CN110423787B

CN110423787B - Preparation method of uniform brown algae trisaccharide

Info

Publication number: CN110423787B
Application number: CN201910736671.7A
Authority: CN
Inventors: 刘伟治; 李智剑; 律倩倩; 张晓华; 何新新
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2019-08-09
Filing date: 2019-08-09
Publication date: 2022-03-04
Anticipated expiration: 2039-08-09
Also published as: CN110423787A

Abstract

The present invention provides a method for degrading alginate in combination with algin lyase, which is to degrade alginate by algin lyase AlyV and AlyF to prepare alginate; wherein the amino acid sequence of algin lyase AlyV is SEQ ID NO: 1, The sequence of the encoding gene is SEQ ID NO:2; the amino acid sequence of the alginate lyase AlyF is SEQ ID NO:3, and the sequence of the encoding gene is SEQ ID NO:4. Wherein the molar ratio of alginate lyase AlyV and AlyF is 1:10-10:1, and the most preferred molar ratio is 1:1. The invention adopts the enzymatic preparation technology, which is green and environmentally friendly; the combined use of dual enzymes with different substrate binding specificities improves the substrate conversion efficiency; and the combined use of the dual enzymes greatly improves the uniformity of the product.

Description

Preparation method of uniform brown algae trisaccharide

Technical Field

The invention belongs to the technical field of oligosaccharide preparation, and particularly relates to a preparation method of uniform brown algae trisaccharide.

Background

Algin is a non-homopolymerizing linear molecule formed by alpha-L-guluronic acid (G) and beta-D-mannuronic acid (M) through 1, 4-glycosidic bonds according to different combination modes. Alginate lyase by beta-eliminationThe 1, 4-glycosidic bond between the monomers is catalyzed to form an unsaturated double bond between C4 and C5 at the non-reducing end, which double bond has a maximum absorbance at 235 nm. In enzyme activity assays, mainly by OD₂₃₅The production of unsaturated double bonds was detected by UV absorption and the homogeneity of the degradation products was determined by Thin Layer Chromatography (TLC).

The research finds that the degradation product of the algin, namely the alginate oligosaccharide, has various biological activities. And intensive research finds that the activity of the brown alga oligosaccharide is closely related to the polymerization degree of the brown alga oligosaccharide. For example, the brown algae oligosaccharide with the polymerization degree of 5(DP5) is found to have better activity for inhibiting osteosarcoma cells than other brown algae oligosaccharides with the polymerization degree.

At present, the brown algae oligosaccharide is mainly obtained by degrading algin through a chemical method and an enzymatic method. Compared with a chemical method, the enzymatic preparation has the advantages of environmental friendliness, strong substrate specificity and the like, and is a main method for preparing uniform brown alginate oligosaccharides in the future. Therefore, the alginate lyase used for the preparation of alginate oligosaccharides has received extensive attention from researchers. However, the currently used alginate lyase still has the problems of low single enzyme degradation efficiency and poor product uniformity, which limits the wide application of homogeneous alginate-oligosaccharide preparation technology based on an enzyme method and becomes a bottleneck restricting the industrial development.

Disclosure of Invention

The invention aims to provide a method for degrading algin by combining algin lyase, thereby making up the defects of the prior art.

The method for degrading algin by using the algin lyase in a combined manner, which is provided by the invention, is characterized in that algin lyase AlyV and AlyF are used for degrading algin to prepare fucoidan trisaccharide;

wherein the amino acid sequence of the alginate lyase AlyV is SEQ ID NO. 1, and the sequence of the coding gene is SEQ ID NO. 2;

wherein the amino acid sequence of the alginate lyase AlyF is SEQ ID NO. 3, and the sequence of the coding gene is SEQ ID NO. 4.

Wherein the molar ratio of the alginate lyase AlyV to the alginate lyase AlyF is 1:10-10:1, and the most preferable molar ratio is 1: 1.

The invention adopts an enzymatic preparation technology, and is green and environment-friendly; the dual enzymes with different substrate binding specificities are combined, so that the substrate conversion efficiency is improved; meanwhile, the dual enzymes are combined, so that the uniformity of the product is greatly improved.

Drawings

FIG. 1: alignment chart of AlyV and homologous protein sequence;

FIG. 2: SDS-PAGE detection of purified AlyV;

FIG. 3: SDS-PAGE detection result chart of purified AlyF;

FIG. 4: distribution graphs of Al products degraded by AlyV and AlyF in each proportion group;

FIG. 5: plots of relative substrate degradation rates for AlyV and AlyF;

FIG. 6: AlyV and AlyF10:10 set of product isolation diagrams.

Detailed Description

The terms referred to for the present invention are explained as follows:

1. algin: algin is a non-homopolymerizing linear molecule formed by alpha-L-guluronic acid (G) and beta-D-mannuronic acid (M) through 1, 4-glycosidic bonds according to different combination modes.

2. Alginate lyase: catalyzing 1, 4-glycosidic bonds among the monomers through beta-elimination reaction to form unsaturated double bonds between C4 and C5 at non-reducing ends, wherein the double bonds have maximum light absorption values at 235 nm;

the present invention will be described in detail with reference to examples.

Example 1: cloning of the genes AlyV and AlyF

1 cloning of the Gene

1.1 Gene cloning of AlyV

Firstly, genome sequencing and homologous protein sequence analysis are carried out on Vibrio maritima Vibrio pelagis with algin degradation activity to obtain gene sequence information of AlyV (the amino acid sequence is SEQ ID NO: 1; and the sequence of the coding gene is SEQ ID NO: 2). Sequence analysis showed that AlyV is an uncharacterized novel alginate lyase belonging to the family of polysaccharide lyases 7 (PL-7). From the CAZy database, the sequences of three proteins, A9mT, AlxM and AlyVOA, among the members characterized in the PL-7 family are most similar to AlyV with homology of 47.1%, 43.4% and 42.8%, respectively, and the amino acid sequence alignment is shown in FIG. 1. The three homologous alginate lyase enzymes of AlyV all have the substrate specificity of polyM, A9mT and AlyVOA are from Vibrio, and AlxM is from photobacterium.

The AlyV gene is obtained by amplifying Vibrio pelagius genome and is constructed in pET-28a expression vector.

Upstream primer AlyV-F: 5' -CGGGATCCGCAAATGCTTCAGATAAGGCTGCTC-3 ', downstream primer AlyV-R: 5' -CCGCTCGAGTTAACGAATTACTGGCTCGCTTTCTAC-3' was subjected to PCR amplification, and the reaction system and conditions are shown in Table 1.

Table 1: AlyV gene amplification PCR system

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 30 s; annealing at 65 ℃ for 30 s; stretching at 72 ℃ for 60 s; 30 cycles; final extension at 72 ℃ for 10 min.

The amplified target gene is detected by agarose gel electrophoresis, purified by Cycle-Purekit (OMEGA), subjected to double enzyme digestion by using restriction enzyme BamH I/Xho I and then connected to a pET-28a vector, wherein the enzyme digestion conditions are 30 ℃ and 20h, and the system is shown in Table 2.

Table 2: AlyV PCR product double enzyme digestion system

Composition of	Volume of
		AlyV PCR product (79 ng/. mu.l)	20μl
10×K buffer	3μl
		BamH I	1μl
Xho I	1μl
		Double distilled water	5μl

After double enzyme digestion, the gene fragment is purified by using Cycle-Pure Kit. The pET-28a vector is cut by the same enzyme cutting system, and the Gel Extraction Kit (OMEGA) recovers vector fragments after agarose Gel electrophoresis detection. The gene fragment and vector fragment were ligated at 16 ℃ for 4h under the conditions shown in Table 3.

Table 3: AlyV gene fragment connection system

The ligation product is transformed into E.coli BL21(DE3) receptor cells, and the gene is sequenced after PCR identification to obtain positive clones.

1.2 Gene cloning of AlyF

Vibrio sp.lentdidus OU02 genome DNA is used as a template, an upstream primer AlyF-F: 5'-CGGGATCCTGTACCACCCAAGAAAAAACAGC-3' and a downstream primer AlyV-R: 5'-CCGCTCGAGTTACTTAGTTGTGTTCTCAAGTAC-3' are used for PCR amplification, the reaction system is as shown in Table 1, and the conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 s; annealing at 58 ℃ for 30 s; stretching at 72 ℃ for 2 min; 30 cycles; final extension at 72 ℃ for 10 min.

By the same method as 1.1, the target gene AlyF is purified, subjected to double enzyme digestion (BamH I/XhoI), connected to a pET-32a vector and sequenced to obtain a positive clone. Wherein Aly has the amino acid sequence of SEQ ID NO. 3; the sequence of the coding gene is SEQ ID NO. 4.

2 recombinant expression of protein

The recombinant expression methods of pET-28a-AlyV and pET-32a-AlyF are basically the same.

1) Activation of Glycerol bacterium

50mL of LB (Kan) containing pET-28a-AlyV and pET-32a-AlyF Escherichia coli BL21(DE3) (500. mu.L) were added to each of 1% of the inoculated amount⁺) And 50mLLB (Amp)⁺) Shaking and culturing overnight (12h) in liquid culture medium at 37 deg.C to obtain seed liquid.

2) Amplification culture and IPTG induced expression

The seed solutions were inoculated with 1L LB (Kan) in an amount of 1% (10mL) respectively⁺) And 1L LB (Amp)⁺) In liquid medium, shake-culturing at 37 deg.C to OD₆₀₀When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.2mM, and the mixture was cultured with shaking at 16 ℃ for 20 hours.

3) Collecting the thallus

The cultured bacterial solution was centrifuged at 3000g for 30min at 4 ℃ and the supernatant was discarded. pET-28a-AlyV resuspended cells with 20mL lysate A (20mM Tris-HCl pH8.0, 500mM NaCl, 10mM imidazole), pET-32a-P-AlyF resuspended cells with 20mL lysate B (20mM Tris-HCl pH7.5, 500mM NaCl, 10mM imidazole), centrifuged at 4 ℃ for 10min at 5000g, the supernatant was discarded, and the cells were stored at-80 ℃.

3 separating and purifying

3.1 separation and purification of AlyV

1) Crushing of thallus

Resuspending thallus in 20mL of lysate A, and performing thallus disruption by using an ultrasonication instrument for 2s at an interval of 4s for 20 min. The bacterial suspension was centrifuged at 12000g at 4 ℃ for 30min to obtain the supernatant.

2) Affinity chromatography

After the thalli are subjected to ultrasonic treatment, the supernatant and 2ml of Ni-NTA gel are combined for 1 hour at 4 ℃ in a rotating way and then flow out. Flow washing was performed with 20 column volumes of lysate A, followed by elution with 2 column volumes of buffers containing 100, 200, 500mM imidazole (20mM Tris-HCl pH8.0, 500mM NaCl), respectively. SDS-PAGE detects the protein content of each eluted component.

3) Dialysis

And dialyzing the elution component with higher purity and concentration of the target protein into 20mM Tris-HCl pH8.0 and 500mM NaCl buffer solution for 2h to complete the purification of AlyV.

4) Protein concentration determination

OD of the enzyme solution was measured using 20mM Tris-HCl pH8.0 and 500mM NaCl buffer as a blank₂₈₀The value is obtained. Predicting the extinction coefficient (Abs) of the AlyV protein by https:// web. expasy. org/protparam/website; the protein concentration was then calculated.

The formula for calculating the protein concentration is: a (absorbance) ═ epsilon (extinction coefficient) × cm (optical path length) × M (protein concentration).

The SDS-PAGE of purified AlyV is shown in FIG. 2.

According to the measurement result of protein concentration, the fermentation efficiencies of AlyV and AlyF are 72mg/ml and 57mg/ml respectively.

3.2 separation and purification of AlyF

1) Crushing of thallus

Resuspending the thallus in 20mL of lysate B, and carrying out thallus disruption by using an ultrasonicator for 2s at an interval of 4s for 20 min. The bacterial suspension was centrifuged at 12000g at 4 ℃ for 30min to obtain the supernatant.

2) Affinity chromatography

The procedure was as in 3.1(1), with the pH of all buffers being changed to 7.5.

3) Enzyme digestion by dialysis

Dialyzing the elution component with higher purity and concentration of the target protein into 20mM Tris-HCl pH7.5 and 500mM NaCl buffer solution, and adding protease according to the mass ratio of 100:1 for dialysis enzyme digestion. After 2h, SDS-PAGE detects that the fusion protein is completely digested, the mixed enzyme solution flows through Ni-NTA gel again, the Trx label is removed, and SDS-PAGE detects that the purification of AlyF is completed.

4) Protein concentration determination

The procedure was as in (3.1) (4).

The SDS-PAGE of purified AlyF is shown in FIG. 3.

Example 2: combined degradation of algin

1. Synergistic study of enzyme activity

The reaction conditions of this example are as follows:

substrate 2mg/ml AL, buffer 20mM Tris-HCl (pH 8.0), 500mM NaCl, reaction at 30 ℃ for 1 h.

AlyV and AlyF have polyM and polyG specific alginate lyase activities, respectively, but when both are used alone, the Alginate (AL) cannot be completely cleaved into alginate oligosaccharides, but the AL can be completely degraded by using a combined reaction of AlyV and AlyF, as shown in FIG. 4, in which the "10: 0" lane and the "0: 10" lane are respectively AlyV and AlyF alone.

The final concentration of the "AlyV + AlyF" group was set to 10nM AlyV +10nM AlyF, the "AlyV" group was set to 20nM AlyV, and the "AlyF" group was set to 20nM AlyF.

Δ OD in the "AlyV + AlyF" group₂₃₅The value was 100%, by calculating the Δ OD of the "AlyV" group and the "AlyF" group₂₃₅The ratio of the values to them gives the relative substrate degradation rates for AlyV and AlyF alone to degrade AL. The results are shown in fig. 5, the relative substrate degradation rates of the AlyV and the AlyF are 72% and 37%, respectively, and the combined use efficiency is obviously reduced compared with the combined use efficiency of the AlyV and the AlyF; fully indicates that the utilization efficiency of the substrate is greatly improved by combining the two enzymes.

2. Determining the optimal ratio of AlyV to AlyF

When the substrate was 2mg/ml AL, degradation reaction was carried out using the respective proportion groups (molar ratio) in Table 4, with a buffer of 20mM Tris-HCl (pH 8.0), 500mM NaCl, 4. mu.L of a mixed enzyme solution (AlyV and AlyF enzyme concentrations were both 2. mu.M) at different proportions, at 30 ℃ for 1 hour, TLC detection of the final product (Merck Silica gel 60F254), n-butanol as a developing reagent: formic acid: water-4: 6: 1(v: v: v), 2g of diphenylamine, 2ml of aniline, 100ml of acetone, 10ml of phosphoric acid and 1ml of concentrated hydrochloric acid are contained in the color developing agent, and the mixture is heated by an alcohol lamp to develop color.

And comprehensively comparing the initial speed and the product distribution of each group of reactions to determine the optimal proportion of AlyV to AlyF to degrade AL. The TLC detection results of the products of each group are shown in FIG. 6.

The results show that when AlyV and AlyF are combined to degrade AL, the substrate degradation of the AlyV/AlyF 10:5 and 10:10 groups is complete, and the trisaccharide ratio in the product is high (fig. 6), wherein the reaction rate of the AlyV/AlyF 10:10 group is the highest (table 4), so that the optimal molar ratio of the AlyV to the AlyF to the AL degradation is 1: 1.

Table 4: initial reaction rate of AlyV to AlyF in each proportion group

AlyV:AlyF	V(OD₂₃₅/min)
		10:0	0.0925±0.0008
10:1	0.0845±0.0018
		10:2	0.0955±0.0010
10:5	0.1151±0.0010
		10:10	0.1256±0.0018
5:10	0.0829±0.0004
		2:10	0.0532±0.0008
1:10	0.0418±0.0003
		0:10	0.0338±0.0005

The products of the 10:10 group were separated using Superdex Peptide 10/30GL in mobile phase 0.2mM NH₄HCO₃The flow rate was 0.3ml/min, the UV235 response signal and the TLC detection results of each component were shown in FIG. 6, and the ratio of products with different degrees of polymerization was quantified by calculating the peak area of each component, the results are shown in Table 5.

Table 5: AlyV and AlyF are shown in the content table of each component of 10:10 group products

Composition of	Content (%)
		DP4	7.1
DP3	77.6
		DP2	15.3

Wherein DP2, DP3, DP4 are fucoidan, fucoidan and fucoidan, respectively.

In conclusion, when the molar ratio of AlyV to AlyF is 1:1, the degradation speed of AL reaches the maximum value, the AL can be completely degraded into oligosaccharide, the polymerization degree of the product is uniform, and the trisaccharide proportion is 77.6%.

Sequence listing

<110> China oceanic university

<120> preparation method of uniform fucoidan

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gttgatattg cgttcaaaca caacgtggtt gataactcac aaacacctga atttgtgagt 1320

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gtcgagaatg gcctgaatgc ttaccaaggt gagggcgcag atgtgtctaa actctcagtt 1500

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Claims

1. A method for degrading algin by using an algin lyase is characterized in that the algin lyase with an amino acid sequence of SEQ ID NO. 1 and the algin lyase with an amino acid sequence of SEQ ID NO. 3 are used for degrading the algin to prepare fucotriose.

2. The method as claimed in claim 1, wherein the alginate lyase has the amino acid sequence of SEQ ID NO. 1 and the gene encoding the alginate lyase has the sequence of SEQ ID NO. 2.

3. The method as claimed in claim 1, wherein the alginate lyase has the amino acid sequence of SEQ ID NO. 3 and the gene encoding the alginate lyase has the sequence of SEQ ID NO. 4.

4. The method of claim 1, wherein the molar ratio of the alginate lyase with the amino acid sequence of SEQ ID NO. 1 to the alginate lyase with the amino acid sequence of SEQ ID NO. 3 is 1:10-10: 1.

5. The method of claim 1, wherein the molar ratio of the alginate lyase having the amino acid sequence of SEQ ID NO. 1 to the alginate lyase having the amino acid sequence of SEQ ID NO. 3 is 1: 1.