CN109136207A - A kind of method of recombination bacillus coli production phospholipase D - Google Patents
A kind of method of recombination bacillus coli production phospholipase D Download PDFInfo
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- CN109136207A CN109136207A CN201810843255.2A CN201810843255A CN109136207A CN 109136207 A CN109136207 A CN 109136207A CN 201810843255 A CN201810843255 A CN 201810843255A CN 109136207 A CN109136207 A CN 109136207A
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Abstract
The invention discloses a kind of methods of recombination bacillus coli production phospholipase D, phospholipase D is placed under rigorous type promoter control, the region par of pSC101 is inserted into expression plasmid, the inheritance stability of plasmid is kept using Escherichia coli recA mutant strain simultaneously, growth period is enriched with the cell culture high density containing plasmid, induction period saturation induction, temperature is reduced simultaneously and applies alkali metal salt stress, alleviate PLD to the cytotoxicity of host, inhibit cell cracking, the generated time for extending PLD, to improve the expression of PLD.
Description
Technical field
The invention belongs to genetic engineerings and technical field of microbial fermentation, and in particular to a kind of recombination bacillus coli production phosphorus
The method of lipase D.
Background technique
Phospholipase D (EC 3.1.4.4, PLD), using phosphatide as substrate, acts on phosphodiester bond, according to the difference of receptor
(water and alcohol) occurs hydrolysis and turns phosphatidyl reaction.Wherein, it by turning phosphatidyl reaction, can be introduced in substrate phosphatide
Various alcohol radicals generate the phosphatide of multiple biological activities and medical value, are widely used in food and pharmaceutical industry.Such as
When substrate is phosphatidyl choline (PC), and receptor is serine, glycerol and ethanol amine, it is phosphatidyl silk ammonia that PLD converts PC respectively
Sour (PS), phosphatidyl glycerol (PG) and phosphatidyl-ethanolamine (PE) (and Iwasaki 2013).Wherein, with
Aging of population arrive, PS is as brain health nutritional supplement, by lasting concern, and in succession by U.S. FDA, Japan
HBM and China national health State Family Planning Commission are authenticated.Compared to the PS for extracting from bovine brain and plant, pass through by substrate of Soybean PC
The PS of PLD biological enzyme preparation, avoids food safety and the low problem (Mor é et al.2014) of plant source content.Into one
Step, some new structure and function phosphatide turn phosphatidyl reaction by PLD and are synthesized, for example, phosphatidyl batyl alcohol
(Arranz-Mart í nez et al.2017), phosphatidyl glucose (Song et al.2012), cuorin analog
(Muller et al.2012), phosphatidyl tyrosol (Yamamoto et al.2011；Casado et al.2013), phosphatidyl
Terpenes (Yamamoto et al.2008a；Yamamoto et al.2008b；Gliszczy ń ska et al.2016) and phosphatide
Acyl serinol (Dippe et al.2008), it is some in them that there is anticancer and antioxidant activity.
In industrial application and laboratory research, for turning the PLD of phosphatidyl reaction mainly from plant (cucumber, cabbage
And peanut) and actinomyces (predominantly streptomycete).Compare other sources, they show it is higher turn phosphatidyl reactivity,
And source is easy to get.Dippe etc. (Dippe et al.2008) compares PLDcab(source cabbage) and PLDstr(source
Streptomyces sp.) catalyze and synthesize the yield and purity of opposed polarity head phosphatide；In all reactions, PLD is comparedcab,
PLDstrIt shows and considerably higher turns phosphatidyl reactivity.
Be limited to phospholipase D source deficiency and price it is high (streptomyces PLD, >=150units/mg, 6516.9$/
1000U, Sigma), industrial extensive use is restricted.The maximum output of PLD is by Japanese scholars Ogino etc. at present
(Ogino et al.2004) became lead streptomycete secreting, expressing in recombination by technique for gene engineering in 2004 and obtains, up to 5.5
×104U·L-1(118mg·L-1)；The country, 2013, Zhang Ying (jade-like stone 2013) used for reference similar technology, became lead strepto- in recombination
Bacterium obtains comparable expression quantity (58U/mL)；Recent five years, other domestic researcher's breeding strain excellents and Optimal Medium, PLD
Yield 103U·L-1Left and right.And in Escherichia coli and yeast expression system, it recombinates PLD and shows serious cytotoxicity,
The problem of causing plasmid instability, cell cracking, generated time short and enzyme leakage, it is difficult to realize that high density fermentation is expressed
(Iwasaki et al.1995；Mishima et al.1997；Zambonelli et al.2003).
Summary of the invention
It is an object of the invention in place of overcome the deficiencies in the prior art, provide a kind of recombination bacillus coli production phosphatide
The method of enzyme D is inserted into stability region in expression plasmid, and plasmid is kept to stablize heredity in Escherichia coli recA mutant strain,
Growth period cultivates high density, and induction period using the saturation induction of rigorous type Arabinose promoter, reduces temperature and applies alkali gold
Belong to salt stress, to alleviate PLD to the cytotoxicity of host, inhibits cell cracking, extend the generated time of PLD, be greatly improved
The expression of PLD, is finally reached 1.1 × 106U·L-1(748mg·L-1), enzyme activity yield be before 20 times of maximum output.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of method of recombination bacillus coli production phospholipase D, comprising:
1) using the plasmid pUC57-par of the par regional sequence containing the pSC101 as shown in SEQ ID No.1 as template,
Expand par sequence；To contain Arabinose promoter PBADPlasmid pBADK be template, expand plasmid pBADK skeleton；With above-mentioned
Obtained par sequence and pBADK skeleton are expanded respectively as primer and template, expands and converts, obtain carrier pBADKP；Using
Streptomyces antibioticus PLD gene order containing the codon optimization as shown in SEQ ID No.2
The plasmid pUC57-PLD and the carrier pBADKP of (GenBank accession no.MH237968), pass through Nco I and Xba
I double digestion is connected and is converted to Escherichia coli recA-Defect strain obtains the recombinant bacterium containing pBADKP-PLD；
2) incubation step 1) the obtained recombinant bacterium containing pBADKP-PLD, (high density refers to cell pair to culture high density
The middle and later periods of number growth, carbon source will be exhausted, such as reach OD600It is 5~6)；Then it supplements the nutrients, saturation induction is adjusted
Saving temperature is 16~22 DEG C, and addition alkali metal salt to concentration is 0.05~0.75M, and inducing expression obtains phospholipase D.
In one embodiment: in the step 1), amplification par sequence is using the primer 1 as shown in SEQ ID No.3 and such as
Primer 2 shown in SEQ ID No.4.
In one embodiment: in the step 1), amplification plasmid pBADK skeleton uses the primer 3 as shown in SEQ ID No.5
And the primer 4 as shown in SEQ ID No.6.
In one embodiment: in the step 1), using par sequence and pBADK skeleton as primer and template, using
POE-PCR is expanded and is converted to E.coli DH5 α.
In one embodiment: in the step 1), Escherichia coli recA-Defect strain is E.coli TOP10 (recA-), it obtains
Recombinant bacterium be TOP10/pBADKP-PLD.
In one embodiment: in the step 2), alkali metal salt is at least one of sodium salt, sylvite, lithium salts.
In one embodiment: in the step 2), alkali metal salt NaCl, concentration be 0.15~0.75M, preferably 0.35~
0.65M。
In one embodiment: in the step 2), inducing temperature is 17~19 DEG C, preferably 18 DEG C.
In one embodiment: in the step 2), nutrition is the glycerol and yeast powder that mass ratio is 1~1.25:1, such as 1:1
Or 1.25:1, final concentration of 20~30gL of glycerol-1。
In one embodiment: in the step 2), saturation induction be add inducer arabinose to concentration be 0.03~
0.04%OD600 -1。
The technical program compared with the background art, it has the following advantages:
The region par of pSC101 and recA-Guarantee plasmid stabilisation heredity, the Arabinose promoter P rigorously regulated and controledBADSuppression
Cell cracking processed and promotion PLD expression；Growth period is enriched with the cell high density containing plasmid, is conducive to improve yield；Induction
Phase supplements the nutrients, saturation induces, and promotes cell to keep optimal physiological status, to greatest extent producing enzyme；Alkali metal is added in induction period
Salt stress and low temperature stress are all conducive to alleviate PLD to the toxicity of cell, inhibit cell cracking, extend PLD generated time and rush
It is expressed into PLD.Present invention determine that composite optimization strategy so that plasmid stability be 100%, cell cracking complete inhibition, surpass
It crosses 99% recombination PLD and is maintained at intracellular, cell cracking and enzyme leakage are totally constrained in induction period, and PLD generated time is by document
2~3h of repayment extends to 36h, output increased to 1.1 × 106U·L-1(748mg·L-1), enzyme activity yield is prior art report
90 times of the bacillus coli expression level in road, 20 times of the streptomycete expression reported before being.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is for illustrating the influence that promoter expresses phospholipase D.
Fig. 2 is used to illustrate influence of the inducing temperature to cell growth and phospholipase D expression.
Fig. 3 is used to illustrate influence of the salt to cell growth and phosphatide expression of enzymes.Potassium phosphate (A), NaCl (B), KCl (C) and
LiCl(D)。
Fig. 4 is for illustrating that batch culture produces phospholipase D in 3L fermentor under salt stress.(A) timing diagram, (B) were induced
Journey SDS-PAGE analysis, swimming lane 1~6 respectively represent induction 0h, 6h, 12h, for 24 hours, 36h, 70h.Swimming lane 7 is the PLD of purifying.
Fig. 5 is pBAD/gIIIC plasmid map used in the embodiment of the present invention.
Fig. 6 is pBADK plasmid map used in the embodiment of the present invention.
Fig. 7 is pBADKP plasmid map used in the embodiment of the present invention.
Fig. 8 is pBADKP-PLD plasmid map used in the embodiment of the present invention.
Fig. 9 is pET22KP-PLD plasmid map used in the embodiment of the present invention.
Figure 10 is pLACKP-PLD plasmid map used in the embodiment of the present invention.
Figure 11 is pUC57-par plasmid map used in the embodiment of the present invention.
Figure 12 is pUC57-PLD plasmid map used in the embodiment of the present invention.
Specific embodiment
The contents of the present invention are illustrated below by embodiment:
Remarks: material as used in the following examples and instrument spy are explained as follows, but it is not limited to this.
Main experimental material:
(original plasmid derives from the plasmid pBAD/gIIIC of Invitrogen company to plasmid pBADK, by AmpRResistant gene
It is substituted for the Kan as shown in SEQ ID No.7RAfter obtain pBADK, and contain rigorous type Arabinose promoter PBAD, Guzman
Et al.1995), plasmid pBADKP-PLD (contains rigorous type Arabinose promoter PBADWith the region par), plasmid pET22KP-
PLD (contains T7 promoter PT7With the region par), plasmid pLACKP-PLD (contains rigorous type Lac operon Plac/ara-1, Lutz
And Bujard 1997 and the region par), (area par containing the pSC101 as shown in SEQ ID No.1 plasmid pUC57-par
Domain sequence, it is fully synthetic by raw work biology (Shanghai) Co., Ltd., obtain the matter containing the par sequence as shown in SEQ ID No.1
Grain pUC57-par) and the plasmid pUC57-PLD (Streptomyces containing the codon optimization as shown in SEQ ID No.2
Antibioticus PLD gene order, it is fully synthetic by raw work biology (Shanghai) Co., Ltd., it obtains containing such as SEQ ID No.2
Shown in PLD sequence plasmid pUC57-PLD).E.coli DH5 α is purchased from Sangon Biotech (Shanghai) Co., Ltd.,
E.coli TOP10(recA-) purchased from silent winged scientific and technological (China) Co., Ltd of generation that of match.Related plasmids map is shown in attached drawing, wherein black
Color filling mark indicates promoter (PBAD、PT7And Plac/ara-1), be positioned at signal peptide sequence (the pelB signal of periplasmic space
Sequence and Gene III secretion signal sequence), the region par, KanRResistant gene passes through Nco I
With Xba I insertion PLD, designed for the myc epitope label of West bolting experiment and for Ni2+Affinitive layer purification
6 × His histidine tag, the two label is located at the C-terminal of PLD.
The building of 1. recombinant bacterium TOP10/pBADKP-PLD of embodiment
With plasmid pUC57-par (the par regional sequence containing the pSC101 as shown in SEQ ID No.1) for template, lead to
Cross primer 1 and primer 2 (table 1), PCR amplification par sequence；To contain Arabinose promoter PBADPlasmid pBADK be template,
Pass through primer 3 and primer 4 (table 1), PCR amplification plasmid pBADK skeleton；Obtained par sequence and pBADK are expanded with above-mentioned two sections
Skeleton carries out POE-PCR respectively as primer and template, and product directly converts to E.coli DH5 α, bacterium colony PCR and verifies and survey
Sequence obtains carrier pBADKP；The Streptomyces of the codon optimization as shown in SEQ ID No.2 will be contained
The plasmid pUC57-PLD and carrier pBADKP of antibioticus PLD gene order pass through Nco I and Xba I double digestion, T4
Ligase connection, conversion to E.coli TOP10 (recA-), bacterium colony PCR is verified and is sequenced, and obtains recombinant bacterium TOP10/
pBADKP-PLD.The reaction system and thermal cycle conditions of PCR and POE-PCR is shown in Table 2, table 3, table 4 and table 5 respectively.
1 primer sequence of table
2 PCR reaction system of table
3 PCR thermal cycle conditions of table
4 POE-PCR reaction system of table
5 POE-PCR thermal cycle conditions of table
The influence that 2. promoter of embodiment expresses phospholipase D.
Four kinds of promoter PT7、Plac/ara-1 a、Plac/ara-1 bAnd PBADIt is used to investigate the expression of phospholipase D, start intensity
It is sequentially reduced.Wherein Plac/ara-1 aStarted by IPTG inducing moiety, Plac/ara-1 bStarted completely by IPTG and arabinose.Recombination
Bacterium TOP10/pBADKP-PLD induces 16h at 25 DEG C, as a result as shown in Figure 1.Under saturation induction, in the expression of PLD,
Plac/ara-1 bAnd PBADIt is better than PT7And Plac/ara-1 a, wherein PBADIt generates maximum PLD enzyme activity and compares yield.In Induction Process,
PLD enzyme activity under all promoters nearly all reaches maximum value after inducing 1h；After inducing 16h, enzymatic activities proportion
Respectively 100%, 25%, 34% and 26%；In whole process, the increases of enzymatic activities is with the reduction of biomass, wherein BLR
(DE3)/pET22KP-PLD (promoter PT7) biomass reduce it is most fast.These results indicate that the toxicity of PLD causes PLD to close
At the time is short and cell cracking, promoter is stronger, and extracellular PLD enzyme activity is higher, it is likely that due to cell cracking is accelerated.It is weaker
Rigorous type promoter (Plac/ara-1And PBAD) it is more suitable for expression PLD, generate higher enzyme activity and less extracellular amount.
3. inducing temperature of embodiment is grown to cell and the influence of phospholipase D expression
In this experiment, TOP10/pBADKP-PLD is cultivated to OD for 37 DEG C in TB culture medium600It is 5~6, then exists respectively
At 12 DEG C, 18 DEG C, 22 DEG C, 25 DEG C, 30 DEG C and 37 DEG C, inducing expression 16h investigates inducing temperature to TOP10/pBADKP-PLD table
Up to the influence of PLD and biomass, as a result as shown in Figure 2.Inducing temperature significantly affects the expression and cell growth of PLD.16~22
DEG C expression it is higher, highest expression appears in 18 DEG C, and 18 DEG C of ratio yield is 3.8 times of 30 DEG C, while 92%
PLD appear in it is intracellular.Meanwhile when temperature is greater than 18 DEG C, PLD expression is reduced.Cell is obviously wanted when being grown in appropriate low temperature
Be better than temperature it is high when, this reflects the toxicity that certain low temperature reduces PLD to cell, while cytolysis reduces.
4. salt stress of embodiment is grown to cell and the influence of phosphatide expression of enzymes
TOP10/pBADKP-PLD is cultivated to OD for 37 DEG C in TB culture medium600It is 5~6, is separately added into various concentration
Salt (potassium phosphate, NaCl, KCl and LiCl), 18 DEG C of inducing expression 16h, as a result as shown in Figure 3.Increase the dense of salt in induction period
Degree, hence it is evident that increase the expression of PLD, while alleviating PLD to the toxicity of cell, increase the biomass of cell.Wherein NaCl is the most aobvious
It writes, compared with the control group, enzyme activity highest improves 5 times, and biomass increased at 0.15~0.75M NaCl, optium concentration
For 0.35~0.65M.
Phospholipase D is expressed in 3L fermentor under 5. salt stress of embodiment
The a small amount of thallus of TOP10/pBADKP-PLD glycerol cryopreservation tube is taken, is seeded to 10mL LB culture medium, 37 DEG C, 200rpm,
Overnight incubation.It by 1% inoculum concentration, is forwarded in 200mL TB culture medium, 37 DEG C, 200rpm, cultivates 6~7h.Then it is seeded to
In 3L fermentor containing fermentation medium, temperature is controlled at 37 DEG C, to cell culture to OD600It is 50~60, glycerol has consumed, this
When adjust cultivation temperature to 18 DEG C.Into fermentor, sodium chloride, arabinose, glycerol and yeast powder are added respectively, wherein chlorination
The final concentration of 0.4M of sodium, the final concentration of 0.035%OD of arabinose600 -1, the mass ratio of glycerol and yeast powder is 1.25:1,
The final concentration of 25gL of glycerol-1.Terminate when Induction Process continues to biomass to begin to decline or PLD enzyme activity declines.As a result such as
Shown in Fig. 4, PLD synthesis continue for 32h, with glycerol consumption and cell growth.This shows that phase coupling is expressed in cell growth and PLD
It closes, during expression, the toxicity of PLD is overcome well.Final highest producing enzyme is up to 1.1 × 106U·L-1(748mg·L-1), born of the same parents
Outer producing enzyme is 1.6 × 104U·L-1, the 1.5% of total enzyme activity is only accounted for, showing cell cracking, there is no occur.
The above is only the preferred embodiment of the present invention, the range implemented of the present invention that therefore, it cannot be limited according to, i.e., according to
Equivalent changes and modifications made by the invention patent range and description, should still be within the scope of the present invention.
Sequence table
<110>Xiamen University
<120>a kind of method of recombination bacillus coli production phospholipase D
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gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct 240
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gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa 540
gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca 600
cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc 660
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Claims (10)
1. a kind of method of recombination bacillus coli production phospholipase D, it is characterised in that: include:
1) using the plasmid pUC57-par of the par regional sequence containing the pSC101 as shown in SEQ ID No.1 as template, amplification
Par sequence；To contain Arabinose promoter PBADPlasmid pBADK be template, expand plasmid pBADK skeleton；With above-mentioned amplification
Obtained par sequence and pBADK skeleton expands and converts, obtain carrier pBADKP respectively as primer and template；Using containing
The plasmid of the Streptomyces antibioticus PLD gene order of the codon optimization as shown in SEQ ID No.2
PUC57-PLD and the carrier pBADKP are connected and are converted to Escherichia coli recA by Nco I and Xba I double digestion-It lacks
Strain is fallen into, the recombinant bacterium containing pBADKP-PLD is obtained；
2) incubation step 1) the obtained recombinant bacterium containing pBADKP-PLD, cultivate high density；Then it supplements the nutrients, saturation lures
It leads, adjusting temperature is 16~22 DEG C, and addition alkali metal salt to concentration is 0.05~0.75M, and inducing expression obtains phospholipase D.
2. the method for recombination bacillus coli production phospholipase D according to claim 1, it is characterised in that: the step 1)
In, amplification par sequence uses the primer 1 as shown in SEQ ID No.3 and the primer 2 as shown in SEQ ID No.4.
3. the method for recombination bacillus coli production phospholipase D according to claim 1, it is characterised in that: the step 1)
In, amplification plasmid pBADK skeleton uses the primer 3 as shown in SEQ ID No.5 and the primer 4 as shown in SEQ ID No.6.
4. the method for recombination bacillus coli production phospholipase D according to claim 1, it is characterised in that: the step 1)
In, using par sequence and pBADK skeleton as primer and template, is expanded and converted to E.coli using POE-PCR
DH5α。
5. the method for recombination bacillus coli production phospholipase D according to claim 1, it is characterised in that: the step 1)
In, Escherichia coli recA-Defect strain is E.coli TOP10 (recA-), obtained recombinant bacterium is TOP10/pBADKP-PLD.
6. the method for recombination bacillus coli production phospholipase D according to claim 1, it is characterised in that: the step 2)
In, alkali metal salt is at least one of sodium salt, sylvite, lithium salts.
7. the method for recombination bacillus coli production phospholipase D according to claim 1, it is characterised in that: the step 2)
In, alkali metal salt NaCl, concentration is 0.15~0.75M.
8. the method for recombination bacillus coli production phospholipase D according to claim 1, it is characterised in that: the step 2)
In, inducing temperature is 17~19 DEG C.
9. the method for recombination bacillus coli production phospholipase D according to claim 1, it is characterised in that: the step 2)
In, nutrition is the glycerol and yeast powder that mass ratio is 1~1.25:1, final concentration of 20~30gL of glycerol-1。
10. the method for recombination bacillus coli production phospholipase D according to claim 1, it is characterised in that: the step 2)
In, saturation induction is that addition inducer arabinose to concentration is 0.03~0.04%OD600 -1。
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| CN110904072A (en) * | 2019-11-21 | 2020-03-24 | 天津科技大学 | Novel phospholipase D and method for preparing functional phospholipid by using same |
| CN111363733A (en) * | 2020-03-10 | 2020-07-03 | 天津科技大学 | Heat-resistant phospholipase D mutant and preparation method thereof and method for synthesizing functional phospholipid |
| CN114921395A (en) * | 2022-05-25 | 2022-08-19 | 厦门大学 | Recombinant escherichia coli constructed by CRISPR-Cas9 technology and application thereof in preparation of phospholipase D |
| CN116855474A (en) * | 2023-06-12 | 2023-10-10 | 天津科技大学 | Phospholipase mutant and preparation and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904072A (en) * | 2019-11-21 | 2020-03-24 | 天津科技大学 | Novel phospholipase D and method for preparing functional phospholipid by using same |
| CN111363733A (en) * | 2020-03-10 | 2020-07-03 | 天津科技大学 | Heat-resistant phospholipase D mutant and preparation method thereof and method for synthesizing functional phospholipid |
| CN111363733B (en) * | 2020-03-10 | 2022-04-08 | 天津科技大学 | Heat-resistant phospholipase D mutant and preparation method thereof and method for synthesizing functional phospholipid |
| CN114921395A (en) * | 2022-05-25 | 2022-08-19 | 厦门大学 | Recombinant escherichia coli constructed by CRISPR-Cas9 technology and application thereof in preparation of phospholipase D |
| CN114921395B (en) * | 2022-05-25 | 2024-05-03 | 厦门大学 | Recombinant escherichia coli constructed by CRISPR-Cas9 technology and application thereof in preparation of phospholipase D |
| CN116855474A (en) * | 2023-06-12 | 2023-10-10 | 天津科技大学 | Phospholipase mutant and preparation and application thereof |
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