CN105154458B - A kind of new endo-type alginate lyase gene and engineering bacteria and application - Google Patents
A kind of new endo-type alginate lyase gene and engineering bacteria and application Download PDFInfo
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Abstract
The invention discloses a kind of method that restructuring algin catenase and its engineering bacteria and hydrolysis prepare brown alga oligose, and according to the new isolated strains Vibrio sp.BZM 1 from marine alga, screening obtains alginate lyase gene-AlgM；The expression vector pHBM905BDM AlgM containing the gene are built, convert Pichia pastoris GS115 competent cell, obtain single copy gene engineering bacteria；The genetic engineering bacterium of structure culture checking multicopy, high efficient expression Prepare restructuring algin catenase, and produce brown alga oligose with the enzyme hydrolysis sodium alginate.Restructuring alginate lyase gene engineering bacteria GS115/AlgM4 (CCTCC No containing four copies：M2015500) expression is higher.Present invention restructuring algin catenase activity is up to 31000U/mL.
Description
Technical field
The present invention relates to a kind of new endo-type algin catenase AlgM gene order.The invention further relates to the restructuring
The structure of the pichia yeast genetic engineering bacteria of algin catenase and the method using Pichia anomala expression algin catenase.
The invention further relates to the method for preparing brown alga oligose using restructuring algin catenase hydrolysis higher concentration sodium alginate.
Background technology
Brown alga oligose is a kind of functional oligose of latest generation, is obtained using separation degradation technique leading in the world
Alginate oligosaccharide of the degree of polymerization below 20, the line style oligomerization being made up of beta-D-mannuronic acid and α-L- guluronic acids
Thing.Alga oligosaccharides molecular weight is low, water-soluble strong, and stability is high, experiment and it is clinical have been found that alga oligosaccharides have immunological regulation,
Lowering blood pressure and blood fat, hypoglycemic, antitumor, anticoagulation and a variety of physiologically actives such as antiviral, are also used as diabetes, fertilizer
The effect of fat disease, carcinoma of the colon and rectum, habitual constipation patient food.Led in drug development, development of functional food, green agriculture etc.
Domain has wide development prospect.
In recent years, the research on oligosaccharide is more and more, and the existing oligomeric sugar product of several functions puts goods on the market, but mesh
Preceding most of oligomeric sugar products belong to medium-sized oligosaccharide, and active ingredient is less than 50%.At present mainly using acid degradation method and
Oxidation degradation method degraded algin, can obtain serial algin oligosaccharide of the degree of polymerization below 20.And degraded for enzyme edman degradation Edman
The research that algin obtains brown alga oligose just starts to walk, it is important to which the activity of currently acquired algin catenase is universal not
Height, in order to improve product purity, it is necessary to handed in subsequent technique using the purification process that complex operation cost is too high, such as ion
Colour changing spectrometry, gel filtration chromatography etc..Enzyme edman degradation Edman degraded algin obtains brown alga oligose, it is important to algin catenase.
The algin catenase of high vigor how is found, improves the effect of hydrolysis so that the purity of product improves, and makes subsequent purification work
Sequence is easy and effective, can produce high-purity oligosaccharide at low cost, it appears particularly important.
Algin catenase can be divided into two major classes by the difference at its degraded alginate position and fragment：Selectivity cracking 1,
α-the L- of lyases (ECA.2.2.3) and selectivity the cracking Isosorbide-5-Nitrae glucosides key connection of the beta-D-mannuronic acid of 4 glucosides key connections
The lyases (EC4.2.2.11) of guluronic acid.They are respectively acting on the mannuronic acid section and gulose aldehyde of alginate
Sour section, the glycosidic bond of algin is cracked by β-elimination reaction, C is produced in non-reducing end4,5Unsaturated double-bond and 230~
240nm has strong absorption.Algin catenase wide material sources, including marine algae, sea mollusk, peronium animal body are interior and more
Kind microorganism (including marine bacteria, soil bacteria and fungi).The country is now with many related scientific research units, as Chinese Sea is big
, the Institute of Oceanology of the Chinese Academy of Sciences, institute of microbiology of Shandong University, Ocean Research Center of Zhongshan, Zhejiang University, Shang Haihai
Foreign university etc. has carried out the research of algin catenase and its related content extensively, but the fermentation enzymatic activity reported is not high, enzyme
It is low to solve product purity, production cost is high-leveled and difficult with industrialization.
Algin is a kind of material extracted in brown alga class algae.Its main component is that polymannuronic acid and poly are ancient
The high-molecular compound that sieve uronic acid is formed.Algin is widely present in bulk kelp, sea-tangle, kelp, pelvetia silquosa, bladder-wrack and horse
In the cell membrane of hundreds of brown alga such as tail algae.Manually cultivated kelp in China very universal, yield is also very big, so in Chinese people
Work cultivates kelp just into the primary raw material of extraction algin.The manufacturing and processing enterprise of algin be concentrated mainly on Qingdao,
The ground such as Yantai and Weihai, Lianyungang of Jiangsu.Brown alga oligose is made through enzymatic hydrolysis using algin catenase in algin, is had very
Good market prospects, the general algin catenase poor activity in general production, hydrolysis efficiency is low, and concentration of substrate is low, it is impossible to
Adapt to the requirement of production.
The content of the invention
The technical problem to be solved by the invention is to provide one kind restructuring algin catenase, its engineering bacteria and hydrolysis system
The method of standby brown alga oligose, high efficient expression is screened with genomic library technology from new isolated strains Vibrio sp.BZM-1
Alginate lyase gene AlgM, build the alginate lyase gene of restructuring, realize that restructuring alginate lyase gene exists
High efficiency stable expression in Pichia pastoris, and prepared using the restructuring algin catenase hydrolysis higher concentration sodium alginate of expression
Brown alga oligose.
Technical scheme is as follows.
Endo-type algin catenase AlgM gene order, it is characterised in that：
(1), according to the bacterial strain Vibrio sp.BZM-1 newly separated from marine alga, the genomic library of the bacterium is established, is screened
Obtain alginate lyase gene-AlgM；And sequencing is carried out, the GenBank accession number of the gene is KM655840, should
545 amino acid of gene code, core sequence encode 521 amino acid；
(2) primer, is designed according to core sequence：Joint GTCA and GGCCA, primer sequence are added respectively at 5 ' ends of primer
For：
AlgM-F：GTCAATGAAACATAAAATCGTCAAAACATTA
AlgM-R：GGCCATTATTTACCTTGATAGGTGCCGTG；
And the alginate lyase gene is expanded by PCR：Amplification condition is 94 DEG C of 3min, 94 DEG C of 30s, 55 DEG C of 30s,
72 DEG C of 1min, totally 30 circulations, last 72 DEG C of 3min.
The structure of expression vector containing restructuring alginate lyase gene AlgM, it is characterised in that：Restructuring algin
Lyase gene AlgM is cloned into new yeast expression vector pHBM905BDM, and expanding algin by PCR first cracks
Enzyme gene AlgM sequences, under dTTP protections, after polymerizeing ferment treatment with T4DNA, then orientation is inserted into through Cpo I and Not I
Double digestion is removed on the carrier pHBM905BDM of kan resistant genes, is obtained recombinant plasmid, is named as pHBM905BDM-AlgM.
It is a kind of to utilize the engineering bacteria for recombinating algin catenase preparation, it is characterised in that：Deposit number CCTCC No：
M2015500, entitled pichia pastoris phaff GS115/AlgM4.
The method that described engineering bacteria hydrolysis prepares brown alga oligose, it is characterised in that：
(1), ferment algin catenase AlgM：Bacterial strain Pichia pastoris GS115/AlgM4 is placed in 100ml BMGY cultures
In base, 28 DEG C~30 DEG C, 260rpm cultivates 40~50h, to OD600For 20~30,5000rpm, 5min are centrifuged in room temperature, is collected
Thalline；
(2), thalline is transferred in 100ml BMMY culture mediums, 28 DEG C~30 DEG C, 260rpm cultures, added every 12h
Methanol；It is crude enzyme liquid to obtain supernatant；
(3), brown alga oligose is prepared using obtained crude enzyme liquid hydrolysis sodium alginate：The concentration 10% of setting hydrolysis substrate~
30%, add the 100ml pH that temperature is 30~40 DEG C and make solvent in 8~9 alkaline water, a small amount of addition substrate, is progressively passed on one side
The amount for increasing substrate reaches predetermined amount, a small amount of on one side progressively to add crude enzyme liquid 1000ul-3000ul, adds substrate and enzyme amount ratio
For 1g:100ul, uniform stirring；Keeping temperature is stable at 30~40 DEG C, until hydrolysis concentration reaches every 100ml alginic acids containing 20g
Sodium；
(4) after the completion of, hydrolyzing, reaction solution is subjected to centrifugation and removes residue, takes supernatant to filter, ultrafiltration, carried out vacuum spraying and do
It is dry, obtain brown alga oligose finished product.
The method that described engineering bacteria hydrolysis prepares brown alga oligose, it is characterised in that：Substrate and enzyme amount ratio are 1g:50-
100ul。
The method for preparing brown alga oligose using described engineering bacteria hydrolysis, it is characterised in that：The substrate is algin, brown
One or any ratio in alginic acid and alginate it is two or more.
The preparation method of described engineering bacteria, it is characterised in that：
(1), the screening of the Pichia yeast engineering of expression restructuring algin catenase：Restriction enzyme Sal I will be used
The pHBM905BDM-AlgM of linearisation, Pichia pastoris GS115 competent cell is converted, obtains the recombinant bacterial strain of single copy number；
(2), the recombinant bacterial strain of single copy number of acquisition is seeded on the flat board containing 1.0% sodium alginate, therefrom
The maximum bacterial strain of hydrolysis circle is filtered out, extracts the conversion subgenom；
(3), based on Biobrick methods, carrier pHBM905BDM is passed through into digestion and connection, obtains multiple copy expression
The recombinant plasmid of box structure tandem sequence repeats；
(4), recombinant plasmid that multiple copies are expressed to the repetition of boxlike structures in series linearizes, and converts Pichia pastoris GS115
Competent cell, obtain the recombinant bacterial strain containing controllable copy number；Copy number n=2,3,4 ..., n are natural number；By four copies
Recombinant bacterial strain is named as pichia pastoris phaff GS115/AlgM4.
The positive effect of the present invention is：The algin catenase base of high efficient expression is screened by building genomic library
Cause, the gene integration obtained into Pichia pastoris high expression algin catenase (enzymatic activity is up to 31000U/ml) and
For bifunctional enzyme, the guluronic acid fragment of the enzyme principal degradation algin, the mannuronic acid fragment for the algin that can also degrade.
Have comparative advantage status in the enzyme system, and when substrate is sodium alginate, hydrolysis concentration of substrate reaches 20%, adjusts pH to 8-9,
Enzymolysis product purity may be up to more than 85%, and production cost is low.
Brief description of the drawings
Fig. 1 is genomic library construction process.First according to the new isolated strains of algin catenase AlgM sources marine alga
Vibrio sp.BZM-1, establish the genomic library of the bacterium.The genomic DNA of the bacterium is extracted first, uses restriction enzyme
Sau3A I do partially digested, Ago-Gel recovery 4-10kb fragment () see figure 1., then under the conditions of existing for dGTP,
Fragment 30min is handled in 30 DEG C with Klenow fragment, is exactly that these fragments are mended dGTP, then glue reclaim these fragments
() see figure 2., while by carrier pBZM201 restriction enzyme Sap I linearization for enzyme restriction, and this fragment is reclaimed as carrier
(), the fragment for mending dGTP is connected 24- for 16 DEG C with the pBZM201 carriers of Sap I linearization for enzyme restriction through T4DNA ligases see figure 3.
48h, Escherichia coli Xl10-gold competent cells are then converted, so as to obtain the genomic library of the bacterium () see figure 4..
Fig. 2 is Pichia pastoris recombinant expression carrier building process.First, high-fidelity enzymatic amplification mesh is used by design of primers
Genetic fragment AlgM after (see figure 1.), Ago-Gel recovery purpose fragment, then under the conditions of existing for dTTP, use
T4DNA polymerases handle fragment 30min () in 12 DEG C see figure 2., generate cohesive end, while by expression vector
PHBM905BDM carries out double digestion through restriction enzyme Cpo I and Not I, produces two single-stranded viscosity with fixed base
End (), Ago-Gel recovery purpose fragment see figure 3..Kan resistant genes finally will be removed through Cpo I and Not I double digestions
Fragment after the pHBM905BDM carriers of fragment polymerize ferment treatment with T4DNA is connected 2-3h for 16 DEG C through Solution I ligases,
Convert Escherichia coli Xl10-gold competent cells, amicillin resistance LB plate screening transformants, through plasmid size ratio
Correct recombinant plasmid pHBM905BDM-AlgM () is obtained see figure 4. to, PCR identification, digestion identification and sequencing identification.
Fig. 3 is recombinant plasmid pHBM905BDM-AlgM through digestion and connection repeatedly, you can obtains multiple copy expression cassettes
The process for the recombinant plasmid that formula structures in series repeats.
PHBM905BDM-AlgM plasmids are largely extracted with plasmid extraction kit, are then carried out with EcoR I and BamH I
Double digestion, 37 DEG C, 3h, the fragment of glue reclaim purifying about 6.1kb sizes standby () as carrier see figure 1..Simultaneously will
PHBM905BDM-AlgM plasmids carry out double digestion with EcoR I and Spe I, and 37 DEG C, 3h, glue reclaim purifies about 3.0kb sizes
Fragment 1 () see figure 2., then pHBM905BDM-AlgM plasmids are subjected to double digestion with Xba I and BamH I, 30 DEG C, 3h, glue reclaim
Purify the fragment 2 () of about 3.0kb sizes see figure 3..Then, the carrier of linearisation and fragment 1, fragment 2 are pressed 1:2:2 mixing are equal
It is even, Escherichia coli Xl10-gold competent cells are then converted, 16-20h is incubated overnight, selects transformant, be incubated overnight 16h,
Extract plasmid in a small amount again, electrophoresis detection is carried out with the plasmid and double digestion checking obtains correct 2 copy and expresses box structure string
Join the recombinant plasmid () repeated see figure 4., the method for building the recombinant plasmid that more multiple copy expression cassette formula structures in series repeats, according to
This analogizes.
Fig. 4 is the SDS-PAGE detections that Pichia pastoris GS115/AlgM4 expresses algin catenase.Swimming lane M is protein
Molecular weight marker, swimming lane 1 are GS115-pHBM905BDM empty carrier postinduction samples, swimming lane 2-12 difference Pichia pastoris GS115/
The copy inductions of AlgM4 tetra- 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 214h, 240h, 264h sample.
Fig. 5 is that the SDS-PAGE of the Pichia anomala expression algin catenase with multiple copy expression cassette formula structure is detected.
Swimming lane M is molecular weight protein marker, and swimming lane 1 is GS115-pHBM905BDM empty carrier postinduction samples, and swimming lane 2-4 is red to finish
The copy inductions of yeast GS115/AlgM3 tri- 96h, 120h, 144h sample, swimming lane 5-7 are that Pichia pastoris GS115/AlgM4 tetra- is copied
Shellfish induce 96h, 120h, 144h sample, swimming lane 8-10 be Pichia pastoris GS115/AlgM5 five copy induction 96h, 120h,
144h sample.
Embodiment
With example, the present invention is further described below.
The present invention step be：
First, the screening of alginate lyase gene and sequencing
According to the algin catenase AlgM sources new isolated strains Vibrio sp.BZM-1 of marine alga, the gene of the bacterium is established
Group library, screening obtain alginate lyase gene --- AlgM；And sequencing is carried out, the GenBank accession number of the gene
For KM655840,545 amino acid of the gene code, core sequence encodes 521 amino acid.
Then primer is designed according to core sequence.The gene is expanded by PCR.Primer is designed, in 5 ' end difference of primer
Plus joint GTCA and GGCCA, primer sequence is：
AlgM-F：GTCAATGAAACATAAAATCGTCAAAACATTA
AlgM-R：GGCCATTATTTACCTTGATAGGTGCCGTG
Amplification condition is 94 DEG C of 3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, totally 30 circulations, last 72 DEG C of 3min.
2nd, the structure of the expression vector containing restructuring alginate lyase gene AlgM
Restructuring alginate lyase gene AlgM is cloned into new yeast expression vector pHBM905BDM by the present invention
(specifically structure is shown in patent of invention 201210591987.X to the expression vector), expands alginate lyase gene by PCR first
AlgM sequences, under dTTP protections, after polymerizeing ferment treatment with T4DNA, then orientation is inserted into through Cpo I and Not I double digestions
On the carrier pHBM905BDM for removing kan resistant genes, recombinant plasmid is obtained, is named as pHBM905BDM-AlgM.
3rd, the screening of the Pichia yeast engineering of expression restructuring algin catenase
The pHBM905BDM-AlgM that will be linearized with restriction enzyme Sal I, convert Pichia pastoris GS115 competence
Cell, you can obtain the recombinant bacterial strain of single copy number.
The recombinant bacterial strain of single copy number of acquisition is seeded on the flat board containing 1.0% sodium alginate, therefrom screened
Go out the maximum bacterial strain of hydrolysis circle, extract the conversion subgenom, be correctly inserted into by PCR method and sequence measurement identification brown
The positive colony of phycocolloid lyase gene.
Based on Biobrick methods, by carrier pHBM905BDM by digestion and connection repeatedly, you can obtain multiple copy
The recombinant plasmid that shellfish expression boxlike structures in series repeats.Then, linearized, convert Pichia pastoris GS115 competent cell,
The recombinant bacterial strain containing controllable copy number can be obtained.Copy number n=2,3,4 ..., n are natural number.
Have now been found that, the restructuring algin catenase hydrolysis effect of four copies is best, and enzyme activity reaches 31,000U/ml.
By the preferable four copies recombinant bacterial strain of the effect above, pichia pastoris phaff GS115/AlgM4 is named as.
The genetic engineering bacterium was deposited in Chinese classical collection culture collection on the 31st in August in 2015, preserving number is
CCTCC No：M 2015500, entitled pichia pastoris phaff GS115/AlgM4, (Pichiapastoris GS115/
AlgM4).Preservation address：Wuhan, China.Hereinafter referred to as AlgM4.
The bacteria characteristic morphological feature of AlgM4 bacterial strains：Pichia bacterium cell is spherical in shape, oval, elongated, even
You are tapered, but does not form pinnacle.Colony colour is milky or cream color, vegetative propagation, is bred as changeable budding.
Physiological and biochemical property：Pichia pastoris can utilize the particular matters such as methanol, oil, ammonium salt to grow, optimum temperature 28-
30℃.Strains A lgM4 is integrated with restructuring algin catenase base in addition to Pichia pastoris physiological and biochemical property on its chromosome
Cause, energy efficient secretory expression algin catenase, the enzyme is bifunctional enzyme, and the enzyme is existing to the special of guluronic acid (PG)
Property also has polymannuronate (PM) specificity.
4th, shake flask fermentation algin catenase AlgM is utilized
By strains A lgM4 be placed in 100mlBMGY (2.0%Tryptone, 1.0%Yeast extrace, 0.34%YNB,
1.0% (NH4)2SO4, 100mmol/L kaliumphosphate buffers pH6.0,1.0% glycerine) and in culture medium, 28 DEG C -30 DEG C, 260rpm
40-50h is cultivated, to OD600For 20-30,5000rpm, 5min are centrifuged in room temperature, collects thalline；Thalline is transferred to 100ml
BMMY (2.0%Tryptone, 1.0%Yeast extrace, 0.34%YNB, 1.0% (NH4)2SO4, 100mmol/L potassium phosphates
PH of buffer 6.0,1.0% methanol) in culture medium, 28 DEG C -30 DEG C, 260rpm cultures, adding methanol every 12h, (dosage is training
Support matrix product 1.0%)；Expression time is 24h-240h.Expression supernatant analysis expression is taken, wherein expression time is 144h
When expression it is optimal, gained supernatant is crude enzyme liquid.In 500ml shake flask fermentation 144h, algin catenase is surveyed with DNS methods
Enzymatic activity is up to 31,000U/ml.
5th, the high restructuring algin catenase hydrolysis sodium alginate expressed obtained using this method prepares brown alga oligose
Method
The concentration 10%-30% of setting hydrolysis substrate, addition temperature are alkali of 30-40 DEG C of the 100ml pH between 8 to 9
Property water make solvent, a small amount of addition substrate on one side, progressively incrementally the amount of substrate reaches predetermined amount, while progressively adds on a small quantity on one side
(it is 1g to add substrate and enzyme amount ratio to enzyme liquid 1000ul-3000ul:100ul), uniform stirring.
Keeping temperature is stable (30-40 DEG C), until hydrolysis concentration reaches 20% i.e. per 100ml sodium alginates containing 20g.Hydrolysis
Rate extends and stepped up with enzymolysis time, and the influence of enzyme concentration and enzymolysis time to percent hydrolysis is to be mutually related, for up to
To certain percent hydrolysis, enzyme concentration is bigger, and enzymolysis time is shorter：Enzyme concentration is fewer, and enzymolysis time is longer.This experiment is in order to control
Percent hydrolysis is in the range of 85%, in 100ml reaction systems, concentration of substrate 20%, enzyme concentration take respectively 1000ul, 2000ul,
Under the conditions of 3000ul, corresponding enzymolysis time control is within 16-14h, 14-10h, 10-8h.After the completion of hydrolysis, by reaction solution
Carry out centrifugation and remove residue, take supernatant to filter, ultrafiltration, carry out vacuum spray drying, produce brown alga oligose finished product, degradation product purity reaches
85%.
The present invention establishes the bacterium according to the algin catenase AlgM sources new isolated strains Vibrio sp.BZM-1 of marine alga
Genomic library, screening obtain alginate lyase gene --- AlgM；It and other sources are found after being compared according to Blast
Alginate lyase gene (Pseudoalteromonas sp.CY24alginate lyase (alyPI) gene and Vibrio
Sp.A9m alg gene for alginate lyase) nucleotide identity highest only up to 71%, the amino acid of derivation is consistent
Property highest only up to 83%, illustrates that the gene is a new gene, GenBank accession number is KM655840, the sequence of AlgM genes
It is as follows：
The sequence of coding is：MKHKIVKTLLASSVLFAVGCTSNGNDTSNLHPQSETGAPLLTPVAIEASSHDGNGP
DRLFDQDINTRWSANGDGEWAVLDYGSVHEFDAVRAAFSKGNERKSKFDILVSTDGKTWTPVLQNQESSGGVIGYER
FEFSPVQARYVKYVGHGNTSNGWNSVTELAAVKCGVNACPSNQIITPAVIAAEQGLIAQQKEAEKARQAARKDLRKG
NFGVPAVYPCQTTVKCAKTALPVPTGLPTTPKAGNKPSQNFDLTSWYLSQPFDHDNNNRPDDVSEWDLANGYEHPDV
FYTAKDGGLVFKSFVKGVRTSPNTKYARTEMREMLRRGDTSIPTKGVNKNNWVFSSAPVADQKAAGGVDGVMEATLK
IDHTTTTGEAGEVGRFIIGQIHDQDDEPIRLYYRKLPNQEKGTVYFAHENTLKGTDQYFDLVGGMTGEIGDDGIALG
EKFSYRIAVKGNTLTVTVMRDGKPDAKQVVDMSQSGYDVGGKYMYFKAGVYNQNITGEMDDYVQATFYKLEKSHGTY
QGK。
Embodiment one
1st, the first step screening algin catenase AlgM genes of the inventive method：
According to the algin catenase AlgM sources new isolated strains Vibrio sp.BZM-1 of marine alga, the gene of the bacterium is established
Group library, the genomic DNA of the bacterium is extracted first, partially digested, agarose gel recovery 4- is of restriction enzyme Sau3A I
10kb fragment, these fragments are mended dGTP, then glue reclaim 3-7kb fragments, while by carrier pBZM201 restriction enzymes
Sap I linearization for enzyme restriction, and this fragment is reclaimed as carrier, benefit base 4-10kb fragment and Sap I linearization for enzyme restriction
PBZM201 carriers connect 24h at 16 DEG C, Escherichia coli Xl10-gold competent cells are then converted, so as to obtain the base of the bacterium
Because of a group library (see Fig. 1).20h is cultivated on the LB culture medium flat plates containing sodium alginate and ampicillin, finally with 5%
Cetyl pyridinium solution submergence 10min or so, the bacterium colony for occurring hydrolyzing circle is extracted DNA, then convert Escherichia coli
Xl10-gold competent cells, cultivated on the LB culture medium flat plates containing sodium alginate and ampicillin, corresponding points contain
The LB culture medium flat plates of sodium alginate and ampicillin, the corresponding bacterium colony for larger hydrolysis circle occur is punctured and serves extra large Sani
Bioisystech Co., Ltd is sequenced, so as to obtain alginate lyase gene --- AlgM；And sequencing is carried out, the sequence
Gene GenBank accession number is KM655840,545 amino acid of the gene code, and core sequence encodes 521 amino acid.
2nd, plasmid pHBM905BDM is subjected to double digestion through restriction enzyme Cpo I and Not I, produces two with solid
Determine the single-stranded cohesive end () of base, Ago-Gel reclaims the carrier see figure 3.；Secondly according to AlgM gene orders, use
GeneTool Software for Design AlgM-F：5’GTCAATGAAACATAAAATCGTCAAAACATTA 3 ' and AlgM-R：5’GGCCATwo primers of TTATTTACCTTGATAGGTGCCGTG 3 ', enter performing PCR using high-fidelity DNA polymerase and expand, obtain
AlgM (1.6kb) genetic fragment, after the fragment Ago-Gel is reclaimed, under the conditions of existing for dTTP, with T4DNA polymerases
Fragment 30min is handled in 12 DEG C, the cohesive end of pairing, glue reclaim fragment are thought in generation with pHBM905BDM carriers.Then will
Carrier is connected 2-3h for 16 DEG C with fragment through Solution I ligases, converts Escherichia coli Xl10-gold competent cells, ammonia benzyl
Penicillin resistance LB plate screening transformants, compared through plasmid size, PCR is identified, digestion identification and sequencing identification are recombinated
Plasmid.Random recombinant plasmid of selecting serves extra large Sani Bioisystech Co., Ltd sequencing, and correct recombinant plasmid name will be sequenced
For pHBM905BDM-AlgM (such as Fig. 2).
3rd, by recombinant plasmid pHBM905BDM-AlgM restriction enzyme Sal I linearization for enzyme restriction, the enzyme after glue reclaim
Cut product and Pichia pastoris GS115 competent cell is converted by PEG1000 methods, be then coated with histidine defect culture medium MD and put down
Plate, 28 DEG C are cultivated 3 days, obtain recombinant conversion.Random picking transformant corresponding points contain BMGY and containing sodium alginate
BMMY culture medium flat plates, add methanol induction 2-3 days, then the BMMY culture medium flat plates of sodium alginate with 5% cetyl
Pyridine solution submergence 10min or so, the bacterium colony on the corresponding BMGY culture medium flat plates for the bacterium colony for occurring hydrolyzing circle is extracted it
Genomic DNA.Using this genomic DNA as template, using AlgM-F and AlgM-R, this enters performing PCR identification, amplification to primer as primer
The transformant for obtaining about 1.6kb products is positive colony, and correct transformant is identified by Function Identification and PCR, final true
It is set to positive colony.
4th, by recombinant plasmid pHBM905BDM-AlgM by digestion and connection repeatedly, you can obtain multiple copy expression
The recombinant plasmid of box structure tandem sequence repeats.The recombinant plasmid of different copy numbers is linear with restriction enzyme Sal I digestions
Change, the digestion products after recovery convert Pichia pastoris GS115 competent cell by PEG100 methods, are then coated with histidine defect
Culture medium MD flat boards, 28 DEG C are cultivated 3 days, obtain recombinant conversion of different copy numbers.Copy number n=2,3,4 ..., n are certainly
So number.
It will verify that the Pichia pastoris correctly containing four copy expression cassette structures is named as Pichia pastoris GS115/AlgM4
(Pichia sp GS115/AlgM4), the preserving number for sending preservation are CCTCC No：M 2015500.The structure stream of the recombinant plasmid
Journey (such as Fig. 3).
Embodiment two
By build three copies, four copies, five copies, six copies and the Pichi strain of seven copy expression cassette structures
It is inoculated in respectively in 100mlBMGY culture mediums, 28 DEG C -30 DEG C, 260rpm culture 40-50h, to OD600For 20-30, in room temperature from
The heart 5000rpm, 5min, collect thalline；Thalline is transferred in 100ml BMMY culture mediums, 28 DEG C -30 DEG C, 260rpm cultures,
Methanol the 1.0% of culture volume (dosage be) is added every 12h, it is upper when taking highest expression quantity respectively after induced expression
Clear liquid carries out SDS-PAGE electrophoresis, analyzes the Pichi strain protein expression situation of different copy expression cassette structures, takes three
The Pichi strain expression time of copy, four copies and five copy expression cassette structures is 96h, 120h and 144h supernatant,
Each sample takes 8 microlitres of progress SDS-PAGE electrophoresis of supernatant, finds in 144h, the Pichia pastoris of four copy expression cassette structures
The zymotic fluid of bacterial strain shows that band is most wide most bright (see Fig. 4), consistent (see Fig. 5) with the obtained result of zymotic fluid survey enzyme activity.
Embodiment three
It is prepared by the restructuring algin catenase crude enzyme liquid obtained using shaking flask in example two, hydrolysis various concentrations sodium alginate
Brown alga oligose.
Raw material：Sodium alginate
The concentration for preparing hydrolysis substrate respectively is 10%, 15%, 20%, 25%, 30%；With 30-40 DEG C of 100ml of temperature
Alkaline waters of the pH between 8 to 9 makees solvent, adds appropriate sodium alginate, stirring, be configured to concentration of substrate be respectively 10%,
15%th, 20%, 25%, 30% reaction solution 100ml, then in substrate and enzyme liquid ratio 1g:100ul, be separately added into 1000ul,
1500ul, 2000ul, 2500ul, 3000ul crude enzyme liquid, adjust pH to 8-9.Hydrolysis time is respectively 16,14,12,10,8 small
When.Reaction solution is subjected to centrifugation and removes residue, takes supernatant to filter, ultrafiltration, vacuum spray drying is carried out, produces brown alga oligose finished product.
It is 85% or so to calculate yield.
Algin catenase that the present invention expresses and be bifunctional enzyme, have comparative advantage status in the enzyme system, works as external source
The expression quantity of albumen reaches highest, then tends to higher expression, and the enzyme activity of algin catenase is kept for 5-8 days and base
This does not reduce (see Fig. 5), and having now been found that the algin catenase of the Pichia anomala expression of four copy expression cassette structures has higher work
Property (see Fig. 4), enzymatic activity are up to 31000U/ml.
The substrate that the present invention degrades is：Algin, alginic acid and alginate etc..
Algin catenase enzyme activity and other documents and the enzyme of the algin catenase in patent of invention in the present invention
Vigour is as follows：
First, the enzyme activity of algin catenase is shown in Table 1 in the patent of invention announced.
Table 1
2nd, the enzyme activity of the of a relatively high algin catenase of the enzymatic activity reported in non-patent literature is shown in Table 2.
Table 2
3rd, conclusion is contrasted.
The enzyme activity of algin catenase in the present invention reaches 31000U/ml, is far longer than the algin reported at present
The enzyme activity of lyases.
Claims (2)
1. a kind of utilize the engineering bacteria for recombinating algin catenase preparation, it is characterised in that：Deposit number CCTCC No：
M2015500, entitled pichia pastoris phaff GS115/AlgM4.
2. the method for preparing brown alga oligose using the engineering bacteria hydrolysis described in claim 1, it is characterised in that：
（1）, fermentation algin catenase AlgM：Bacterial strain Pichia pastoris GS115/AlgM4 is placed in 100ml BMGY culture mediums,
28 DEG C~30 DEG C, 260rpm cultivates 40~50h, to OD600For 20~30,5000rpm, 5min are centrifuged in room temperature, collects thalline；
（2）, thalline is transferred in 100 ml BMMY culture mediums, 28 DEG C~30 DEG C, 260rpm cultures, first is added every 12h
Alcohol；It is crude enzyme liquid to obtain supernatant；
（3）, using obtain crude enzyme liquid hydrolysis sodium alginate prepare brown alga oligose：%~30 of concentration 10 of setting hydrolysis substrate
%, add the 100ml pH that temperature is 30~40 DEG C and make solvent in 8~9 alkaline water, a small amount of addition substrate, is progressively incremented by one side
The amount of substrate reaches predetermined amount, a small amount of on one side progressively to add crude enzyme liquid 1000ul-3000ul, adds substrate and enzyme amount ratio is
1g:100ul, uniform stirring；Keeping temperature is stable at 30~40 DEG C, until hydrolysis concentration reaches every 100ml alginic acids containing 20g
Sodium；
（4）, after the completion of hydrolysis, reaction solution is subjected to centrifugation and removes residue, takes supernatant to filter, ultrafiltration, carry out vacuum spray drying, obtain
To brown alga oligose finished product.
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| CN109295043A (en) * | 2018-10-19 | 2019-02-01 | 中国科学院天津工业生物技术研究所 | A kind of novel algin catenase, preparation method and application |
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