CN104726357B - The bacillus amyloliquefaciens mutagenic strain and phospholipase C of one plant of generation phosphatidase - Google Patents
The bacillus amyloliquefaciens mutagenic strain and phospholipase C of one plant of generation phosphatidase Download PDFInfo
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Abstract
The present invention provides the bacterial strains that one plant is expressed phospholipase C, are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.8499.Preparing resulting phospholipase C using bacterial strain provided by the present invention and preparation method can be used for oil and fat refining, and degumming effect is good, can be widely applied to oil and fat refining, additive, medicine and other fields.
Description
Technical field
The invention belongs to enzymatic degumming field, the specifically bacillus amyloliquefaciens mutagenic bacteria of one plant of generation phosphatidase
Strain.
Background technique
Phosphatidase (Phospholipase, PL) is that the existing enzyme that can hydrolyze glycerophosphatide, hydrolysate are in organism
Various phosphatidic acids and amino alcohol, such as cholamine, choline, serine, ethanol amine.According to the site of phosphatide enzyme hydrolysis glycerophosphatide
(Sehmiel DH, Miller VL.Bacterial Phospholipases and Pathogenesis [J] .Microbes
And Infection, 1999,1:1103-1112) different, phosphatidase can be divided into phospholipase A (phospholipase A,
PLA), phospholipase B (Phospholipase B, PLB), phospholipase C (Phospholipase C, PLC) and phospholipase D
(Phospholipase D, PLD), as shown in Figure 1.Phospholipase C acts on 3 phosphoric acid ester bonds, and product is diglyceride, phosphoric acid
Choline or phosphoethanolamine etc..
Phosphatidase can be widely applied to concise grease, phospholipid modified, feed modifying agent, food industry and medicine etc..
Degumming is an important link during vegetable oil refining, most important to the quality for improving oil, and degumming mainly removes phosphorus
Rouge will affect the deep processing of oil product and the stability of oil product if degumming is not thorough, and lower oil product shelf life.Enzymatic degumming can be with
Overcome the glue removal efficiency of traditional Degumming method low, while the problem of neutral oil loss height etc..And in enzymatic degumming technique, it is existing
Document reports that phosphatidase PLA is used for vegetable oil degumming more.Phosphatidase PLA (including PLA1 and PLA2), can produce polarity after degumming
Lysophosphatide and polarity fatty acid, PLA degumming tech only loses phospholipid molecule total in oil, to reduce refining loss.And phosphorus
Lipase PLC enzyme is reacted by selective hydrolysis phosphate functional group with phosphatide, and diglyceride (DAG) and phosphatide acid groups are generated,
Diglyceride be not required to it is to be removed, so PLC degumming tech only removes phosphate functional by retaining original phospholipid molecule
Group is to reduce refining loss, the problem of there is no the polarity fatty acid that can improve degummed oil acid value generated in PLA degumming tech,
PLC is better than PLA for degumming to be with the obvious advantage.
Degumming is carried out using PLC, PLC enzyme both can be used, can also directly use the fermentation of phospholipase C producing bacterial strain
Liquid.The fermentation liquid for directly using high enzyme activity, can exempt many and diverse purification step, improve the utilization rate of enzyme, be greatly reduced and be produced into
This.But existing phospholipase C producing bacterial strain, is mostly pathogenic bacterial strains, therefore can not directly use existing phospholipase C producing strains
The fermentation liquid of strain directly carries out degumming to grease.Therefore, the high GRAS(U.S. FDA evaluation food addition of phospholipase C enzyme activity is found
The safety indexes of agent) bacterium is at a key factor to solve this problem.
Summary of the invention
It is an advantage of the invention to provide a kind of bacterial strains for expressing phospholipase C.
The bacterial strain of the expression phospholipase C of separation provided by the invention, is named as WBRD00091-D, is preserved in the micro- life of China
Object culture presevation administration committee common micro-organisms center, deposit number are CGMCC No.8499.
A further object of the invention is, provides a kind of method for producing phospholipase C.
The method of production phospholipase C provided by the invention includes: to cultivate bacterial strain above-mentioned so that the bacterial strain or cell are raw
Phospholipase C is produced, phospholipase C is harvested.
In a preferred embodiment of the invention, the culture is to be suitable for the strain growth and/or be suitable for described
The bacterial strain is cultivated under conditions of bacterial strain expression phospholipase C, it is preferred that described to cultivate to be suitable for the condition of the strain growth
The lower culture bacterial strain, to increase the bacterial strain quantity, then then at being suitable for cultivating under conditions of the bacterial strain expression phospholipase C
The bacterial strain, to express the phospholipase C.
In a preferred embodiment of the invention, the culture is in 15 DEG C -55 DEG C, the 50-300rpm culture bacterium
Strain 5-72h.
In a preferred embodiment of the invention, the harvest includes separation and collects containing secretion or be discharged into extracellular
Phospholipase C culture supernatants, thus to obtain the phospholipase C enzyme solution containing phospholipase C；Preferably, the method is also wrapped
It includes and the phospholipase C enzyme solution is concentrated.
A further object of the invention is, provides a kind of phospholipase C.
Phospholipase C provided by the invention is to be made with preceding method.
A further object of the invention is, provides a kind of phospholipase C enzyme solution.
Phospholipase C enzyme solution provided by the invention is to be made with preceding method.
A further object of the invention is, provides a kind of method of degumming.
The method of degumming provided by the invention is to carry out degumming using phospholipase C above-mentioned or phospholipase C enzyme solution, preferably
, the degumming is fat degumming.
A further object of the invention is, provides bacterial strain or phospholipase C or phospholipase C enzyme solution above-mentioned for degumming
Purposes, it is preferred that the degumming is fat degumming.
A further object of the invention is, provides bacterial strain or phospholipase C or phospholipase C enzyme solution above-mentioned and changes for phosphatide
Property, the purposes of food additives, feed modifying agent and medical industry.
Bacterium --- the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) of production phospholipase C of the invention
WBRD00091-D and the enzyme solution obtained using the bacterial strain and phospholipase C can be used for oil and fat refining, and degumming effect is good, due to
Bacillus amyloliquefaciens are GRAS bacterial strains, therefore, are used directly for food industry, securely and reliably, such as can be with safe ready
Be used for oil and fat refining.What equally the phospholipase C in bacillus amyloliquefaciens source can be safe adds for phospholipid modified, food
Agent, feed modifying agent and medical industry etc..
Detailed description of the invention
Fig. 1 is the restriction enzyme site schematic diagram of several phosphatidases.
Fig. 2 is the TLC testing result figure after bacillus amyloliquefaciens phospholipase C processing phosphatidyl choline, wherein swimming lane 1
For the sample of bacillus amyloliquefaciens PLC hydrolytic phosphatide phatidylcholine (PC)；Swimming lane 2 is 1,2 glyceryl dioleates；Swimming lane 3 is 1,3
Glyceryl dioleate；Swimming lane 4 is olein, and swimming lane 5 is phosphatidyl choline.
Fig. 3 is bacillus amyloliquefaciens phospholipase C optimum temperature curve.
Fig. 4 is bacillus amyloliquefaciens phospholipase C optimum pH curve.
Fig. 5 is bacillus amyloliquefaciens phospholipase C temperature stability curve.
Fig. 6 is the pH stability curve of bacillus amyloliquefaciens phospholipase C
Fig. 7 is the influence curve to bacillus amyloliquefaciens phospholipase C activity such as metal ion, EDTA
The bacterial strain is deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms on November 29th, 2013
Center (CGMCC) " (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, postcode 100101),
Deposit number is CGMCC No.8499, classification naming are as follows: bacillus amyloliquefaciens Bacillus amyloliquefaciens.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following embodiment is merely to illustrate this
Invention is not for limiting the scope of the invention.
In the present invention, if without particularly illustrating, percentage (%) or part all refer to the weight relative to composition
Percentage or parts by weight.
In the present invention, if without particularly illustrating, related each component or its preferred ingredient be can be combined with each other
Form new technical solution.
In the present invention, if without particularly illustrating, all embodiments mentioned in this article and preferred implementation side
Formula can be combined with each other to form new technical solution.
In the present invention, if without particularly illustrating, all technical characteristics and preferred feature mentioned in this article can
New technical solution is formed to be combined with each other.
In the present invention, if not opposite explanation, the sum of content of each component is 100% in composition.
In the present invention, if not opposite explanation, the sum of number of each component can be 100 weight in composition
Part.
In the present invention, unless otherwise indicated, numberical range " a-b " indicates the contracting of any real combinings between a to b
Sketch form shows that wherein a and b is real number.Such as numberical range " 0-5 " expression has all listed between " 0-5 " herein
Whole real numbers, " 0-5 " are that the breviary of these combinations of values indicates.
In the present invention, unless otherwise indicated, integer numberical range " a-b " indicates the arbitrary integer combination between a to b
Breviary indicate, wherein a and b is integer.Such as integer numberical range " 1-N " indicates 1,2 ... N, wherein N is integer.
In the present invention, unless otherwise indicated, " a combination thereof " indicates the multicomponent mixture of each element, such as two
Kind, three kinds, four kinds and until maximum possible multicomponent mixture.
If be not specifically stated, term "an" used in this specification refers to "at least one".
If be not specifically stated, the benchmark of percentage (including weight percent) of the present invention is all the combination
The total weight of object.
" range " disclosed herein is in the form of lower and upper limit.It can be respectively one or more lower limits and one
Or multiple upper limits.Given range is defined by a selected lower limit and a upper limit.Selected lower and upper limit limit
The boundary of special range is determined.All ranges that can be defined in this way comprising and can combine, i.e., any lower limit
It can combine to form a range with any upper limit.For example, the range of 60-120 and 80-110 are listed for special parameter, reason
Solution is that the range of 60-110 and 80-120 is also to expect.In addition, if the minimum zone value 1 and 2 listed, and if list
Maximum magnitude value 3,4 and 5, then below range can all expect: 1-3,1-4,1-5,2-3,2-4 and 2-5.
Herein, unless otherwise indicated, the ratio or weight of each component all refer to dry weight.
Herein, unless otherwise indicated, each reaction all carries out at normal temperatures and pressures.
Herein, unless otherwise indicated, each reaction step can be carried out sequentially, can also be carried out out of order.Example
Such as, other steps be may include between each reaction step, and can also be with reversed order between reaction step.Preferably, originally
Reaction method in text is that sequence carries out.
The present inventor obtains a bacillus amyloliquefaciens (Bacillus by mutagenic and breeding
Amyloliquefaciens) WBRD00091-D, through detecting, which is able to produce that have provided phosphatidyl choline (PC) active
Phospholipase C, the phospholipase C degumming effect are good.Since bacillus amyloliquefaciens are GRAS bacterial strains, it can be used for food
Industry securely and reliably, such as can be used for oil and fat refining with safe ready.Equally, bacillus amyloliquefaciens provided by the invention
The phospholipase C in source, acceptable safety are used for phospholipid modified, food additives, feed modifying agent and medical industry etc..
For example, prepare phosphatidase modified phospholipid, make phosphatide nutritive value, emulsifiability and color, in terms of greatly improve and mention
Height, to expand application range and use value of the phosphatide in food, health care product industry significantly；In food additives side
Face, phospholipase C of the invention can be used for baking, can make dough formed colloidal complex, it is possible to reduce starch retrogradation, adjust and
Improve the stability of dough, therefore also very extensive baking industrial application；It, will be provided by the invention in terms of medical research
Phospholipase C can be used for preventing and treating the infection of various pathogens as vaccine, develop antiplatelet with phospholipase C provided by the invention
Assemble class drug, cardiovascular and cerebrovascular disease etc. can be treated or prevented, phospholipase C provided by the invention can also be applied to antineoplastic
The development etc. of object.
The present invention provides a kind of bacterial strains of the expression phospholipase C of mutagenic and breeding, are named as WBRD00091-D, are preserved in
China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are CGMCC No.8499.
The present invention provides a kind of methods for producing phospholipase C.
The method of production phospholipase C provided by the invention includes: culture bacterial strain CGMCC No.8499 above-mentioned so that described
Bacterial strain or cell produce phospholipase C, harvest phospholipase C.
The conventional culture methods of bacillus amyloliquefaciens can be used in the cultural method of bacterial strain CGMCC No.8499, such as make
It is cultivated with LB culture medium, YPD culture medium or PDA culture medium.
The culture of bacterial strain CGMCC No.8499 can be suitable for the strain growth and/or be suitable for the bacterial strain expression phosphorus
The bacterial strain is cultivated under conditions of lipase C, it is preferred that the culture of the bacterial strain CGMCC No.8499 be suitable for the bacterial strain
The bacterial strain is cultivated under conditions of growth, to increase the bacterial strain quantity, then then at being suitable for bacterial strain expression phospholipase C
Under the conditions of cultivate the bacterial strain, to express the phospholipase C.
The condition of culture of bacterial strain CGMCC No.8499 can be, but not limited to are as follows: cultivate institute in 15 DEG C -55 DEG C, 50-300rpm
State bacterial strain 5-72h, it is preferred that culture bacterial strain CGMCC No.849916-48h.
The phospholipase C of production can be secreted into extracellular culture medium by bacterial strain CGMCC No.8499.At of the invention one
In preferred embodiment, the harvest phospholipase C includes separation and the culture for collecting containing secretion or being discharged into extracellular phospholipase C
Object supernatant, thus to obtain the phospholipase C enzyme solution containing phospholipase C.
In one of embodiment, phospholipase C enzyme solution of the invention is concentrated, phospholipase C enzyme can be improved in this way
The phospholipase C activity of liquid.Enzyme solution of the invention can be used directly.It can be using various methods known to enzyme engineering field from originally
Phospholipase C is further separated and purified in the enzyme solution of invention, thus to obtain phospholipase C of the invention.
The present invention also provides a kind of phospholipase C, phospholipase C provided by the invention is to be made using preceding method.
The present invention also provides a kind of phospholipase C enzyme solution, phospholipase C enzyme solution provided by the invention is with preceding method system
?.In a preferred embodiment of the invention, which is containing secreting or be discharged on the culture of extracellular phospholipase C
Clear liquid.In another preferred embodiment of the invention, phospholipase C enzyme solution of the invention is concentrated, can be mentioned in this way
The phospholipase C activity of high phosphorus lipase C enzyme solution.
The present invention also provides a kind of methods of degumming.
The method of degumming provided by the invention is to carry out degumming using phospholipase C above-mentioned or phospholipase C enzyme solution, due to solution
Bacillus amyloliquefaciens are GRAS bacterial strains, and therefore, phospholipase C of the invention or phospholipase C enzyme solution can be used for grease, especially edible
With the degumming of grease.
The present invention also provides the purposes that bacterial strain above-mentioned or phospholipase C or phospholipase C enzyme solution are used for degumming, it is preferred that
The degumming is the degumming of grease, especially edible fats.
A further object of the invention is, provides bacterial strain or phospholipase C or phospholipase C enzyme solution above-mentioned and changes for phosphatide
Property, the purposes of food additives, feed modifying agent and medical industry.
In following embodiments of the invention, the culture medium that uses:
LB culture medium (unit gL-1): tryptone 10, yeast extract 5, sodium chloride 10, pH7.0.
The fermentation medium for producing phospholipase C for bacillus amyloliquefaciens forms (unit wt%): sucrose 0.5, peptone
0.5, yeast extract powder 1.0, beef extract 0.5, K2HPO40.05, MgSO4·7H2O 0.05, CaCl20.05, MnSO4·H2O
0.1, pH7.0；
LB agar plate: 1.8% agar is added in LB culture medium.
Lecithin agar plate:
A: yeast extract powder 0.5%, peptone 0.5%, K2HPO40.1%, KCl 0.5%, anhydrous magnesium sulfate 0.05%, L-
Arginine 0.5%, agar 1.8%, pH are natural.
B: lecithin mixture 3g/45ml, 115 DEG C of 15min sterilizings.
After sterilizing, A 110ml and B 90ml mixing is taken then to make plate.
Skimmed milk agar plate:
Component A: K2HPO40.1%, KCl 0.5%, epsom salt 0.05%, agar powder 2%, pH7.5,113 DEG C go out
Bacterium 30 minutes
Component B: 20% skimmed milk power, pH is naturally, 115 DEG C sterilize 15 minutes
After sterilizing, component A 150ml and component B 50ml are mixed while hot, then make plate.
Glycerin tributyrate plate
Component A: ammonium sulfate 0.1%, yeast extract powder 0.5%, peptone 0.5%, K2HPO40.1%, KCl 0.5%, seven
Water magnesium sulfate 0.05%, agar 1.8%, pH are natural.
Component B: 1.25g Arabic gum is dissolved in 88ml water, is heated while stirring, until be completely dissolved, filter paper filtering,
Half is taken, 6ml glycerin tributyrate, emulsification is added.
Aseptic condition simultaneously takes component B 4.3ml to be added to the mixing of 100ml component A completely in 60 DEG C of heat preservations, then
Make plate.
In following embodiments of the invention, the enzyme activity of phospholipase C uses P- nitrophenylphosphate choline (p-NPPC) method
Detect (Birch M., Robson G., Law D., et al.Evidence of Multiple Extracellular
Phospholipase Activities of Aspergillus fumigates.Infect.Immun.1996,64(3):
751-755.), this method passes through the phosphorus of the catalyzing hydrolysis substrate of phospholipase C using p- nitrophenylphosphate choline as reaction substrate
Acid esters key and generate color products p-nitrophenol (p-NP), which has strong absorb under 405-410nm wavelength.Enzyme activity
Unit of force is defined as: enzyme amount needed for the PLC enzyme activity of 1 unit refers to the p-NP of 1 μm of ol of catalysis release per minute.This method
It is the common method of domestic and international measurement phospholipase C vigor, it is easy to operate, fast and high sensitivity is reacted, is a kind of accurate and efficient
Vigour-testing method.
The breeding of embodiment 1, mutagenic strain
Bacillus amyloliquefaciens GIMT1.192(is purchased from Guangdong Province's Culture Collection (GIMCC)) it is seeded to
In shaking flask containing 50ml LB liquid medium, 180rpm, 30 DEG C of culture 16h.The bacterium solution 1ml of bacillus amyloliquefaciens is taken,
The sterile saline of 2ml is added, measuring the OD value at 600nm at this time is 0.517.
It takes bacillus amyloliquefaciens GIMT1.192 bacterium solution 1ml into the triangle shake bottle with a small amount of bead, is added 30 μ L's
The dithyl sulfate solution of 50wt%.After 28 DEG C of oscillation treatment 17min, the sodium thiosulfate that the 25wt% of 0.4ml is added immediately is molten
Liquid.
Take out the bacteria suspension dilution 10 of 1ml3、104With 105After times, the bacteria suspension 0.1ml of each dilution is taken to be respectively coated on
LB agar plate, lecithin agar plate, tributyrin agar plate and skimmed milk agar plate are trained in 28 DEG C of constant temperature
It supports to growing bacterium colony.
It picks out all appearances, transparent circle or muddy circle from each plate three is any and differ markedly from most of bacterium colony
Bacterium carry out fermented and cultured, in 180rpm, 30 DEG C of culture 16h.
It takes fermentation liquid to be centrifuged 10min in 12000g, centrifugal clear liquid phospholipase C enzyme activity is measured, according to the PLC enzyme of shake flask fermentation
Biopsy survey selects part in 28 DEG C of constant temperature incubations to bacterium colony is grown as a result, the high bacterium colony of picking enzyme activity carries out plating dilutions coating
Bacterium colony as outer appearnce and the discrepant bacterium colony of partial external appearance characteristic carry out shake flask fermentation.Fermentation liquid enzyme activity is measured, from relaying
It is continuous to pick out the high bacterium colony of enzyme activity and carry out continuation breeding.
The bacillus amyloliquefaciens mutagenic fungi of high yield phospholipase C is obtained by continuous 5 passages and breeding, is named as
WBRD00091-D。
WBRD00091-D bacterial strain and GIMT1.192 bacterial strain are subjected to shake flask fermentation with batch, respectively take fermentation liquid 1ml in
After 12000g is centrifuged 10min, the detection of phospholipase C vigor is carried out to its supernatant.The results show that GIMT fermented supernatant fluid enzyme activity
For 1.02u/ml, WBRD00091-D strain fermentation supernatant enzyme activity is 2.18u/ml, and WBRD00091-D bacterial strain produces phospholipase C
Enzyme activity improves 100% or more compared with original strain GIMT1.192.
The bacterial strain is on November 29th, 2013 " in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart " (CGMCC) preservation, deposit number are CGMCC No.8499, classification naming are as follows: bacillus amyloliquefaciens Bacillus
amyloliquefaciens。
The identification of embodiment 2, the extracellular phospholipase C of bacillus amyloliquefaciens WBRD00091-D
With the produced phospholipase C hydrolytic phosphatide phatidylcholine of bacillus amyloliquefaciens WBRD00091-D
(Phosphatidylcholine PC), by 0.5ml enzyme solution, 0.1M pH value 8.0Tris-HCL buffer, the 2.5ml of 2.0ml
2% phosphatidyl choline (be more than 98% phosphatidyl choline using purity, be purchased from Aladdin reagent Co., Ltd) mixing, in
150rpm, 37 DEG C of water-baths, oscillating reactions 24 hours.
After reaction, 1ml is sampled, n-hexane extraction is added in equal volume, oscillation mixes, and 12000rpm is centrifuged 2 minutes, takes
Upper organic phase part out is added into a new pipe.It takes lower layer's water phase to repeat extraction, centrifugally operated, collects, merges twice just
Hexane extract is uncapped, and is placed in draught cupboard organic phase volatilization is complete.Every Guan Zhongjia 15ul isopropanol fills part dissolution.It takes
5ul point chromatoplate.Carry out TLC detection (TLC detection method referring to document Toida J., Arikawa Y., Kondou K.,
Fukuzawa M..Purification and characterization of triacylglycerol lipase from
Aspergillus oryzae.1998.Bioscience Biotechnology Biochemistry, 62 (4): 759-763).
TLC testing result is shown in Fig. 2, occurs diglyceride in hydrolysate, thus determines the enzyme for phospholipase C.
The preparation of the extracellular phospholipase C of embodiment 3, bacillus amyloliquefaciens
(1) fermentation of the extracellular phospholipase C of bacillus amyloliquefaciens
Bacillus amyloliquefaciens WBRD00091-D is seeded in LB culture medium, in 28 DEG C, 180rpm, culture for 24 hours, is obtained
It obtains bacillus amyloliquefaciens WBRD00091-D seed and trains liquid.
Bacillus amyloliquefaciens WBRD00091-D seed liquor is seeded in LB culture medium with 1% inoculum concentration, in 28 DEG C,
180rpm is cultivated for 24 hours.
It takes bacillus amyloliquefaciens WBRD00091-D fermentation culture to be centrifuged 10min in 12000g, collects and obtain centrifugation clearly
Liquid about 45ml detects the phospholipase C enzyme activity of centrifugal clear liquid, and the enzyme activity of the phospholipase C of centrifugal clear liquid is about 2.1u/ as the result is shown
ml。
(2) concentration of bacillus amyloliquefaciens phospholipase C crude enzyme liquid
By the method that manufacturer provides, centrifugal clear liquid is surpassed using the film (Omega, PALL company, the U.S.) of 2-30kDa
Filter concentration obtains concentration enzyme solution about 5.8ml.
Bacillus amyloliquefaciens phospholipase C enzyme activity after detection concentration, the results show that the bacillus amyloliquefaciens after concentration
Phospholipase C enzyme activity is about 9.1u/ml.
Embodiment 4, bacillus amyloliquefaciens WBRD00091-D extracellular phospholipase C part zymologic property
(1) optimum temperature of the extracellular phospholipase C of bacillus amyloliquefaciens
Respectively 25 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 70 DEG C at a temperature of measurement embodiment 3 prepare
The enzyme activity of resulting bacillus amyloliquefaciens phospholipase C.It is other using the PLC enzyme activity of the temperature spot of highest PLC enzyme activity as 100%
The PLC enzyme activity of temperature spot is divided by the highest enzyme activity, so that the opposite enzyme activity of the temperature spot is obtained, using opposite enzyme activity as ordinate,
Temperature spot is abscissa, and the opposite enzyme activity of each temperature spot is sequentially connected with smoothed curve, by these with respect to enzyme activity with smoothed curve
Connection, is as a result shown in Fig. 3.
Fig. 3 is the results show that the optimal reactive temperature of WBRD00091-D01160 PLC is 55 DEG C.
(2) optimum pH of the extracellular phospholipase C of bacillus amyloliquefaciens
The pH value for preparing 0.1M is respectively 3.0,3.5,4.0,4.5,5.0,5.5 and 6.0 acetic acid-acetate buffer body
System；Prepare the Tris-HCl buffer system that the pH value of 0.1M is 6.5,7.0,7.5,8.0,8.5 and 9.0；Prepare 0.1M concentration
The Glycine-NaOH buffer system that pH value is 9.5 and 10.0；Prepare disodium hydrogen phosphate-hydroxide of the pH11.0 of 0.1M
Sodium buffer system.
Respectively pH value be 3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,
In 10.0 and 11.0 300ul buffer system, be added 5ul embodiment 3 prepare concentration enzyme solution, under 50 DEG C of water bath conditions into
The detection of row PLC enzyme activity.
Using the PLC enzyme activity of the pH point of highest PLC enzyme activity as 100%, the PLC enzyme activity of other pH divided by the highest enzyme activity, from
And the opposite enzyme activity of the pH is obtained, using opposite enzyme activity as ordinate, pH value is abscissa, is sequentially connected each pH's with smoothed curve
Opposite enzyme activity, is as a result shown in Fig. 4.
Fig. 4 the results show that WBRD01160 ferment crude enzyme liquid PLC enzyme activity optimum pH between 8.5-9.0.
(3) temperature stability of bacillus amyloliquefaciens phospholipase C
Take 500ul to be distributed into aliquot enzyme solution, be respectively placed in 4 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C,
After keeping the temperature 1h in 55 DEG C, 60 DEG C and 70 DEG C of water-bath, enzyme activity is detected, with the PLC enzyme activity of sample under 4 DEG C of water-baths for 100%,
The PLC enzyme activity of its temperature spot obtains it divided by the enzyme activity with respect to enzyme activity value, and using opposite enzyme activity as ordinate, temperature is cross
Coordinate is connected the opposite enzyme activity of each temperature spot with smoothed curve, as a result sees Fig. 5.
The enzyme solution PLC activity of Fig. 5 WBRD00091-D01160 as the result is shown stands 1 hour, PLC in 4-45 DEG C of water-bath
Enzyme activity is more stable, has almost no change, and enzyme solution remains to the enzyme that reservation is more than 80% after standing 1 hour in 50 DEG C, 55 DEG C of water-baths
Living, enzyme solution stands 1 in 60 DEG C of water-baths still can as a child retain vigor more than 75%.And PLC enzyme activity is then in 70 DEG C of water-baths
Decline is quickly.
(4) stability of the bacillus amyloliquefaciens phospholipase C in different pH environments
The pH value for preparing 0.1M is respectively 3.0,3.5,4.0,4.5,5.0,5.5 and 6.0 acetic acid-sodium acetate buffer solution；
Prepare the Tris-HCl buffer that the pH value of 0.1M is 6.5,7.0,7.5,8.0,8.5 and 9.0；Prepare 0.1M concentration pH value be
9.5 and 10.0 Glycine-NaOH buffer；Prepare disodium hydrogen phosphate-sodium hydrate buffer solution of the pH11.0 of 0.1M.
By the above-mentioned different pH value of phospholipase C concentration enzyme solution and the 0.1M concentration of 2 times of volumes prepared in embodiment 3
Buffer mixed after, be placed in 4 DEG C of environment place 5h after, measure enzyme activity.With three points of the enzyme activity force value of embodiment 3
One of value be 100%, calculate enzyme activity relative value when each pH value, as a result see Fig. 6.
Fig. 6's the results show that the present invention prepares resulting PLC more stable in the range of pH6.5-9.5, pH6.0
With the vigor still when pH10 with 90% or more.
(5) influence of the metal ion to bacillus amyloliquefaciens phospholipase C activity
Respectively prepare 250mM each metal ion (cobalt ions, manganese ion, zinc ion, magnesium ion, calcium ion, copper ion,
Nickel ion, iron ion, ammonium ion, sodium ion, potassium ion) mother liquor, prepare sodium citrate, the EDTA mother liquor of 250mM.By above-mentioned mother
Liquid is added according to the amount of 2.5mM and 5.0mM into reaction system respectively, measures enzyme activity, not add any ion and EDTA
The energy value that sample is surveyed is 100%, as control, calculates the sample enzyme activity value of other additives, as a result sees Fig. 7.
Fig. 7's the results show that the manganese ion of 2.5mM and 5mM, the cobalt ions of 2.5mM have apparent rush to the vigor of the PLC
Into effect, and the cobalt ions and citrate ion of copper ion, zinc ion, iron ion, nickel ion, high concentration, EDTA etc. pair
The PLC has very strong inhibiting effect.
The above results show that the optimum temperature of the produced phospholipase C of bacillus amyloliquefaciens of the present invention is 55 DEG C, most suitable
PH is 8.5-9.0, can be by a certain amount of manganese all having very high stability within 45 DEG C and in the range of pH6.5-9.5
It activates with the cobalt ions of low concentration, is obviously inhibited by zinc from, the cobalt ions of iron ion, copper ion, nickel ion and high concentration.
Claims (13)
1. a kind of bacterial strain of the expression phospholipase C of separation, the bacterial strain is bacillus amyloliquefaciens, is preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, deposit number are CGMCC No.8499, classification naming are as follows: solution starch gemma bar
Bacterium (Bacillus amyloliquefaciens).
2. a kind of method for producing phospholipase C, comprising: cultivate bacterial strain described in claim 1 so that the bacterial strain produces phosphatide
Enzyme C harvests phospholipase C.
3. method according to claim 2, which is characterized in that the culture is to be suitable for the strain growth and/or be suitable for
The bacterial strain is cultivated under conditions of the bacterial strain expression phospholipase C.
4. method as claimed in claim 3, which is characterized in that the culture is to train under conditions of being suitable for the strain growth
The bacterial strain is supported, to increase the bacterial strain quantity, then then at being suitable under conditions of bacterial strain expression phospholipase C described in culture
Bacterial strain, to express the phospholipase C.
5. method according to claim 2, which is characterized in that the culture is in 15 DEG C -55 DEG C, 50-300rpm culture institute
State bacterial strain 5-72h.
6. the method as described in any one of claim 2-5, the harvest includes separation and collects containing secretion or be discharged into
The culture supernatants of extracellular phospholipase C, thus to obtain the phospholipase C enzyme solution containing phospholipase C
7. method as claimed in claim 6, which is characterized in that dense the method also includes being carried out to the phospholipase C enzyme solution
Contracting.
8. a kind of phospholipase C enzyme solution is made with claim 6 or 7 the methods.
9. a kind of method of degumming carries out degumming using the phospholipase C enzyme solution of claim 8
10. method as claimed in claim 9, which is characterized in that the degumming is fat degumming.
11. the purposes that bacterial strain described in claim 1 or the phospholipase C enzyme solution of claim 8 are used for degumming
12. purposes as claimed in claim 11, which is characterized in that the degumming is fat degumming.
13. bacterial strain described in claim 1 or the phospholipase C enzyme solution of claim 8 are used for phospholipid modified, food additives, feeding
Expect the purposes of modifying agent and medical industry.
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