure Build up function.
By recombinant plasmid pPICZ alpha-car-16Linearized, electrotransformation is transferred in Pichia pastoris X33 bacterial strain, through Zeocin Resistance screening obtains transformant, is seeded in BMGY fluid nutrient medium and cultivates 48 h, is then forwarded in BMMY culture medium, addition Methanol induction, additive amount 1%v/v, every 12 h addition are primary.Recon is inoculated with after two-stage seed culture by 5% inoculum concentration, It is primary every 24 h sampling, survey supernatant enzyme activity and thallus weight in wet base (Fig. 1).In 5 L fermentation cylinder for fermentation, 144 h enzyme activities reach Highest is 11.27 U/mL；Thallus weight in wet base increases with longer fermentation times, increases slowly after 144 h, is up to 154.4 g/L。
The design of 2 expression primer of table
The vigour-testing method of carragheen lyases:
Using DNS(3,5- dinitrosalicylic acid) method survey content of reducing sugar.Fermentation liquid is taken to be centrifuged under the conditions of 4 DEG C, 12000 rpm 5 min, collecting supernatant is the crude enzyme liquid that ferments.Take the 0.15% carragheen substrate of 0.1 mL crude enzyme liquid and 0.9 mL in 40 DEG C It reacts 15 min in water-bath, 1.5 mL DNS is added and simultaneously boil 5 min colour developing, while to inactivate enzyme solution group as blank control, Light absorption value is measured under 540 nm, content of reducing sugar is calculated according to above-mentioned D- galactolipin standard curve.
Enzyme activity unit is defined as: under the above-described reaction conditions, catalysis substrate per minute generates needed for 1 μm of ol reduced sugar Enzyme amount as an enzyme activity unit (U).
Enzyme activity calculation formula is as follows:
In formula:
U-enzyme activity, U/mL；
N-enzyme solution extension rate；
D- galactose content, mg corresponding to m-light absorption value；
M-D- galactose molecule amount；
V-enzyme solution volume, mL；
T-reaction time, min.
The enzymatic hydrolysis optimum preparation condition of 3 kappa-carrageenin oligose of embodiment
Carragheen is made into certain density substrate with the PBS buffer solution of corresponding pH, is uniformly dissolved, and the enzyme that embodiment 2 obtains is added Liquid, oscillating reactions certain time under the conditions of certain temperature, boiling water enzyme deactivation immediately 5 min, 13000 rpm are centrifuged 5 min, supernatant Add ethanol solution to final concentration of 70%, 4 DEG C overnight, 13000 rpm are centrifuged 5 min, go to precipitate, and filter, and 45 DEG C of supernatant revolvings are close It is dry, obtain kappa-carrageenin oligose.
Response surface design optimization is carried out on the basis of experiment of single factor.Factor level coding schedule is as shown in table 3, response surface design Experimental design and the results are shown in Table 4.
3 response surface design experimental design factor level coding schedule of table
4 response surface design experimental program of table and result
Regression analysis is carried out using experimental result of the Design-Expert 8.0.5b software to table 4, obtains regression equation:
Y=-284.73+6.69X1+56.05X2+38.70X3+0.95X1X2+0.30X1X3-3.50X2X3-0.14X1 2-8.03X2 2- 16.10X3 2
Show that best enzymatic hydrolysis carragheen prepares the process conditions of carrageenan oligosaccharide using established mathematical model are as follows: pH6.0, temperature 45 DEG C of degree, concentration of substrate 0.7%(g/mL), 1 h of enzymolysis time, carragheen degree of hydrolysis is 49.8%.Under above-mentioned optimal conditions into Row confirmatory experiment, obtaining degree of hydrolysis is 51%.Optimum process confirmatory experiment is consistent with actual experiment result approach, illustrates the model It can show that optimal enzymatic hydrolysis carragheen prepares the process conditions of carrageenan oligosaccharide.
The HPLC of 4 carragheen enzymolysis product of embodiment is analyzed
Constituent analysis is carried out using enzymolysis product kappa-carrageenin oligose of the HPLC to carragheen different time (1 h, 12 h, 24 h) (Fig. 2).Catabolite is kappa-carrageenan disaccharides, tetrose and six sugar, and wherein tetrose is primary product.With prolonging for degradation time Long, six sugar amounts gradually decrease, and the amount of tetrose and disaccharides gradually increases.
HPLC analysis method:
The kappa-carrageenin oligose for taking 5 mg difference enzymolysis times to prepare is dissolved in 500 uL deionized waters, sequentially adds 200 uL 0.2 mol/L AEC solution (AEC is dissolved in methanol), 25 uL 0.5 mol/L NaBH3CN solution and 50 uL glacial acetic acid, 1 h is heated under the conditions of 70 DEG C after mixing.After reaction, it is neutralized with 1 mol/L NaOH, 1 mL methylene chloride is then added With 1 mL distilled water to sample extraction, organic phase is abandoned, retains water phase, in triplicate.Water intaking mutually carries out HPLC analysis.
Marked product condition: detector is UV detector；Ultraviolet detection wavelength is 254 nm；Chromatographic column is C18；Column temperature It is 40 DEG C；
Separation condition: mobile phase A: 10 mmol/L ammonium acetate solutions (pH5.0)；Mobile phase B: acetonitrile.
Elution program: 0 min-60 min, 20%-40%B；Flow velocity is 0.3 mL/min；Sample volume is 20 uL.
The antioxidant activity of 5 kappa-carrageenin oligose of embodiment is analyzed
1, Hydroxyl radical-scavenging ability
The Hydroxyl radical-scavenging ability of kappa-carrageenin oligose, carragheen and Vc is as shown in Figure 3.Kappa-carrageenin oligose and the equal table of Vc Reveal stronger Hydroxyl radical-scavenging ability, and increase along with the increase of concentration, wherein the IC50 of Vc scavenging hydroxyl For 0.0268 mg/mL, kappa-carrageenin oligose is 0.0186 mg/mL；Carragheen there's almost no Hydroxyl radical-scavenging ability.
, ABTS free radical scavenging ability
Kappa-carrageenin oligose to ABTS free radical scavenging ability size as shown in Figure 4.Kappa-carrageenin oligose is clear to ABTS free radical Except rate and concentration are positively correlated, ABTS free radical scavenging activity increases with the increase of concentration；And carragheen does not have removing ABTS The ability of free radical.Kappa-carrageenin oligose is 0.0786 mg/mL, and Vc(IC50 to ABTS radicals scavenging power IC50: 0.0039 mg/mL) differ larger.
, superoxide anion Scavenging activity
Kappa-carrageenin oligose and carragheen are as shown in Figure 5 to the Scavenging activity of ultra-oxygen anion free radical.Kappa-carrageenin oligose is clear Except the ability of ultra-oxygen anion free radical is positively correlated with its concentration, IC50 is 2.583 mg/mL, but and Vc(IC50:0.0226 Mg/mL) Scavenging activity difference is bigger.Carragheen is very weak to the Scavenging activity of ultra-oxygen anion free radical.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>University of Fuzhou
<120>a kind of enzymolysis preparation of functional kappa-carrageenin oligose
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Claims (2)

1. a kind of enzymolysis preparation of functionality kappa-carrageenin oligose, it is characterised in that: described method includes following steps:
(1) carragheen, process conditions are as follows: solid-liquid ratio 1:60w/v, temperature 100 extraction of carragheen: are extracted by raw material of Eucheuma DEG C, pH7, water mention 3 h of time；
(2) exploitation and fermentation preparation of kappa-carrageenan lyases: withZobelliaSp. genomic DNA is template, clones κ- Carrageenaseκ-car16 full length gene, 1638 bp, sequence is as shown in SEQ ID NO.1, construction recombination plasmid pPICZ α-car- 16；By recombinant plasmid pPICZ alpha-car-16Linearized, electrotransformation is transferred in Pichia pastoris X33 bacterial strain, is sieved through Zeocin resistance Choosing obtains transformant, is seeded in BMGY fluid nutrient medium and cultivates 48 h, then is forwarded in BMMY culture medium；Add methanol induction Expression, additive amount 1%v/v, every 12 h addition is primary, and every 24 h takes 1 mL fermentation liquid, and fermentation liquid is in 4 DEG C, 12000 rpm items It is centrifuged 5 min under part, takes supernatant to detect kappa-carrageenan enzyme activity, reaches highest in 144 h enzyme activities, is 11.27 U/mL； Collecting supernatant is the crude enzyme liquid that ferments；
(3) the enzymatic hydrolysis preparation of kappa-carrageenin oligose: carragheen is made into certain density substrate with the PBS buffer solution of corresponding pH, molten Uniformly, the enzyme solution that step (2) obtain, oscillating reactions certain time under the conditions of certain temperature, boiling water enzyme deactivation immediately 5 is added in solution Min, 13000 rpm are centrifuged 5 min, above reset and add ethanol solution to final concentration of 70%, 4 DEG C of overnight, 13000 rpm centrifugation 5 Min goes to precipitate, filtering, and 45 DEG C of supernatant revolvings are close dry, obtains kappa-carrageenin oligose.
2. a kind of enzymolysis preparation of functional kappa-carrageenin oligose according to claim 1, it is characterised in that: step (3) enzymatic hydrolysis condition in are as follows: pH6.0, temperature 45 C, 0.7% g/mL of concentration of substrate, enzymolysis time are 1 h.
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