CN111041013B

CN111041013B - Algin lyase or pectinase and application thereof in cooperative degradation of brown algae

Info

Publication number: CN111041013B
Application number: CN201911422366.7A
Authority: CN
Inventors: 李福利; 公衍军; 苏航
Original assignee: Weifang Macaaji Biotechnology Co ltd
Current assignee: Weifang Macaaji Biotechnology Co ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-11-17
Anticipated expiration: 2039-12-31
Also published as: CN111041013A

Abstract

The invention relates to the technical field of genetic engineering, in particular to an alginate lyase and a pectinase for degrading brown algae and application thereof in the aspect of synergistically degrading brown algae. The basic sequence of the coding gene of the alginate lyase AL1059 is SEQ ID NO.2 in sequence. The base sequence of the coding gene of the pectinase pG1466 is SEQ ID NO.5 in sequence. The invention can utilize a bacillus subtilis expression system to quickly express the algin lyase derived from marine thermophilic bacteria defluvitala phayphophhia sp.Alg1 and the pectinase pG1466 derived from thermophilic bacteria Caldicellulosriptor sp.strain F32.

Description

Algin lyase or pectinase and application thereof in cooperative degradation of brown algae

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an alginate lyase and a pectinase for degrading brown algae and application thereof in the aspect of synergistically degrading brown algae.

Background

Kelp is a representative of a new renewable biomass energy source. Taking brown algae as an example, algin in the cell wall of brown algae is the most main polysaccharide component, and the degradation product of alginate oligosaccharide shows very excellent biological activity in many applications such as blood sugar reduction, blood fat reduction, tumor resistance, immunity improvement, crop quality improvement and the like. At present, medicines, health care products, cosmetics, fertilizers and feed additives which take brown algae oligosaccharide as raw materials are widely applied. At present, there are three main processes for producing brown algae oligosaccharide:

(1) the chemical extraction method adopts organic solvent extraction, chemical hydrolysis (strong acid and strong alkali) and other methods. The method has low cost and rapid reaction. However, chemical raw materials such as strong acid, strong base and organic solvent used in the process can cause serious damage to the biological activity of the brown alga oligosaccharide, and the activity is poor;

(2) the physical extraction method comprises osmotic shock, high-pressure homogenizing, freezing and pulverizing, and low-temperature blasting. The method has high requirements on equipment, high energy consumption and high production cost, and finally extracted algin is mainly in a macromolecular form and is not beneficial to absorption and utilization of organisms;

(3) the biological enzymolysis method retains the main active components of alginate oligosaccharide to the maximum extent, converts macromolecules in the algin into micromolecules which can be directly absorbed by organisms, and is an environment-friendly and efficient processing technology. However, most of the currently known alginate lyase is medium-low temperature enzyme, has poor thermal stability and low reaction temperature, and is not suitable for large-scale production.

In another aspect, many wild strains capable of expressing alginate lyase are available, but the general problems are low secretion efficiency, low purity, difficult large-scale culture, and complicated genetic manipulation means, which are not conducive to genetic modification. Therefore, at present, more people adopt escherichia coli as an engineering bacterium to carry out heterologous expression on the alginate lyase, but the escherichia coli also faces various adverse factors such as low expression level, high difficulty in subsequent separation and purification and the like. These problems have hindered the industrialization of alginate lyase and have also seriously hindered the wide application of alginate oligosaccharides.

Therefore, pichia pastoris with high secretion efficiency, easy large-scale culture and convenient product purification is used as a host strain at the present stage, the high-efficiency heterologous expression is carried out on the alginate lyase with high heat stability from the source of the marine thermophilic bacteria, and the expression efficiency and the enzyme activity are improved. However, pichia expression systems also have some disadvantages, such as: the fermentation period is long, and the fermentation period of 120-144h is usually required; in addition, there is a partial methanol residue in the methanol induction process, which also limits the application of the methanol induction process in the food industry.

Disclosure of Invention

The invention aims to provide an alginate lyase and a pectinase for degrading brown algae and application thereof in the aspect of synergistically degrading brown algae.

In order to achieve the purpose, the invention adopts the technical scheme that:

an alginate lyase, wherein the base sequence of the coding gene of the alginate lyase AL1059 is SEQ ID NO.2 in sequence.

The algin lyase is a coding gene which has at least 95 percent of homology with a base sequence shown in SEQ ID NO.2 and has activity.

The amino acid sequence coded by the alginate lyase is SEQ ID NO. 3.

The base sequence of the coding gene of pG1466 of the pectinase is SEQ ID NO.5 in sequence.

The pectinase is an encoding gene which has at least 95 percent of homology with the base sequence shown in SEQ ID NO.5 and has activity.

The amino acid sequence coded by the pectinase is SEQ ID NO. 6.

A plasmid comprising said alginate lyase.

A plasmid comprising said pectinase.

The plasmid vector is pHT43, pUB110, pE194, pUCX05-bgaB, pWB980, pHP13, pBE2, pHP13, pHP13-43, pHT01, pHT304, pMK3, pHCMC05, pMA5, pHY300PLK, pMUTIN4 or pBT 502.

A strain comprising the alginate lyase.

A strain comprising said pectinase.

The host strain is Bacillus subtilis.

An application of an alginate lyase, an application of the alginate lyase and the pectinase in degrading kelp.

Preferably, the algin lyase, the pectinase and the protease are applied to degradation of the kelp.

The adding amount of each enzyme is that 1 percent of algin, 0.5 percent of pectinase and 0.5 percent of protease are added into the kelp powder in turn. The protease is an acid protease, is a protein hydrolase, can hydrolyze animal and plant proteins under an acidic condition, and can hydrolyze the proteins into small peptides and amino acids through endo-and exo-cleavage (enzyme activity 60000u/g, available from Ningxia Xisheng industry group Limited).

The application of pectinase, the algin lyase and the pectinase are applied to degrading kelp.

The invention has the advantages that: the invention can utilize a bacillus subtilis expression system to quickly express the algin lyase derived from marine thermophilic bacteria defluvitala phayphophhia sp.Alg1 and the pectinase pG1466 derived from thermophilic bacteria Caldicellulosriptor sp.strain F32.

Drawings

FIG. 1 is a schematic diagram of the prediction of the gene functions of alginate lyase AL1059 and pectinase pG1466 provided in the embodiments of the present invention;

FIG. 2 is the agarose gel electrophoresis diagram of pHT43 expression vector, alginate lyase AL1059, and pectinase pG1466 provided by the embodiment of the invention; wherein, 1 is pHT43 expression vector, 2 is alginate lyase AL1059, 3 is pectase pG1466 agarose gel electrophoresis picture

FIG. 3 is a plasmid map of recombinant plasmids pHT43-AL1059 and pHT43-pG1466 provided by the embodiment of the present invention;

FIG. 4 is a diagram of colony PCR validation agarose gel electrophoresis provided by an embodiment of the invention; wherein A is AL 1059A; and B is pG 1466.

FIG. 5 shows the optimum temperature and pH values of alginate lyase AL1059 and pectinase pG1466 according to an embodiment of the present invention; wherein A is the optimum temperature of the alginate lyase AL1059, B is the optimum temperature pH of the alginate lyase AL1059, C is the optimum temperature of the pectinase pG1466, and D is the optimum pH of the pectinase pG1466

FIG. 6 is a graph showing the activity of the enzyme and OD600nm in the AL1059 and pG1466 fermentation process in the 3L fermenter according to the embodiment of the present invention as a function of time; wherein A is a time-varying graph of the enzyme activity and OD600nm of AL1059 fermentation process, and B is a time-varying graph of the enzyme activity and OD600nm of pG1466 fermentation process.

FIG. 7 is a SDS-PAGE analysis of extracellular protein expression in 3L fermentors at different sampling times for AL1059 and pG1466 according to the example of the present invention; wherein A is extracellular protein expression of AL1059 at different sampling time points and B is extracellular protein expression of pG1466 at different sampling time points.

FIG. 8 shows the types of buffers and pH measurements for the kelp enzymolysis process according to the present invention;

FIG. 9 is the optimum temperature measurement of the kelp enzymolysis process provided by the embodiment of the invention;

fig. 10 is a diagram illustrating the content determination of reducing sugar in the kelp enzymolysis liquid after different enzymolysis times in the kelp enzymolysis process according to an embodiment of the present invention;

FIG. 11 is a standard curve for determining protein content by BCA method provided in the examples of the present invention;

FIG. 12 is a standard curve of the content of mannitol determined by HPLC method provided in the example of the present invention;

FIG. 13 is a schematic diagram illustrating a DNS method for determining glucuronic acid labeled yeast and determining the enzyme activity of alginate lyase;

FIG. 14 shows the time of the mannitol content peak by HPLC;

fig. 15 is a standard curve for determining glucose content by DNS method according to an embodiment of the present invention.

Detailed Description

The invention is further explained below with reference to the figures and examples.

The invention clones the genes of alginate lyase and pectinase into bacillus subtilis mother strain which is a well-known safe strain by using a genetic engineering method. In a 3L fermentation tank, after 42 hours of fermentation of the alginate lyase AL1059, the OD reaches the maximum of 56.6 after 36 hours of fermentation, and the enzyme activity reaches the maximum of 26194.2U/mL after 42 hours of fermentation. The fermentation result of the pectinase pG1466 is shown in FIG 6B, the OD reaches the maximum 68.3 after 42h of fermentation, and the enzyme activity reaches the maximum 6.08U/mL after 48h of fermentation. Obtaining the bacillus subtilis strain capable of preparing the alginase and the pectinase. The generated recombinant algin lyase and pectinase can be matched with protease for degrading brown algae, and after the degradation of the compound enzyme is carried out for 12 hours, the total protein content in the enzymatic hydrolysate is 20.6mg/L, the alginic acid content is 5.16%, the mannitol content is 12g/L, and the reducing sugar content is 20 g/L. The degradation process is mild and efficient, and has good industrial application potential

Examples 1,

According to the AL1059 gene sequence in the genome DNA of the marine thermophilic bacterium D.phaphyphila sp.Alg1 strain and pectinase pG1466 derived from thermophilic bacterium Caldicellulosriptor sp.strain F32, codon optimization and gene synthesis are carried out. By NCBI: (http://www.ncbi.nlm.nih.gov/) Basic Local Alignment Search Tool of website: (http://blast.ncbi.nlm.nih.gov/Blast.cgi)，Prediction and analysis of conserved domains were performed for Al1059 and Pg 1466.

Al1059 is encoded by orf1059 and the gene is 1935bp in length and has two predicted domains, namely AlgLyase superfamily (amino acid sequence 19-329) and Heper _ II _ III superfamily (amino acid sequence 346-585). A truncated protein of only AlgLyase superfamily (amino acid sequence 19-329) is selected for expression, an enzyme expressed by the gene is named AL1059, and the base sequence is shown as SEQ ID NO.1 (the result of the structure prediction schematic diagram is shown as figure 1).

Pg1466 is encoded by orf1466, the total length of the gene is 1341bp, and the gene is shownThe resulting enzyme designated pG1466 is Polygalacturonase Polygalactaturonase, having Pgu1superfamily domain (the results of the structure prediction scheme are shown in FIG. 1). Since the host strain has its own Codon preference, Codon-optimization software Codon-Adaptation (A) is usedhttp://www.jcat.de/Start.jsp) Optimizing an AL1059 original sequence by taking Bacillus subtilis as a target strain; sending a corresponding sequence to Huada gene company for gene synthesis, wherein the gene sequence after codon optimization of alginate lyase AL1059 is shown as SEQ ID NO. 2; the codon-optimized gene sequence of the pectinase pG1466 is shown in SEQ ID No. 5.

The original gene sequence of the algin lyase AL1059 is shown in SEQ ID NO. 1.

SEQ ID NO.1：

TTATTAAATCATTATAGGAAAGTTCTCCCTTACAGAACTAGAGGATATCATCAAGATGAAATAGGACCAATTATACCTGAAGACACTTCAGAAAAAGCAAATAAATTTTTAAATAGAGAATTTGATTTAGCAGGATATACTGTAAATATGAAAGATAAATTAAATTGGTATGCTACACCTACTGGAGATTTAGAATGGAATGGTGGTTTAGTAAGACATGGTCATTTTGTTGTTATGGCCAATGAATATATAAGAACATCTGATGAAAGATTTGCAAAAGAAATTATTGACCAAATGCTTGATTATATTGAAAATGTTCCTGTATATGACCCAACAGACAAACCATATCTTGAATATAAAAAATCAACTTGGAGGCCTTTTGAAGCAGCAGCTAGAATGGGAGAAACATGGCCTGAAGCTTTAGGGAAAATAATTAATTCAAAATCAATGACAGCAGAAGCTTTTGCAAAAATATTATTATCTACTTATGAACATGCAAAATTTATTAGAAAATACCATTGGAAAACTGGAAATCATGCAGTAGGAGAAGTTGCATCATTAGGGATAACATCTATATTTTACAGTGAATTTAAAGAAGCAGATACTTGGCGTCAATATGCAGTAGATTTTTTAATGAATATGTGGGATAAACTATTTAATAAAGATTATTATACAAATGAGATGTCAGGTGGTTATCACTGGGTTGCTATGAGAAGTTTTTTTGCATTTTATGAAGTAGCAAAGAAAAATGCTCTTGAACATATATTTCCTAATATATATACCGAAAGATTAATTAATGCCTCTTATGCTGAATTATATCAAGAAAAACCAGACTATTCTATTCCAGTTACTAATGATTCAAGTAGCAAAACAAATAGAAAGAAACAATTAGAAAGAATTTATAATCTTTTTAAATTAGAAGAAATTAAATAT

(a) Sequence characteristics:

length: 933

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: DNA

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The codon optimized gene sequence of the algin lyase AL1059 is shown in SEQ ID NO. 2.

SEQ ID NO.2：

CTTCTTAACCATTACCGTAAAGTTCTTCCTTACCGTACACGTGGCTACCATCAAGATGAAATCGGCCCTATCATCCCTGAAGATACATCTGAAAAAGCTAACAAATTCCTTAACCGTGAATTCGATCTTGCTGGCTACACAGTTAACATGAAAGATAAACTTAACTGGTACGCTACACCTACAGGCGATCTTGAATGGAACGGCGGCCTTGTTCGTCATGGCCATTTCGTTGTTATGGCTAACGAATACATCCGTACATCTGATGAACGTTTCGCTAAAGAAATCATCGATCAAATGCTTGATTACATCGAAAACGTTCCTGTTTACGATCCTACAGATAAACCTTACCTTGAATACAAAAAATCTACATGGCGTCCTTTCGAAGCTGCTGCTCGTATGGGCGAAACATGGCCTGAAGCTCTTGGCAAAATCATCAACTCTAAATCTATGACAGCTGAAGCTTTCGCTAAAATCCTTCTTTCTACATACGAACATGCTAAATTCATCCGTAAATACCATTGGAAAACAGGCAACCATGCTGTTGGCGAAGTTGCTTCTCTTGGCATCACATCTATCTTCTACTCTGAATTCAAAGAAGCTGATACATGGCGTCAATACGCTGTTGATTTCCTTATGAACATGTGGGATAAACTTTTCAACAAAGATTACTACACAAACGAAATGTCTGGCGGCTACCATTGGGTTGCTATGCGTTCTTTCTTCGCTTTCTACGAAGTTGCTAAAAAAAACGCTCTTGAACATATCTTCCCTAACATCTACACAGAACGTCTTATCAACGCTTCTTACGCTGAACTTTACCAAGAAAAACCTGATTACTCTATCCCTGTTACAAACGATTCTTCTTCTAAAACAAACCGTAAAAAACAACTTGAACGTATCTACAACCTTTTCAAACTTGAAGAAATCAAATAC

(a) Sequence characteristics:

length: 933

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: DNA

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The sequence alignment results before and after optimization are as follows

The amino acid sequence of the algin lyase AL1059 is shown in SEQ ID NO. 3.

SEQ ID NO.3：

LLNHYRKVLPYRTRGYHQDEIGPIIPEDTSEKANKFLNREFDLAGYTVNMKDKLNWYATPTGDLEWNGGLVRHGHFVVMANEYIRTSDERFAKEIIDQMLDYIENVPVYDPTDKPYLEYKKSTWRPFEAAARMGETWPEALGKIINSKSMTAEAFAKILLSTYEHAKFIRKYHWKTGNHAVGEVASLGITSIFYSEFKEADTWRQYAVDFLMNMWDKLFNKDYYTNEMSGGYHWVAMRSFFAFYEVAKKNALEHIFPNIYTERLINASYAELYQEKPDYSIPVTNDSSSKTNRKKQLERIYNLFKLEEIKY

(a) Sequence characteristics:

length: 311

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The original gene sequence of the pectinase pG1466 is shown in SEQ ID NO. 4.

SEQ ID NO.4：

ATGTTTTTAAATGTTCGTGATTTTGGAGCGGTGGGAAATGGGCAGGTAAAAGATACAGAAGCATTCAAAAAGGCGATTGAAGCAAGTTGGGAGCAAGGTGGTGGGACGGTTTATGTACCTGCTGGAGTTTATCTAACCGGTCCCATTCATCTGAAGAGCAATATAACCCTCTATATAGAAAGTGGGGCTACATTGAAATTTTCCAACGACCTGGATGATTTTCCTTTAGTTTATACAAGATGGGAAGGAGAAGAACAAGAAGCATACTCTCCACTAATTTATGCCGAAAATGCAGAAAACATTGCAGTTGTTGGATTTGGCACAATAGATGGGCAGGGTGAAATGTGGTGGAAACTTCACAGAAATAAAGAACTAAAATATCCAAGACCAAGGACAGTTTGCTTTTACAGGTGCAAAAATGTGACTATTGAAGGAATAAAGATTGTCAATTCACCAAGCTGGACGGTAAACCCAATCGAATGTGAGAATGTCACAGTTCACAATGTCAAGATTCAAAACCCTTATGATTCTCCTAACACAGATGGGATAAATCCTGAGTCTTGCGAAGGTGTTAGAATCTCAAACTGCTACATAGATGTTGGCGATGACTGTGTAACTTTAAAATCCGGAACAGAAGATTGCAAGGTGAGGATTCCTTGTGAGAATATAGCAATTACAAACTGCATAATGGCACATGGTCATGGTGGAATTGTCATTGGCAGTGAAATGAGCGGTGGTGTTAGAAATGTTGTCATTTCAAACTGCATTTTTGAGGGTACAGACAGGGGAATAAGAATAAAAACCCGCCGTGGAAGAGGTGGGATTGTTGAAGATATAAGAGTTTCAAACATTGTGATGAAGAATGTAATCTGTCCATTTGCGTTTTATATGTACTATCACTGTGGCAAAGGCGGAAAAGAAAAGAGAGTTTGGGACAAGTCTCCTTATCCTGTAGACAGTACAACTCCAATTGTAAGAAGAATTTATATAAGCGATGTGATTGTAAGGCAGGCGCGAGCAGCAGCTGGATTTTTGTATGGTCTTACAGAGATGCCAATTGAAGATGTTGTATTTTCGAATGTCACAGTTGAGATGGCACAAAACCCTGAGCCTGAACTTCCTGCTATGATGAGCTATTTAGAACCGATGGCAAAAAGAGGGTTTGTCATAAATACTGTGAGGAATATAAGATTTATGAATGTTACTGTGCTGGAACAGGAAGGTGTTGCTTTTGAACTTAACAATTGTGAAAATGTAGAGTTTTACAGATGCAGGGCAAAAGATACAGCAGATTATTCTAAGATTTTGAGTCTGAATAATACAATGAATCTGATTGCCGAG

(a) Sequence characteristics:

length: 1341

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: DNA

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: strain F32, caldicellulosriptor sp

The codon optimized gene sequence of the pectinase pG1466 is shown in SEQ ID No. 5.

SEQ ID NO.5：

ATGTTCCTTAACGTTCGTGATTTCGGCGCTGTTGGCAACGGCCAAGTTAAAGATACAGAAGCTTTCAAAAAAGCTATCGAAGCTTCTTGGGAACAAGGCGGCGGCACAGTTTACGTTCCTGCTGGCGTTTACCTTACAGGCCCTATCCATCTTAAATCTAACATCACACTTTACATCGAATCTGGCGCTACACTTAAATTCTCTAACGATCTTGATGATTTCCCTCTTGTTTACACACGTTGGGAAGGCGAAGAACAAGAAGCTTACTCTCCTCTTATCTACGCTGAAAACGCTGAAAACATCGCTGTTGTTGGCTTCGGCACAATCGATGGCCAAGGCGAAATGTGGTGGAAACTTCATCGTAACAAAGAACTTAAATACCCTCGTCCTCGTACAGTTTGCTTCTACCGTTGCAAAAACGTTACAATCGAAGGCATCAAAATCGTTAACTCTCCTTCTTGGACAGTTAACCCTATCGAATGCGAAAACGTTACAGTTCATAACGTTAAAATCCAAAACCCTTACGATTCTCCTAACACAGATGGCATCAACCCTGAATCTTGCGAAGGCGTTCGTATCTCTAACTGCTACATCGATGTTGGCGATGATTGCGTTACACTTAAATCTGGCACAGAAGATTGCAAAGTTCGTATCCCTTGCGAAAACATCGCTATCACAAACTGCATCATGGCTCATGGCCATGGCGGCATCGTTATCGGCTCTGAAATGTCTGGCGGCGTTCGTAACGTTGTTATCTCTAACTGCATCTTCGAAGGCACAGATCGTGGCATCCGTATCAAAACACGTCGTGGCCGTGGCGGCATCGTTGAAGATATCCGTGTTTCTAACATCGTTATGAAAAACGTTATCTGCCCTTTCGCTTTCTACATGTACTACCATTGCGGCAAAGGCGGCAAAGAAAAACGTGTTTGGGATAAATCTCCTTACCCTGTTGATTCTACAACACCTATCGTTCGTCGTATCTACATCTCTGATGTTATCGTTCGTCAAGCTCGTGCTGCTGCTGGCTTCCTTTACGGCCTTACAGAAATGCCTATCGAAGATGTTGTTTTCTCTAACGTTACAGTTGAAATGGCTCAAAACCCTGAACCTGAACTTCCTGCTATGATGTCTTACCTTGAACCTATGGCTAAACGTGGCTTCGTTATCAACACAGTTCGTAACATCCGTTTCATGAACGTTACAGTTCTTGAACAAGAAGGCGTTGCTTTCGAACTTAACAACTGCGAAAACGTTGAATTCTACCGTTGCCGTGCTAAAGATACAGCTGATTACTCTAAAATCCTTTCTCTTAACAACACAATGAACCTTATCGCTGAA

(a) Sequence characteristics:

length: 1341

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: DNA

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: strain F32, caldicellulosriptor sp

The amino acid sequence of the pectinase pG1466 is shown in SEQ ID NO. 6.

SEQ ID NO.6：

MFLNVRDFGAVGNGQVKDTEAFKKAIEASWEQGGGTVYVPAGVYLTGPIHLKSNITLYIESGATLKFSNDLDDFPLVYTRWEGEEQEAYSPLIYAENAENIAVVGFGTIDGQGEMWWKLHRNKELKYPRPRTVCFYRCKNVTIEGIKIVNSPSWTVNPIECENVTVHNVKIQNPYDSPNTDGINPESCEGVRISNCYIDVGDDCVTLKSGTEDCKVRIPCENIAITNCIMAHGHGGIVIGSEMSGGVRNVVISNCIFEGTDRGIRIKTRRGRGGIVEDIRVSNIVMKNVICPFAFYMYYHCGKGGKEKRVWDKSPYPVDSTTPIVRRIYISDVIVRQARAAAGFLYGLTEMPIEDVVFSNVTVEMAQNPEPELPAMMSYLEPMAKRGFVINTVRNIRFMNVTVLEQEGVAFELNNCENVEFYRCRAKDTADYSKILSLNNTMNLIAE

(a) Sequence characteristics:

length: 447

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: strain F32, caldicellulosriptor sp

Examples 2,

Bacillus subtilis expression vectors pHT43-AL1059 and pHT43-pG 1466.

According to the gene sequence after codon optimization of AL1059 and pG1466 and the multiple cloning site of the shuttle plasmid pHT43 of the escherichia coli-bacillus subtilis; the pHT43 expression vector is an escherichia coli-bacillus subtilis shuttle plasmid and is used as an inducible vector, the strong promoter Pgrac of pHT43 is obtained by fusing the promoter groESL at the downstream of a lactose operon, and the induced expression can be carried out by using a lactose substitute IPTG in the later application, so that the industrial application is realized. And selecting BamH I and Sma I as enzyme cutting sites, designing corresponding primers, removing 84bp signal peptide from the primers, and adding enzyme cutting sites at two ends respectively. Primers were designed as follows:

AL1059：

a forward primer F: 5' -CGGGATTCCTTCTTAACCATTACCGTAAAGTTCTTCC-3’(BamH I)；

Reverse primer R: 5' -TCCCCCGGGGTATTTGATTTCTTCAAGTTTGAAAAGG-3’(Sma I)；

pG1466：

A forward primer F: 5' -CGGGATTCATGTTCCTTAACGTTCGTGATTTCGGCGCTG-3’(BamH I)；

Reverse primer R: 5' -TCCCCCGGGTTCAGCGATAAGGTTCATTGTGTTGTTAAG-3’(Sma I)；

The restriction endonuclease BamH I site is underlined in the forward primer, the restriction endonuclease Sma I site is underlined in the reverse primer, and the protective base is shown in bold;

1) the gene PCR amplification of AL1059 was carried out using Defluvitalea phaphyphia sp.Alg1 genomic DNA as a template, and the gene PCR amplification PCR reaction system of pG1466 was carried out using Caldicellulosriptor sp.strain F32 genomic DNA as a template as follows:

forward primer (10pM)	1μL
		Reverse primer (10pM)	1μL
Template DNA	1μL
		2×TransStart FastPfu PCR SuperMix	50μL
H₂O	43μL

The PCR amplification conditions were as follows: pre-denaturation, annealing, extension, cycle 33 times, extension. After the PCR is finished, agarose gel electrophoresis is carried out to verify that the molecular weights of AL1059 and pG1466 are about 933bp and 1341bp, which indicates that the amplification is successful, and as shown in figure 2B, 2 is pG1466, and 1 is AL 1059.

2) Plasmids were extracted using the TransGen Biotech plasmid miniprep kit and double digested. And carrying out double enzyme digestion on the amplified target gene fragment and the vector. The treatment was carried out at 37 ℃ overnight for 2 h.

The enzyme digestion system is as follows:

PCR product/plasmid	50μL
		BamH I	1μg/μL
Sma I	1μg/μL
		10 Xdigestion reaction buffer	10μL
H₂O	Make up to 100 mu L

The digested vector is firstly subjected to agarose gel electrophoresis to determine the molecular weight of about 8000bp, and the molecular weight is shown in figure 2A, which indicates that the digestion is successful.

3) Connecting: PCR product purification was performed using Omega PCR purification kit, concentration was determined as large fragment: the molar ratio of the small fragments is 1: 3, and performing ligation by using T4DNA ligase at 16 ℃ for 12h, wherein the ligation system is as follows:

fragment of interest AL1059	2μL
		pHT43	5μL
T₄ DNA ligase	1μL
		10 Xligation reaction buffer	1μL
H₂O	Make up to 10 mu L

4) Transns 1-E1 competent cells (TransGen Biotech) were transformed: the ligation product was added to 50. mu.L of Trans1-T1 competent cells (ligation product was added just after the competent cells had thawed), gently mixed and ice-cooled for 30 min. After the ice bath was completed, the mixture was heat-shocked at 42 ℃ for 30 seconds and then immediately placed on ice for 2 minutes. 0.6ml of LB medium without antibiotics was added, 200 rotations were performed, and incubation was performed at 37 ℃ for 1 hour. After the bacterial liquid was centrifuged at 6000rpm for 1min, 50 to 100. mu.L of the whole was applied to LB plates to which ampicillin was added, and cultured overnight.

5) Analysis of Positive recombinants by PCR method

(1) The selected clones were cultured in 500. mu.L of LB liquid medium at 37 ℃ for 5 to 6 hours.

(2) mu.L of the bacterial solution from 25. mu.L of the reaction system was used as a template for PCR reaction, and the recombinants were identified using the forward and reverse primers AL1059 and pG 1466.

PCR reaction conditions, cycle number 33:

94℃	10min
		94℃	30sec
67℃	30sec
		72℃	60sec
72℃	10min

as a result, amplified bands of about 933bp and 1341bp were obtained, as shown in FIG. 4. After primary verification, the correct plasmids are selected and sent to Qingdao catalpi corporation for sequencing, and the constructed recombinant plasmids pHT43-AL1059, pHT43-pG1466 are further proved to be correct. The plasmid map is shown in FIG. 3.

Example 3 construction of recombinant engineering bacteria of Bacillus subtilis

The correct plasmids obtained in example 2 were subjected to overnight scale-up culture, after which plasmids pHT43-AL1059 and pHT43-pG1466 were extracted with a plasmid miniprep kit (TransGen Biotech), respectively, and the extracted plasmids were transformed into Bacillus subtilis expression strain WB800, respectively. The transformation method comprises the following steps:

1) inoculating the bacillus subtilis expression strain WB800 into 5 microliter to 5mL LB culture medium, culturing the strain overnight at 220rpm and 37 ℃;

2) inoculating 0.2ml of overnight cultured seeds to 20ml of RHAF culture medium, and performing shake culture at 37 ℃ and 250 rpm;

3) detecting that the OD600nm of the culture reaches 0.4 (the bacillus subtilis WB800 generally grows for 3-3.5h), then centrifuging at 6000rpm for 10min to collect thalli, and suspending in 2ml of RHAF culture medium;

4) adding lysozyme with final concentration of 0.6mg/ml into the heavy suspension, and incubating at 37 deg.C for 25-30min (microscopic examination shows that the cells are all spherical);

5) after the incubation is finished, centrifuging for 5min by 1000g, collecting protoplast, washing once by using 2ml of RHAF culture medium, and then re-suspending by using 2ml of RHAF culture medium;

6) transferring 0.2ml of the heavy suspension into a new centrifuge tube, respectively adding the constructed plasmids pHT43-AL1059, pHT43-pG1466 and 0.2ml of 35% PEG 8000, incubating at 37 ℃ for 3min, immediately adding 3ml of RHAF culture medium, centrifuging at 1000g for 5min, collecting protoplast, and carrying out heavy suspension precipitation on 1ml of RHAF culture medium;

7) incubating the heavy suspension at 37 ℃ for 90min, coating the heavy suspension on a RHAF culture medium plate without resistance, and culturing at 30 ℃ overnight (the culture time is not too long, about 16h is needed, otherwise, the bacterial concentration is too high, and the plate is not beneficial to coating the rear surface);

8) eluting colonies (the cells are recovered to normal form by microscopic examination) with LB culture medium, and coating on a chloramphenicol resistant LB plate containing 5. mu.g/. mu.L; colonies can be seen after 12-24 h;

the positive clone was verified by colony PCR under the following conditions for 33 cycles

94℃	10min
		94℃	30sec
67℃	30sec
		72℃	60sec
72℃	10min

As a result, amplified bands of about 933bp and 1341bp were obtained.

The formula of the culture medium used in the process is as follows:

LB culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of NaCl;

RHAF culture medium: NH (NH)₄Cl 1g/L，Tris base 12g/L，KCl 0.035g/L，NaCl 0.058g/L，Na₂SO₄·10H₂O,0.30g/L，KH₂PO₄ 0.14g/L，MgCl₂.5H₂O4.26 g/L (added after adjusting pH), and fermenting5g/L of mother powder, 5g/L of peptone and 2g/L of sucrose 68.46g/L of glucose solution. The pH was adjusted to 7.5.

Example 4 expression of AL1059 and pG1466 in recombinant engineered bacteria of Bacillus subtilis

4.1 protein-induced expression of recombinant engineering bacteria of Bacillus subtilis in shake flask

1) The recombinant bacillus subtilis containing the recombinant plasmids pHT43-AL1059 and pHT43-pG1466 obtained in the above example is taken out from a seed-preserving tube, inoculated into LB liquid culture medium containing 5mg/L chloramphenicol according to the inoculation amount of 1 wt% respectively, and cultured overnight at 37 ℃ and 220rpm as a primary seed;

2) the primary seeds of pHT43-AL1059 and pHT43-pG1466 were inoculated at 10 wt% into 500mL flasks containing 100mL LB medium, and induction was initiated by adding 1mM inducer IPTG to the final concentration of 0.8 to 1.0 when the concentration of cells was measured to reach OD600 nm. Performing induction culture at 37 deg.C and 220rpm for 24-30 hr, centrifuging at 8000rpm and 4 deg.C for 10min, and collecting supernatant as crude enzyme solution containing alginate lyase and crude enzyme solution containing pectinase pG 1466.

3) The enzyme activity determination method of the alginate lyase is a 3, 5-dinitrosalicylic acid method. As shown in FIG. 13, the crude enzyme solution obtained in the above example was mixed with 1mL of 1% sodium alginate solution (100 mM HAC-NaAC buffer solution, pH 5.8) in an EP tube, and a blank control was performed by replacing the enzyme solution with the same volume of distilled water. Reacting at 70 deg.C for 5min, cooling, taking out 100 μ L, adding 125 μ L of 3, 5-dinitrosalicylic acid developer, boiling in water bath for 5min for color development, cooling, adding 200 μ L of distilled water, mixing, measuring absorbance (zero adjustment with blank) at 550nm, and defining the activity unit (U) of alginate lyase as 1 μ g of glucuronic acid generated by catalyzing hydrolysis of sodium alginate per minute. The enzyme activity was determined to be 336.5U/mL.

4) Determination of optimum reaction temperature and pH of alginate lyase AL 1059:

the enzyme activities of the alginate lyase AL1059 in different temperature ranges (30, 40, 50, 60, 65, 70, 75, 80, 85, 90 ℃) were measured under the condition of pH7.0 value by using 0.2% sodium alginate as a substrate, and the optimal reaction temperature was determined, and the results are shown in FIG. 5A. The optimum reaction pH values of the protein were determined at 70 ℃ in different pH ranges (200 mM acetate-sodium acetate buffer pH4, 5, 5.2, 5.6, 5.8; enzyme activity of phosphate buffer pH6, 6.4, 7, 7.4, 8; Tris-HCl pH7.1, 7.5, 8.1, 8.5). The results are shown in FIG. 5B.

5) The enzyme activity of pectinase is determined by OD235nm method, and the amount of unsaturated alginate oligosaccharide produced is determined by spectrophotometry at OD235nm when 0.2% polygalacturonic acid (polygalacturonic acid) is used as substrate to determine protease activity. The change in OD235nm was measured in an ultraviolet spectrophotometer with cyclic heating in a water bath. The cleavage of polygalacturonic acid in 1 minute to yield 1. mu. mol of unsaturated polygalacturonic acid is defined as one unit of enzyme activity.

6) The optimal reaction temperature of the pectinase is respectively as follows: the optimum reaction temperature was determined by measuring the enzyme activity of 0.2% polygalacturonic acid (polygalacturonic acid) at its pH7.0 in various temperature ranges (30, 40, 50, 60, 65, 70, 75, 80, 85, 90 ℃), and the results are shown in FIG. 5C. The optimum reaction pH values of the protein were determined at 70 ℃ in different pH ranges (200 mM acetate-sodium acetate buffer pH4, 5, 5.2, 5.6, 5.8; enzyme activity of phosphate buffer pH6, 6.4, 7, 7.4, 8; Tris-HCl pH7.1, 7.5, 8.1, 8.5). The results are shown in FIG. 5D.

4.2 protein-induced expression of recombinant engineering bacteria of Bacillus subtilis in 3L fermentation tank

1) Taking out the recombinant bacillus subtilis containing recombinant plasmids pHT43-AL1059 and pHT43-pG1466 from a seed-preserving tube, respectively inoculating the recombinant bacillus subtilis into LB liquid culture medium containing 5mg/L chloramphenicol according to the inoculation amount of 1wt per thousand, and culturing at 37 ℃ and 220rpm overnight to be used as a primary seed;

2) adding 130 μ L of the obtained primary seeds into 500mL conical flasks filled with 130mL of LB medium, and culturing at 37 deg.C and 220rpm for 12h to obtain secondary seeds;

3) the secondary seeds obtained above were inoculated, respectively, in an amount of 10 wt% using a 3-L fermenter (BioFlo115, New Brunswick Scientific Co., Edison, N.J.) in an amount of 1.2L as the initial charge of fermentation medium.

The fermentation medium was as follows: yeast powder 20g/L, Na₂SO₃2g/L, 30g/L of corn starch, 10g/L of glucose and MgSO₄·7H₂O 1g/L,(NH₄)₂–H-citrate 1g/L,(NH₄)₂SO₄2.68g/L,NaH₂PO₄·H₂O 4g/L,K₂HPO₄14.6g/L, and 3ml/L of trace elements.

Wherein the trace elements comprise the following: CaCl₂ 0.5g/L,ZnSO₄·7H₂O 0.18g/L,MnSO₄·H₂O 0.1g/L,Na₂-EDTA 10.05g/L,FeCl₃ 8.35g/L,CuSO₄·5H₂O 0.16g/L,CoCl₂·6H₂O 0.18g/L。

The supplementary culture medium comprises: 40mL/L of the above-mentioned trace elements, 500g/L of glucose, (NH)₄)₂HPO₄ 63.36g/L,MgSO₄·7H₂O 7.89g/L。

4) The process control parameters are as follows, the initial temperature is maintained at 37 ℃, the dissolved oxygen is gradually reduced after inoculation, when the dissolved oxygen is reduced to 30 percent, the ventilation volume (0.5-5L/min) is manually adjusted, and the DO is controlled to be 20-30 percent by adopting a mode of correlating the rotating speed with the dissolved oxygen (the rotating speed is between 200 and 800 rpm). After 6h, at an OD600nm of between 5 and 6, IPTG was added to a final concentration of 1mM, while the fermentation temperature was lowered to 35 ℃ and induction was started. After the glucose in the initial culture medium is exhausted, feeding is started to maintain the glucose concentration in the system to be 1-5g/L and the DO to be 20-30%. pH with ammonia and 20% (v/v) H₃PO₄The fermentation period is kept at 7.0 for 48h, and sampling is carried out every 6h to determine OD and enzyme activity.

The fermentation result of AL1059 is shown in FIG. 6A, OD reached to 56.6 at the maximum after 42h of fermentation, and the enzyme activity reached to 26194.2U/mL at the maximum after 42h of fermentation. The results of extracellular protein SDS-PAGE are shown in FIG. 7A, and 1, 2, 3 and 4 represent the extracellular protein expression conditions of 12h, 24h, 36h and 48h respectively.

The fermentation result of pG1466 is shown in FIG 6B, the OD reaches the maximum of 68.3 after 42h of fermentation, and the enzyme activity reaches the maximum of 6.08U/mL after 48h of fermentation. The results of extracellular protein SDS-PAGE are shown in FIG. 7B, and 1, 2, 3 and 4 represent the extracellular protein expression conditions of 12h, 24h, 36h and 48h respectively.

Example 5 synergistic degradation of kelp

The cell wall of kelp contains about 40% of algin, and the other main components are protein, laminarin, fucoidin, etc. which are intertwined to form a compact structure. The existing method mainly uses a certain enzyme to treat the kelp, so that a single enzyme method cannot play a good role.

The following degradable sea-tangle systems are used:

(1) taking 15 wt% of kelp powder as a substrate, putting the substrate in a constant temperature shaking table at 50 ℃ and 200rpm for swelling for 2 hours until the substrate becomes colloidal solid and no bulk dry kelp powder exists, indicating that the swelling is complete.

(2) Then adding 1% of alginate lyase, 0.5% of pectinase and 0.5% of protease into the swollen kelp powder in sequence, wherein the pH of the system is 7.0, and the pH of the system is 0.2M Tris-HCl buffer solution. Continuously putting the mixture into a constant temperature shaking table at 50 ℃ and 200rpm to start enzymolysis.

1) Adopting alginate lyase AL1059 and pectinase pG1466 to synergistically degrade the kelp:

when 15 wt% of kelp powder was used as a substrate, the degradation rate of kelp powder was 88.3% when only 1% of alginate lyase and 0.5% of pectinase (0.2M Tris-HCl buffer solution with a system pH of 7.0) were added.

2) Alginate lyase AL1059, pectinase pG1466 and protease synergistically degrade kelp

When 15 wt% of kelp powder is used as a substrate, 1% of alginate lyase by mass of the substrate, 0.5% of pectinase by mass of the substrate and 0.5% of protease by mass of the substrate are added, and the degradation rate of the kelp powder can reach 92.3% when the system pH is 7.0 and 0.2M Tris-HCl buffer solution is adopted.

The three enzymes are used for synergistic degradation under the following optimization conditions:

(1) in order to obtain the optimal enzymolysis conditions, the optimal reaction buffer solution and the optimal pH (the type and gradient of the pH buffer solution: 0.2M for each of the following buffers) in the enzymolysis process of the complex enzyme are further determined by taking the reducing sugar in the kelp enzymolysis solution as an indexThe washing solution KAC-HAC has pH of 4.5, KAC-HAC has pH of 5, KAC-HAC has pH of 5.5, NaAC-HAC has pH of 4.5, NaAC-HAC has pH of 5, NaAC-HAC has pH of 5.5, and Na₂CO₃pH4.5、Na₂CO₃pH5、Na₂CO₃pH5.5), optimum temperature (40 ℃, 50 ℃, 60 ℃) and time required for enzymatic reaction (see FIGS. 8 and 9). The results are shown in FIG. 8, which shows that the optimum buffer for enzymatic hydrolysis is acetic acid-sodium acetate buffer, the optimum pH is 5.5, and the optimum temperature is 50 ℃ as shown in FIG. 9. The content of the reducing sugar released after the enzymolysis reaction is 12 hours is 15.96g/L, and the maximum content of the reducing sugar after the enzymolysis reaction is 24 hours is 17.11 g/L. The enzymolysis time for 12h reaches 94.5 percent of the maximum content, and the enzymolysis time for 12h is proper in comprehensive consideration.

(2) Performing compound enzymolysis on the kelp according to the obtained optimal conditions on the content of the rest nutrient components in the kelp enzymolysis liquid after enzymolysis, and measuring alginic acid, total protein and mannitol in the kelp enzymolysis liquid after 12 hours of enzymolysis.

The alginic acid content is determined by a sulfuric acid-carbazole method, the standard curve is determined by sodium alginate, the protein content is detected by a BCA method, the mannitol content is detected by an HPLC method, an Agilent 1260HPLC system is used, the model of a chromatographic column is Hi-Plex H300 multiplied by 7.7mM, a differential refraction detector is used, the mobile phase is 5mM sulfuric acid, the flow rate is 0.7mL/min, the retention time is 30min, and the column temperature is controlled at 55 ℃. The time to peak of mannitol was determined to be 10.552min (see FIG. 14). The protein content is shown in FIG. 11, the mannitol content is shown in FIG. 12, and the reducing sugar content is shown in FIG. 15. The detection shows that the total protein content in the enzymolysis liquid is 20.6mg/L, the alginic acid content is 5.16%, the mannitol content is 12g/L, and the reducing sugar content is 20 g/L.

Sequence listing

<110> Weifang Mi card Azi Biotech Co., Ltd

<120> an algin lyase or pectinase and its application in synergistic degradation of brown algae

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 933

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<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttattaaatc attataggaa agttctccct tacagaacta gaggatatca tcaagatgaa 60

ataggaccaa ttatacctga agacacttca gaaaaagcaa ataaattttt aaatagagaa 120

tttgatttag caggatatac tgtaaatatg aaagataaat taaattggta tgctacacct 180

actggagatt tagaatggaa tggtggttta gtaagacatg gtcattttgt tgttatggcc 240

aatgaatata taagaacatc tgatgaaaga tttgcaaaag aaattattga ccaaatgctt 300

gattatattg aaaatgttcc tgtatatgac ccaacagaca aaccatatct tgaatataaa 360

aaatcaactt ggaggccttt tgaagcagca gctagaatgg gagaaacatg gcctgaagct 420

ttagggaaaa taattaattc aaaatcaatg acagcagaag cttttgcaaa aatattatta 480

tctacttatg aacatgcaaa atttattaga aaataccatt ggaaaactgg aaatcatgca 540

gtaggagaag ttgcatcatt agggataaca tctatatttt acagtgaatt taaagaagca 600

gatacttggc gtcaatatgc agtagatttt ttaatgaata tgtgggataa actatttaat 660

aaagattatt atacaaatga gatgtcaggt ggttatcact gggttgctat gagaagtttt 720

tttgcatttt atgaagtagc aaagaaaaat gctcttgaac atatatttcc taatatatat 780

accgaaagat taattaatgc ctcttatgct gaattatatc aagaaaaacc agactattct 840

attccagtta ctaatgattc aagtagcaaa acaaatagaa agaaacaatt agaaagaatt 900

tataatcttt ttaaattaga agaaattaaa tat 933

<210> 2

<211> 933

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cttcttaacc attaccgtaa agttcttcct taccgtacac gtggctacca tcaagatgaa 60

atcggcccta tcatccctga agatacatct gaaaaagcta acaaattcct taaccgtgaa 120

ttcgatcttg ctggctacac agttaacatg aaagataaac ttaactggta cgctacacct 180

acaggcgatc ttgaatggaa cggcggcctt gttcgtcatg gccatttcgt tgttatggct 240

aacgaataca tccgtacatc tgatgaacgt ttcgctaaag aaatcatcga tcaaatgctt 300

gattacatcg aaaacgttcc tgtttacgat cctacagata aaccttacct tgaatacaaa 360

aaatctacat ggcgtccttt cgaagctgct gctcgtatgg gcgaaacatg gcctgaagct 420

cttggcaaaa tcatcaactc taaatctatg acagctgaag ctttcgctaa aatccttctt 480

tctacatacg aacatgctaa attcatccgt aaataccatt ggaaaacagg caaccatgct 540

gttggcgaag ttgcttctct tggcatcaca tctatcttct actctgaatt caaagaagct 600

gatacatggc gtcaatacgc tgttgatttc cttatgaaca tgtgggataa acttttcaac 660

aaagattact acacaaacga aatgtctggc ggctaccatt gggttgctat gcgttctttc 720

ttcgctttct acgaagttgc taaaaaaaac gctcttgaac atatcttccc taacatctac 780

acagaacgtc ttatcaacgc ttcttacgct gaactttacc aagaaaaacc tgattactct 840

atccctgtta caaacgattc ttcttctaaa acaaaccgta aaaaacaact tgaacgtatc 900

tacaaccttt tcaaacttga agaaatcaaa tac 933

<210> 3

<211> 311

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Leu Leu Asn His Tyr Arg Lys Val Leu Pro Tyr Arg Thr Arg Gly Tyr

1 5 10 15

His Gln Asp Glu Ile Gly Pro Ile Ile Pro Glu Asp Thr Ser Glu Lys

20 25 30

Ala Asn Lys Phe Leu Asn Arg Glu Phe Asp Leu Ala Gly Tyr Thr Val

35 40 45

Asn Met Lys Asp Lys Leu Asn Trp Tyr Ala Thr Pro Thr Gly Asp Leu

50 55 60

Glu Trp Asn Gly Gly Leu Val Arg His Gly His Phe Val Val Met Ala

65 70 75 80

Asn Glu Tyr Ile Arg Thr Ser Asp Glu Arg Phe Ala Lys Glu Ile Ile

85 90 95

Asp Gln Met Leu Asp Tyr Ile Glu Asn Val Pro Val Tyr Asp Pro Thr

100 105 110

Asp Lys Pro Tyr Leu Glu Tyr Lys Lys Ser Thr Trp Arg Pro Phe Glu

115 120 125

Ala Ala Ala Arg Met Gly Glu Thr Trp Pro Glu Ala Leu Gly Lys Ile

130 135 140

Ile Asn Ser Lys Ser Met Thr Ala Glu Ala Phe Ala Lys Ile Leu Leu

145 150 155 160

Ser Thr Tyr Glu His Ala Lys Phe Ile Arg Lys Tyr His Trp Lys Thr

165 170 175

Gly Asn His Ala Val Gly Glu Val Ala Ser Leu Gly Ile Thr Ser Ile

180 185 190

Phe Tyr Ser Glu Phe Lys Glu Ala Asp Thr Trp Arg Gln Tyr Ala Val

195 200 205

Asp Phe Leu Met Asn Met Trp Asp Lys Leu Phe Asn Lys Asp Tyr Tyr

210 215 220

Thr Asn Glu Met Ser Gly Gly Tyr His Trp Val Ala Met Arg Ser Phe

225 230 235 240

Phe Ala Phe Tyr Glu Val Ala Lys Lys Asn Ala Leu Glu His Ile Phe

245 250 255

Pro Asn Ile Tyr Thr Glu Arg Leu Ile Asn Ala Ser Tyr Ala Glu Leu

260 265 270

Tyr Gln Glu Lys Pro Asp Tyr Ser Ile Pro Val Thr Asn Asp Ser Ser

275 280 285

Ser Lys Thr Asn Arg Lys Lys Gln Leu Glu Arg Ile Tyr Asn Leu Phe

290 295 300

Lys Leu Glu Glu Ile Lys Tyr

305 310

<210> 4

<211> 1341

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgtttttaa atgttcgtga ttttggagcg gtgggaaatg ggcaggtaaa agatacagaa 60

gcattcaaaa aggcgattga agcaagttgg gagcaaggtg gtgggacggt ttatgtacct 120

gctggagttt atctaaccgg tcccattcat ctgaagagca atataaccct ctatatagaa 180

agtggggcta cattgaaatt ttccaacgac ctggatgatt ttcctttagt ttatacaaga 240

tgggaaggag aagaacaaga agcatactct ccactaattt atgccgaaaa tgcagaaaac 300

attgcagttg ttggatttgg cacaatagat gggcagggtg aaatgtggtg gaaacttcac 360

agaaataaag aactaaaata tccaagacca aggacagttt gcttttacag gtgcaaaaat 420

gtgactattg aaggaataaa gattgtcaat tcaccaagct ggacggtaaa cccaatcgaa 480

tgtgagaatg tcacagttca caatgtcaag attcaaaacc cttatgattc tcctaacaca 540

gatgggataa atcctgagtc ttgcgaaggt gttagaatct caaactgcta catagatgtt 600

ggcgatgact gtgtaacttt aaaatccgga acagaagatt gcaaggtgag gattccttgt 660

gagaatatag caattacaaa ctgcataatg gcacatggtc atggtggaat tgtcattggc 720

agtgaaatga gcggtggtgt tagaaatgtt gtcatttcaa actgcatttt tgagggtaca 780

gacaggggaa taagaataaa aacccgccgt ggaagaggtg ggattgttga agatataaga 840

gtttcaaaca ttgtgatgaa gaatgtaatc tgtccatttg cgttttatat gtactatcac 900

tgtggcaaag gcggaaaaga aaagagagtt tgggacaagt ctccttatcc tgtagacagt 960

acaactccaa ttgtaagaag aatttatata agcgatgtga ttgtaaggca ggcgcgagca 1020

gcagctggat ttttgtatgg tcttacagag atgccaattg aagatgttgt attttcgaat 1080

gtcacagttg agatggcaca aaaccctgag cctgaacttc ctgctatgat gagctattta 1140

gaaccgatgg caaaaagagg gtttgtcata aatactgtga ggaatataag atttatgaat 1200

gttactgtgc tggaacagga aggtgttgct tttgaactta acaattgtga aaatgtagag 1260

ttttacagat gcagggcaaa agatacagca gattattcta agattttgag tctgaataat 1320

acaatgaatc tgattgccga g 1341

<210> 5

<211> 1341

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgttcctta acgttcgtga tttcggcgct gttggcaacg gccaagttaa agatacagaa 60

gctttcaaaa aagctatcga agcttcttgg gaacaaggcg gcggcacagt ttacgttcct 120

gctggcgttt accttacagg ccctatccat cttaaatcta acatcacact ttacatcgaa 180

tctggcgcta cacttaaatt ctctaacgat cttgatgatt tccctcttgt ttacacacgt 240

tgggaaggcg aagaacaaga agcttactct cctcttatct acgctgaaaa cgctgaaaac 300

atcgctgttg ttggcttcgg cacaatcgat ggccaaggcg aaatgtggtg gaaacttcat 360

cgtaacaaag aacttaaata ccctcgtcct cgtacagttt gcttctaccg ttgcaaaaac 420

gttacaatcg aaggcatcaa aatcgttaac tctccttctt ggacagttaa ccctatcgaa 480

tgcgaaaacg ttacagttca taacgttaaa atccaaaacc cttacgattc tcctaacaca 540

gatggcatca accctgaatc ttgcgaaggc gttcgtatct ctaactgcta catcgatgtt 600

ggcgatgatt gcgttacact taaatctggc acagaagatt gcaaagttcg tatcccttgc 660

gaaaacatcg ctatcacaaa ctgcatcatg gctcatggcc atggcggcat cgttatcggc 720

tctgaaatgt ctggcggcgt tcgtaacgtt gttatctcta actgcatctt cgaaggcaca 780

gatcgtggca tccgtatcaa aacacgtcgt ggccgtggcg gcatcgttga agatatccgt 840

gtttctaaca tcgttatgaa aaacgttatc tgccctttcg ctttctacat gtactaccat 900

tgcggcaaag gcggcaaaga aaaacgtgtt tgggataaat ctccttaccc tgttgattct 960

acaacaccta tcgttcgtcg tatctacatc tctgatgtta tcgttcgtca agctcgtgct 1020

gctgctggct tcctttacgg ccttacagaa atgcctatcg aagatgttgt tttctctaac 1080

gttacagttg aaatggctca aaaccctgaa cctgaacttc ctgctatgat gtcttacctt 1140

gaacctatgg ctaaacgtgg cttcgttatc aacacagttc gtaacatccg tttcatgaac 1200

gttacagttc ttgaacaaga aggcgttgct ttcgaactta acaactgcga aaacgttgaa 1260

ttctaccgtt gccgtgctaa agatacagct gattactcta aaatcctttc tcttaacaac 1320

acaatgaacc ttatcgctga a 1341

<210> 6

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Phe Leu Asn Val Arg Asp Phe Gly Ala Val Gly Asn Gly Gln Val

1 5 10 15

Lys Asp Thr Glu Ala Phe Lys Lys Ala Ile Glu Ala Ser Trp Glu Gln

20 25 30

Gly Gly Gly Thr Val Tyr Val Pro Ala Gly Val Tyr Leu Thr Gly Pro

35 40 45

Ile His Leu Lys Ser Asn Ile Thr Leu Tyr Ile Glu Ser Gly Ala Thr

50 55 60

Leu Lys Phe Ser Asn Asp Leu Asp Asp Phe Pro Leu Val Tyr Thr Arg

65 70 75 80

Trp Glu Gly Glu Glu Gln Glu Ala Tyr Ser Pro Leu Ile Tyr Ala Glu

85 90 95

Asn Ala Glu Asn Ile Ala Val Val Gly Phe Gly Thr Ile Asp Gly Gln

100 105 110

Gly Glu Met Trp Trp Lys Leu His Arg Asn Lys Glu Leu Lys Tyr Pro

115 120 125

Arg Pro Arg Thr Val Cys Phe Tyr Arg Cys Lys Asn Val Thr Ile Glu

130 135 140

Gly Ile Lys Ile Val Asn Ser Pro Ser Trp Thr Val Asn Pro Ile Glu

145 150 155 160

Cys Glu Asn Val Thr Val His Asn Val Lys Ile Gln Asn Pro Tyr Asp

165 170 175

Ser Pro Asn Thr Asp Gly Ile Asn Pro Glu Ser Cys Glu Gly Val Arg

180 185 190

Ile Ser Asn Cys Tyr Ile Asp Val Gly Asp Asp Cys Val Thr Leu Lys

195 200 205

Ser Gly Thr Glu Asp Cys Lys Val Arg Ile Pro Cys Glu Asn Ile Ala

210 215 220

Ile Thr Asn Cys Ile Met Ala His Gly His Gly Gly Ile Val Ile Gly

225 230 235 240

Ser Glu Met Ser Gly Gly Val Arg Asn Val Val Ile Ser Asn Cys Ile

245 250 255

Phe Glu Gly Thr Asp Arg Gly Ile Arg Ile Lys Thr Arg Arg Gly Arg

260 265 270

Gly Gly Ile Val Glu Asp Ile Arg Val Ser Asn Ile Val Met Lys Asn

275 280 285

Val Ile Cys Pro Phe Ala Phe Tyr Met Tyr Tyr His Cys Gly Lys Gly

290 295 300

Gly Lys Glu Lys Arg Val Trp Asp Lys Ser Pro Tyr Pro Val Asp Ser

305 310 315 320

Thr Thr Pro Ile Val Arg Arg Ile Tyr Ile Ser Asp Val Ile Val Arg

325 330 335

Gln Ala Arg Ala Ala Ala Gly Phe Leu Tyr Gly Leu Thr Glu Met Pro

340 345 350

Ile Glu Asp Val Val Phe Ser Asn Val Thr Val Glu Met Ala Gln Asn

355 360 365

Pro Glu Pro Glu Leu Pro Ala Met Met Ser Tyr Leu Glu Pro Met Ala

370 375 380

Lys Arg Gly Phe Val Ile Asn Thr Val Arg Asn Ile Arg Phe Met Asn

385 390 395 400

Val Thr Val Leu Glu Gln Glu Gly Val Ala Phe Glu Leu Asn Asn Cys

405 410 415

Glu Asn Val Glu Phe Tyr Arg Cys Arg Ala Lys Asp Thr Ala Asp Tyr

420 425 430

Ser Lys Ile Leu Ser Leu Asn Asn Thr Met Asn Leu Ile Ala Glu

435 440 445

Claims

1. The application of the algin lyase and the pectinase in degrading the kelp is characterized in that: the base sequence of the alginate lyase is SEQ ID NO. 2; the base sequence of the pectinase is SEQ ID NO. 5.

2. Use according to claim 1, characterized in that: the application of alginate lyase, pectinase and protease in degrading kelp.