CN103525745A - Recombinant escherichia coli, method for preparing phospholipase C and method for degumming soybean crude oil - Google Patents
Recombinant escherichia coli, method for preparing phospholipase C and method for degumming soybean crude oil Download PDFInfo
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Abstract
The invention discloses recombinant escherichia coli, a method for preparing phospholipase C and a method for degumming soybean crude oil. The method for preparing phospholipase C (PLC) is finished by the following steps: (1) cloning by taking genome deoxyribonucleic acid (DNA) of staphylococcus aureus of which the preservation number is GIM1.142 (ATCC6538) as a template to obtain a phospholipase C gene, wherein a base sequence of phospholipase C gene is shown in SEQ ID NO:1 (2) cloning the phospholipase C gene obtained in the step (1) to a pET-28a(+) expression carrier, and building recombinant expression plasmids; (3) transforming the recombinant expression plasmids obtained in the step (2) into escherichia coli BL21(DE3) competent cells, so as to obtain the recombinant escherichia coli BL21(28a-S.a PLC). In the method for degumming soybean crude oil, phospholipase C produced by the recombinant escherichia coli is taken as a catalyst, so that the enzyme source is convenient, the yield is high, the fermentation technology is simple, and the cost is low. Compared with the traditional enzyme degumming, the method for degumming soybean crude oil by adopting phospholipase C has the advantages that the reaction time can be shortened; loss of neutral oil is reduced; the refining yield is improved. Therefore, the degumming method disclosed by invention has a certain application prospect in the field of the oil production industry.
Description
Technical field
The present invention relates to biotechnology and fat degumming field, particularly a strain recombination bacillus coli is prepared method and this enzyme catalysis crude oil of soybean Degumming method of Phospholipase C.
Background technology
Phospholipase C (phospholipase C, PLC) can catalytic hydrolysis phosphatide Sn-3 position ester bond produce the head group of triglyceride (DAG) and phosphorylation.According to the substrate specificity of PLC, mainly the PLC of bacterial origin can be divided into 2 large classes: phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine preference type Phospholipase C (PC-PLC).Hydrolysate InsP3 (IP3) and the triglyceride (DAG) of PLC play an important role in information transmission and cellular metabolism, and the former can impel Ca
2+from calcium storehouse, discharge; DAG energy activated protein kinase K (PKC), thus a series of endocellular functions triggered, as: cell proliferation, signal space distribution etc.PLC is to resisting cardiovascular disease, and platelet aggregation-against and the aspect such as antitumor have potential value.Along with going deep into of PLC research, its range of application is also constantly being widened, and expands to the fields such as chemical industry and food-processing from field of medicaments, for example: the ceramide that PLC processing sphingomyelin generates is widely used as the moisturizing composition of makeup; PLC can improve surface ag(e)ing phenomenon that the dough/pasta of freezing preservation produces when baking, relax pears leather jacket epidermis; PLC can also be used to do enzymatic degumming, further improves the efficiency of oil and fat refining etc.
The bacterium that produces PLC is a lot, for example: bacillus cereus (Bacillus cereus), bacillus thuringiensis (Bacillus thuringiensis), clostridium perfringens (Clostridium perfringens), Pseudomonas aeruginosa (Pseudomonas aeruginosa), streptococcus aureus (Staphyloccocus aureus), single listeria spp (Listeria monocytogenes), mycobacterium tuberculosis (Mycobacterium tuberculosis) etc. of increasing.As far back as the eighties in last century, abroad just have and utilize genetic engineering bacterium to express the report of PLC: nineteen eighty-two, the people such as Vasil have built strain P.aeruginosa PAO2003 (pVB81)-CT, and the enzyme work of PLC that this bacterial strain produces reaches 442.8U/ml; The people such as Cota-Gomez in 1997 have built E.coli BL21 (DE3) pGEM-PLCHR, and this bacterial strain produces the work of PLC enzyme can reach 24.1U/mg.Domestic PLC research is started late, for example: 2003, the normal high 15 strain Phospholipid hydrolase superior strains that filter out of king; And four strain bacterium have wherein been carried out to classification and identified; 2005, Chen Tao etc. on this basis, to high yield Phospholipid hydrolase bacterial strain B.cereus ShenZhen754-1 carried out ultraviolet ray and
60co-gamma-ray and mutagenesis is processed, and has obtained 3 strain high productive mutants, and phospholipase activity reaches respectively 14.878,16.450,16.400U/ml.2013, Zhao Jin magnitude successfully screened a Pseudomonas aeruginosa strain, and built the recombination bacillus coli that produces PLC, and enzyme work can reach 722.89U/ml.
Coming unstuck is the important step in soybean oil refining process, is directly connected to the quality of refining oil quality.Colloid in soybean oil is mainly phosphatide, therefore the high meeting of phospholipids content affect oil storage, refining and processing, the phosphorus content of soybean oil need be reduced to below 10ppm, could meet the requirement of oil and fat refining.Enzymatic degumming reaction conditions is gentle, economic environmental protection, and reduced the loss of neutral oil, and the PLC only a small amount of moisture of need that comes unstuck, and can remove nonhydratable phosphatide, can greatly reduce the quantity discharged of waste water.The people such as Meng Qingfei have carried out the research of PLC hydrolyzed soy oil phosphatide, make the refining yield of crude oil improve 3.08%.Purifine company has for oil prodution industry comes unstuck and the PLC product of refining, in crude oil of soybean, the mass percent of phosphatide is 1.76%, Purifine PLC can be hydrolyzed wherein 70%, the DAG80% of generation can stay in final product, has been equivalent to increase by 0.99% output.
PLC expression amount in animal and plant body is very limited, and extraction and sepn process loaded down with trivial details, cost is high.Microbe-derived, particularly the PLC in the genetic engineering bacterium source after restructuring have with short production cycle, the advantage such as production cost is low, and output is large.But also face some unavoidable problems simultaneously, as: the original strain that produces PLC is pathogenic bacterium mostly, is applied in food aspect and has potential safety hazard; Compared to traditional acid system, come unstuck, enzymatic degumming still faces the problem that cost is higher, if can realize the cost that effective immobilization of PLC can reduce oil and fat refining greatly; In addition, the PLC in common micro-organisms source is all undesirable to acid and hot tolerance, has greatly limited its application in foodstuffs industry, therefore need screening or transform out the PLC with stronger acidproof and resistance toheat.
Summary of the invention
Problem in view of existing in above-mentioned and/or existing enzyme genetically engineered and enzyme engineering field, has proposed the present invention.
Therefore, one of them object of the present invention is to provide a kind of recombination bacillus coli.
For solving the problems of the technologies described above, according to an aspect of the present invention, the invention provides following technical scheme: a kind of recombination bacillus coli BL21(DE3)-pET28a-Sa-PLC, deposit number is CCTCC No.M2013300.
As recombination bacillus coli of the present invention (Escherichia coli) BL21(DE3)-pET28a-Sa-PLC, a kind of preferred version of CCTCC No.M2013300, wherein: by following method, made:
(1) take the genomic dna that deposit number is the streptococcus aureus of GIM1.142 (ATCC6538) is template, and clone obtains phospholipase C gene, and its base sequence is as shown in SEQ ID NO:1;
(2) phospholipase C gene of (1) gained is cloned on pET-28a (+) expression vector, builds the expression plasmid that obtains restructuring;
(3), by recombinant plasmid transformed e. coli bl21 (DE3) competent cell of (2) gained, obtain recombination bacillus coli BL21(DE3)-pET28a-Sa-PLC.
Intestinal bacteria provided by the present invention (Escherichia coli) BL21(DE3)-pET28a-Sa-PLC, Chinese Typical Representative culture collection center (CCTCC), deposit number on June 28th, 2013, have been preserved in: CCTCC No.M2013300, address: China, Wuhan, Wuhan University.
Another object of the present invention is to provide a kind of recombination bacillus coli BL21(DE3 that utilizes)-pET28a-Sa-PLC carries out as fermentation strain the method that liquid fermenting is prepared Phospholipase C.
According to another aspect of the present invention, the invention provides the method for preparing Phospholipase C, it be take this recombination bacillus coli and carries out liquid fermenting as fermentation strain, prepares Phospholipase C.
As a kind of preferred version of preparing the method for Phospholipase C of the present invention, wherein: comprise,
(1) by recombination bacillus coli BL21(DE3)-pET28a-Sa-PLC is inoculated in the seed culture medium of sulfur acid kantlex 50mg/mL, and in 37 ℃, 180～200r/min shake-flask culture is to logarithmic phase, as seed liquor;
(2) seed liquor described in (1) is inoculated in fermention medium by 1%～5% inoculum size, in 37 ℃, 180～200r/min shake-flask culture is to OD600=0～1.2, add again IPTG to final concentration 0.001～0.1mM, in 18～37 ℃, inducing culture 3～30h under 180～200r/min condition;
(3) gained fermented liquid in (2) is centrifugal, collect bacterial sediment, with damping fluid, resuspended and ultrasonication cell, centrifugal by cytoclasis liquid, gets supernatant, is the Phospholipase C crude enzyme liquid preparing.
As a kind of preferred version of preparing the method for Phospholipase C of the present invention, wherein: the described seed culture based component of step (1) is: NaCl10g/L, peptone 10g/L, yeast extract 5g/L, pH7.2～7.4.
As a kind of preferred version of preparing the method for Phospholipase C of the present invention, wherein: the described fermentation culture based component of step (2) is: peptone 12g/L, yeast extract 24g/L, glycerine 0.4% (v/v), KH
2pO
40～170mM, K
2hPO
40～720mM.
As a kind of preferred version of preparing the method for Phospholipase C of the present invention, wherein: the resuspended damping fluid of described step (3) is the Tris-HCl of 50mM pH7.2, wherein MgCl
2concentration be 0～10mM.
A further object of the present invention is to provide the method for crude oil of soybean being carried out to degumming process, the method energy Reaction time shorten, and reduced the loss of neutral oil, improve refining yield.
According to a further aspect of the invention, the invention provides a kind of method that crude oil of soybean comes unstuck, comprise,
(1) crude oil of soybean is heated to 60～95 ℃, adds the citric acid of mass concentration 35%～55%, the pass of citric acid volume and quality of crude oil is 1.2mL/kg, acid treatment 20min under 300r/min stirring velocity;
(2) crude oil of soybean of handling in (1) is cooled to 40～60 ℃, adding mass concentration is that 4% NaOH solution regulation system pH is between 4～6;
(3) in the ratio of 400～2000U/kg crude oil of soybean, add Phospholipase C as prepared in claim 3 method, then add distilled water, it is heavy by 2%～5% that the cumulative volume of Phospholipase C enzyme liquid and distilled water accounts for oil, mixes;
(4) under 300r/min stirring velocity, carry out enzymatic degumming, the reaction times is 0.5～4h;
(5) will the system of coming unstuck boil the 10min enzyme that goes out, under 10000r/min rotating speed, centrifugal 10min, retains upper strata oil sample, completes and comes unstuck.
The present invention adopts Phospholipase C that recombination bacillus coli produces as catalyzer, and enzyme source is convenient, and output is high, and zymotechnique is simple and cost is low; Phospholipase C comes unstuck and compares with traditional enzymatic degumming, can Reaction time shorten, and reduced the loss of neutral oil, and improve refining yield, in oil prodution industry field, there is certain application prospect.
Embodiment
A lot of details have been set forth in the following description so that fully understand the present invention, but the present invention can also adopt other to be different from alternate manner described here and implement, those skilled in the art can do similar popularization without prejudice to intension of the present invention in the situation that, so the present invention is not subject to the restriction of following public specific embodiment.
Embodiment 1:
Seed culture based component is: NaCl10g/L, peptone 10g/L, yeast extract 5g/L, pH7.4,121 ℃, sterilizing 20min.
Fermentation culture based component is: peptone 12g/L, yeast extract 24g/L, glycerine 0.4% (v/v), KH
2pO
4170mM, K
2hPO
4720mM, 121 ℃, sterilizing 20min.
By recombination bacillus coli BL21(DE3)-pET28a-Sa-PLC is inoculated in the seed culture medium of sulfur acid kantlex 50mg/mL, and in 37 ℃, 200r/min shake-flask culture is to logarithmic phase, as seed liquor; Seed liquor is inoculated in the 20mL fermention medium of sulfur acid kantlex 50mg/mL by 5% inoculum size, in 37 ℃, 200r/min shake-flask culture is to OD again
600=0.6, then add IPTG to final concentration 0.05mM, in 30 ℃, inducing culture 8h under 200r/min condition; By fermented liquid, in 4 ℃, the centrifugal 10min of 8000r/min, collects thalline, with the resuspended thalline of Tris-HCl of 50mM pH7.2, and ultrasonication 10min, by ultrasonic rear cell debris, in 4 ℃, the centrifugal 10min of 10000r/min, gets supernatant; By p-NPPC method, measuring enzyme work is 38.23U/mL.
Embodiment 2:
Seed culture based component is: NaCl10g/L, peptone 10g/L, yeast extract 5g/L, pH7.4,121 ℃, sterilizing 20min.
Fermentation culture based component is: peptone 12g/L, yeast extract 24g/L, glycerine 0.4% (v/v), KH
2pO
4170mM, K
2hPO
4720mM, 121 ℃, sterilizing 20min.
By recombination bacillus coli BL21(DE3)-pET28a-Sa-PLC is inoculated in the seed culture medium of sulfur acid kantlex 50mg/mL, and in 37 ℃, 180r/min shake-flask culture is to logarithmic phase, as seed liquor; Seed liquor is inoculated in the 20mL fermention medium of sulfur acid kantlex 50mg/mL by 1% inoculum size, in 37 ℃, 180r/min shake-flask culture is to OD again
600=1.0, then add IPTG to final concentration 0.005mM, in 37 ℃, inducing culture 5h under 180r/min condition; By fermented liquid, in 4 ℃, the centrifugal 10min of 8000r/min, collects thalline, with the resuspended thalline of Tris-HCl of 50mM pH7.2, and ultrasonication 15min, by ultrasonic rear cell debris, in 4 ℃, the centrifugal 10min of 10000r/min, gets supernatant; By p-NPPC method, measuring enzyme work is 48.59U/mL.
Embodiment 3:
Seed culture based component is: NaCl10g/L, peptone 10g/L, yeast extract 5g/L, pH7.4,121 ℃, sterilizing 20min.
Fermentation culture based component is: peptone 12g/L, yeast extract 24g/L, glycerine 0.4% (v/v), 121 ℃, sterilizing 20min.
By recombination bacillus coli BL21(DE3)-pET28a-Sa-PLC is inoculated in the seed culture medium of sulfur acid kantlex 50mg/mL, and in 37 ℃, 200r/min shake-flask culture is to logarithmic phase, as seed liquor; Again seed liquor is inoculated in the 20mL fermention medium of sulfur acid kantlex 50mg/mL by 3% inoculum size, directly adds IPTG to final concentration 0.001mM, in 30 ℃, inducing culture 8h under 180r/min condition; By fermented liquid, in 4 ℃, the centrifugal 10min of 8000r/min, collects thalline, with the resuspended thalline of Tris-HCl of pH7.2, and ultrasonication 15min, by ultrasonic rear cell debris, in 4 ℃, the centrifugal 10min of 10000r/min, gets supernatant; By p-NPPC method, measuring enzyme work is 73.04U/mL.
Embodiment 4:
Seed culture based component is: NaCl10g/L, peptone 10g/L, yeast extract 5g/L, pH7.4,121 ℃, sterilizing 20min.
Fermentation culture based component is: peptone 12g/L, yeast extract 24g/L, glycerine 0.4% (v/v), 121 ℃, sterilizing 20min.
Recombination bacillus coli BL21 (28a-S.a PLC) is inoculated in the seed culture medium of sulfur acid kantlex 50mg/mL, and in 37 ℃, 200r/min shake-flask culture is to logarithmic phase, as seed liquor; Again seed liquor is inoculated in the 20mL fermention medium of sulfur acid kantlex 50mg/mL by 5% inoculum size, directly adds IPTG to final concentration 0.001mM, in 30 ℃, inducing culture 10h under 180r/min condition; By fermented liquid, in 4 ℃, the centrifugal 10min of 8000r/min, collects thalline, with containing 10mM MgCl
2the resuspended thalline of Tris-HCl of pH7.2, ultrasonication 15min, by ultrasonic rear cell debris, in 4 ℃, the centrifugal 10min of 10000r/min, gets supernatant; By p-NPPC method, measuring enzyme work is 151.55U/mL.
Embodiment 5:
Crude oil of soybean is heated to 80 ℃, adds the citric acid of mass concentration 35%, the pass of citric acid volume and quality of crude oil is 1.2mL/kg, acid treatment 20min under 300r/min stirring velocity; Crude oil is cooled to 40 ℃, and adding mass concentration is that 4% NaOH solution regulation system pH is 4.2; Ratio in 800U/kg oil adds Phospholipase C, then adds distilled water, and it is heavy by 2% that the cumulative volume of Phospholipase C enzyme liquid and distilled water accounts for oil, mixes; Under 300r/min stirring velocity, carry out enzymatic degumming, the reaction times is 1h; Will the system of coming unstuck boil the 10min enzyme that goes out, under 10000r/min rotating speed, centrifugal 10min, retains upper strata oil sample, completes and comes unstuck.According to GB GB/T5537-2008, get 5g oil sample and carry out Analysis of phosphorus contents.The phosphorus content of soybean oil is by the near 25.12ppm of 266ppm.
Embodiment 6:
Crude oil of soybean is heated to 70 ℃, adds the citric acid of mass concentration 45%, the pass of citric acid volume and quality of crude oil is 1.2mL/kg, acid treatment 20min under 300r/min stirring velocity; Crude oil is cooled to 50 ℃, and adding mass concentration is that 4% NaOH solution regulation system pH is 4.7; Ratio in 1200U/kg oil adds Phospholipase C, then adds distilled water, and it is heavy by 2% that the cumulative volume of Phospholipase C enzyme liquid and distilled water accounts for oil, mixes MgCl in water
2concentration be 10mM; Under 300r/min stirring velocity, carry out enzymatic degumming, the reaction times is 2h; Will the system of coming unstuck boil the 10min enzyme that goes out, under 10000r/min rotating speed, centrifugal 10min, retains upper strata oil sample, completes and comes unstuck.According to GB GB/T5537-2008, get 5g oil sample and carry out Analysis of phosphorus contents.The phosphorus content of soybean oil is by the near 3.5ppm of 266ppm.
Embodiment 7:
Crude oil of soybean is heated to 70 ℃, adds the citric acid of mass concentration 45%, the pass of citric acid volume and quality of crude oil is 1.2mL/kg, acid treatment 20min under 300r/min stirring velocity; Crude oil is cooled to 60 ℃, and adding mass concentration is that 4% NaOH solution regulation system pH is 4.5; Ratio in 1000U/kg oil adds Phospholipase C, then adds distilled water, and it is heavy by 2% that the cumulative volume of Phospholipase C enzyme liquid and distilled water accounts for oil, mixes; Under 300r/min stirring velocity, carry out enzymatic degumming, the reaction times is 1h; Will the system of coming unstuck boil the 10min enzyme that goes out, under 10000r/min rotating speed, centrifugal 10min, retains upper strata oil sample, completes and comes unstuck.According to GB GB/T5537-2008, get 5g oil sample and carry out Analysis of phosphorus contents.The phosphorus content of soybean oil is by the near 19.49ppm of 266ppm.
Embodiment 8:
Crude oil of soybean is heated to 70 ℃, adds the citric acid of mass concentration 45%, the pass of citric acid volume and quality of crude oil is 1.2mL/kg, acid treatment 20min under 300r/min stirring velocity; Crude oil is cooled to 45 ℃, and adding mass concentration is that 4% NaOH solution regulation system pH is 5.2; Ratio in 1600U/kg oil adds Phospholipase C, then adds distilled water, and it is heavy by 2% that the cumulative volume of Phospholipase C enzyme liquid and distilled water accounts for oil, mixes; Under 300r/min stirring velocity, carry out enzymatic degumming, the reaction times is 2h; Will the system of coming unstuck boil the 10min enzyme that goes out, under 10000r/min rotating speed, centrifugal 10min, retains upper strata oil sample, completes and comes unstuck.According to GB GB/T5537-2008, get 5g oil sample and carry out Analysis of phosphorus contents.The phosphorus content of soybean oil is by the near 7.06ppm of 266ppm.
It should be noted that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (8)
1. recombination bacillus coli BL21(DE3)-pET28a-Sa-PLC, deposit number is CCTCC No.M2013300.
2. recombination bacillus coli according to claim 1, is characterized in that: by following method, made:
(1) take the genomic dna that deposit number is the streptococcus aureus of GIM1.142 (ATCC6538) is template, and clone obtains phospholipase C gene, and its base sequence is as shown in SEQ ID NO:1;
(2) phospholipase C gene of (1) gained is cloned on pET-28a (+) expression vector, builds the expression plasmid that obtains restructuring;
(3), by recombinant plasmid transformed e. coli bl21 (DE3) competent cell of (2) gained, obtain recombination bacillus coli BL21(DE3)-pET28a-Sa-PLC.
3. with recombination bacillus coli described in claim 1 or 2, prepare the method for Phospholipase C, it is characterized in that: take this recombination bacillus coli carries out liquid fermenting as fermentation strain, prepares Phospholipase C.
4. prepare according to claim 3 the method for Phospholipase C, it is characterized in that: concrete steps are as follows:
(1) by recombination bacillus coli BL21(DE3)-pET28a-Sa-PLC is inoculated in the seed culture medium of sulfur acid kantlex 50mg/mL, and in 37 ℃, 180～200r/min shake-flask culture is to logarithmic phase, as seed liquor;
(2) seed liquor described in (1) is inoculated in fermention medium by 1%～5% inoculum size, in 37 ℃, 180～200r/min shake-flask culture is to OD
600=0～1.2, then add IPTG to final concentration 0.001～0.1mM, in 18～37 ℃, inducing culture 3～30h under 180～200r/min condition;
(3) gained fermented liquid in (2) is centrifugal, collect bacterial sediment, with damping fluid, resuspended and ultrasonication cell, centrifugal by cytoclasis liquid, gets supernatant, is the Phospholipase C crude enzyme liquid preparing.
5. prepare according to claim 4 the method for Phospholipase C, it is characterized in that: the described seed culture based component of step (1) is: NaCl10g/L, peptone 10g/L, yeast extract 5g/L, pH7.2～7.4.
6. prepare according to claim 4 the method for Phospholipase C, it is characterized in that: the described fermentation culture based component of step (2) is: peptone 12g/L, yeast extract 24g/L, glycerine 0.4% (v/v), KH
2pO
40～170mM, K
2hPO
40～720mM.
7. Phospholipase C preparation method according to claim 4, is characterized in that: the resuspended damping fluid of described step (3) is the Tris-HCl of 50mM pH7.2, wherein MgCl
2concentration be 0～10mM.
8. a crude oil of soybean Degumming method, is characterized in that: comprise the steps:
(1) crude oil of soybean is heated to 60～95 ℃, adds the citric acid of mass concentration 35%～55%, the pass of citric acid volume and quality of crude oil is 1.2mL/kg, acid treatment 20min under 300r/min stirring velocity;
(2) crude oil of soybean of handling in (1) is cooled to 40～60 ℃, adding mass concentration is that 4% NaOH solution regulation system pH is between 4～6;
(3) in the ratio of 400～2000U/kg crude oil of soybean, add Phospholipase C as prepared in claim 3 method, then add distilled water, it is heavy by 2%～5% that the cumulative volume of Phospholipase C enzyme liquid and distilled water accounts for oil, mixes;
(4) under 300r/min stirring velocity, carry out enzymatic degumming, the reaction times is 0.5～4h;
(5) will the system of coming unstuck boil the 10min enzyme that goes out, under 10000r/min rotating speed, centrifugal 10min, retains upper strata oil sample, completes and comes unstuck.
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