CN104630176B - Method for increasing yield of phospholipase C produced by fermentation method - Google Patents
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Abstract
The invention provides a fermentation medium for a bacterial strain for producing phospholipase C. The fermentation medium comprises one or more of manganese ions, L-arginine and olive oil, and preferably comprises two or three of manganese ions, L-arginine and olive oil. The fermentation medium provided by the invention can improve the yield of phospholipase C produced by bacterial strain fermentation.
Description
Technical Field
The invention belongs to the field of enzyme degumming, and particularly relates to a method for increasing the yield of phospholipase C produced by a fermentation method.
Background
Phospholipases (PL) are enzymes present in organisms that hydrolyze glycerophospholipids, with the hydrolysis products being various phosphatidic acids and amino alcohols, such as cholestyramine, choline, serine, ethanolamine, etc. Phospholipase enzymes can be classified into Phospholipase A (PLA), Phospholipase B (PLB), Phospholipase C (PLC) and Phospholipase D (PLD) according to their site of hydrolysis of glycerophospholipids (Sehmiel DH, Miller VL. bacterial Phospholipids and Pathogenesis [ J ]. Microband Infection, 1999, 1: 1103-1112). Phospholipase C acts on 3-position phosphate ester bond, and the product is diglyceride, phosphorylcholine or phosphoethanolamine.
The phospholipase can be widely applied to the aspects of oil refining, phospholipid modification, feed modifiers, food industry, medicine and the like. Degumming is an important link in the vegetable oil refining process and is important for improving the quality of oil, the degumming is mainly to remove phospholipid, and if the degumming is not thorough, the deep processing of oil products and the stability of the oil products are influenced, and the shelf life of the oil products is reduced. The enzymatic degumming can overcome the problems of low degumming removal rate, high neutral oil loss and the like of the traditional degumming method. In the enzymatic degumming process, phospholipase PLA is widely used for degumming vegetable oil in the prior literature. Phospholipase PLA (including PLA1 and PLA2) produces polar lysophospholipids and polar fatty acids after degumming, and the PLA degumming process only loses the total phospholipid molecules in the oil to reduce refining losses. And the phospholipase PLC enzyme reacts with phospholipid through selectively hydrolyzing phosphate ester functional groups to generate Diglyceride (DAG) and phosphatidic acid groups, and the diglyceride does not need to be removed, so the PLC degumming process reduces refining loss by retaining original phospholipid molecules and only removing the phosphate ester functional groups, the problem of polar fatty acid which can improve the acid value of the degummed oil and is generated in the PLA degumming process is solved, and the degumming advantage of the PLC is obviously better than that of the PLA.
The existing phospholipase C product is produced by fermentation, so that the problem of improving the yield of the produced phospholipase C is urgently needed to be solved by the technical personnel in the field.
Disclosure of Invention
In a first aspect of the present invention, there is provided a fermentation medium for a phospholipase C producing strain.
The fermentation medium provided by the invention contains one or more of manganese ions, L-arginine and olive oil.
In a preferred embodiment of the present invention, the fermentation medium comprises two or three of manganese ions, L-arginine and olive oil.
In a preferred embodiment of the invention, the fermentation medium comprises manganese ions in the range of 0.59mM to 17.7mM, L-arginine in the range of 0.05 to 2wt%, preferably L-arginine in the range of 0.1 to 2wt%, and/or olive oil in the range of 0.42 to 1.5wt%, preferably olive oil in the range of 0.42 to 1.32 wt%.
In a preferred embodiment of the present invention, the fermentation medium contains manganese ions 2.95mM-11.8mM, L-arginine 0.3-1.5wt%, and/or olive oil 0.66-1.02 wt%.
In a preferred embodiment of the invention, the manganese ions in the fermentation medium are derived from inorganic salts of manganese ions.
In a preferred embodiment of the invention, the manganese ions in the fermentation medium are derived from one or more of manganese sulfate, manganese chloride, manganese acetate, manganese carbonate, manganese nitrate, manganese phosphate.
In a preferred embodiment of the invention, the strain is: bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, Bacillus atrophaeus or Bacillus simplex; in a preferred embodiment of the invention, the strain is: bacillus subtilis CGMCC No.7506, bacillus subtilis GIM1.135, bacillus subtilis GIM1.286, bacillus subtilis GIM1.129, bacillus licheniformis GIM1.182, bacillus licheniformis GIM1.362, bacillus licheniformis CGMCC1.0265, bacillus licheniformis CGMCC No.7878, bacillus licheniformis CGMCC1.0521 and bacillus licheniformis CGMCC 1.0813; bacillus pumilus CGMCC No.7879, Bacillus mojavensis CGMCC No.4961, Bacillus atrophaeus CGMCC No.7881 or Bacillus simplex CGMCC No. 7880.
In a second aspect of the invention, a method for increasing the yield of phospholipase C produced by fermentation of a strain is provided.
The method for improving the yield of the phospholipase C produced by the strain fermentation comprises the step of adding one or more of manganese ions, L-arginine and olive oil into a fermentation medium of the strain.
In a preferred embodiment of the present invention, two or three of manganese ions, L-arginine and olive oil are added to a fermentation medium of the strain.
In a preferred embodiment of the invention, the fermentation medium comprises manganese ions in the range of 0.59mM to 17.7mM, L-arginine in the range of 0.05 to 2wt%, preferably L-arginine in the range of 0.1 to 2wt%, and/or olive oil in the range of 0.42 to 1.5wt%, preferably olive oil in the range of 0.42 to 1.32 wt%.
In a preferred embodiment of the present invention, the fermentation medium contains manganese ions 2.95mM-11.8mM, L-arginine 0.3-1.5wt%, and/or olive oil 0.66-1.02 wt%.
In a preferred embodiment of the invention, the manganese ions in the fermentation medium are derived from inorganic salts of manganese ions.
In a preferred embodiment of the invention, the manganese ions in the fermentation medium are derived from one or more of manganese sulfate, manganese chloride, manganese acetate, manganese carbonate, manganese nitrate, manganese phosphate.
In a preferred embodiment of the invention, the strain is: bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, Bacillus atrophaeus or Bacillus simplex; in a preferred embodiment of the invention, the strain is: bacillus subtilis CGMCC No.7506, bacillus subtilis GIM1.135, bacillus subtilis GIM1.286, bacillus subtilis GIM1.129, bacillus licheniformis GIM1.182, bacillus licheniformis GIM1.362, bacillus licheniformis CGMCC1.0265, bacillus licheniformis CGMCC No.7878, bacillus licheniformis CGMCC1.0521 and bacillus licheniformis CGMCC 1.0813; bacillus pumilus CGMCC No.7879, Bacillus mojavensis CGMCC No.4961, Bacillus atrophaeus CGMCC No.7881 or Bacillus simplex CGMCC No. 7880.
In a third aspect of the invention, a method for improving the yield of phospholipase C produced by fermentation of a strain is provided, wherein the strain is a phospholipase C producing strain fermented by using the culture medium of the first aspect.
In a preferred embodiment of the invention, the strain is: bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, Bacillus atrophaeus or Bacillus simplex; in a preferred embodiment of the invention, the strain is: bacillus subtilis CGMCC No.7506, bacillus subtilis GIM1.135, bacillus subtilis GIM1.286, bacillus subtilis GIM1.129, bacillus licheniformis GIM1.182, bacillus licheniformis GIM1.362, bacillus licheniformis CGMCC1.0265, bacillus licheniformis CGMCC No.7878, bacillus licheniformis CGMCC1.0521 and bacillus licheniformis CGMCC 1.0813; bacillus pumilus CGMCC No.7879, Bacillus mojavensis CGMCC No.4961, Bacillus atrophaeus CGMCC No.7881 or Bacillus simplex CGMCC No. 7880.
The strain WBRD01280 of the invention has been preserved in China general microbiological culture Collection center (CGMCC) in 2013, 4 months and 22 days (China institute of microbiology, academy of sciences, China, institute of microbiology, No. 3, West Lu No. 1 Hospital, Chaozhou, Chaoyang, Beijing), the preservation number is CGMCC No.7506, the classification and the name are: bacillus subtilis.
The strain WBRD00240 disclosed by the invention has been preserved in China general microbiological culture Collection center (CGMCC) in 2013, 7 months and 4 days (China institute of microbiology, national institute of sciences, 3, national institute of sciences, west road, 1, north Chen, south China, Beijing City, the preservation number is CGMCC No.7879, and the classification and the designation are as follows: bacillus pumilus strain.
The strain WBRD01160 disclosed by the invention has been preserved in China general microbiological culture Collection center (CGMCC) in 2013, 7 months and 4 days (CGMCC) (institute of microbiology, China academy of sciences, No. 3, West Lu No. 1 Hospital, Kyoho, Beijing city, and zip code 100101), the preservation number is CGMCC No.7878, and the classification and the name are as follows: bacillus licheniformis.
The strain WBRD1.10062302 of the invention has been deposited in China general microbiological culture Collection center (CGMCC) in 2011 for 17 th 6 th (China institute of microbiology, national institute of sciences, 3 of West Lu No. 1 Hospital, Tokyo, Chaoyang, Japan), the preservation number is CGMCC No.4961, and the classification and the name are as follows: bacillus mojavensis.
The strain WBRD00980 of the invention has been preserved in China general microbiological culture Collection center (CGMCC) in 2013, 7 months and 4 days (China institute of microbiology, national institute of sciences, 3, national institute of sciences, west road, 1, north Chen, south China, Beijing City, the preservation number is CGMCC No.7881, and the classification and the name are as follows: bacillus atrophaeus.
The strain WBRD01120 disclosed by the invention has been preserved in China general microbiological culture Collection center (CGMCC) in 2013, 7 months and 4 days (China general microbiological culture Collection center, China academy of sciences, institute of microbiology, No. 3, West Lu No. 1 institute of North Chen West Lu, Chaozhou, China, Japan, and Japan, the preservation number is CGMCC No.7880, and the classification and the name are as follows: bacillus simplex simpliciens.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
In the present invention, the percentage (%) or parts refers to the weight percentage or parts by weight with respect to the composition, unless otherwise specified.
In the present invention, the respective components referred to or the preferred components thereof may be combined with each other to form a novel embodiment, if not specifically stated.
In the present invention, all embodiments and preferred embodiments mentioned herein may be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, the sum of the contents of the components in the composition is 100% if not indicated to the contrary.
In the present invention, the sum of the parts of the components in the composition may be 100 parts by weight, if not indicated to the contrary.
In the present invention, unless otherwise stated, the numerical range "a-b" represents a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "0 to 5" indicates that all real numbers between "0 to 5" have been listed herein, and "0 to 5" is only a shorthand representation of the combination of these numbers.
In the present invention, unless otherwise indicated, the integer numerical range "a-b" represents a shorthand representation of any combination of integers between a and b, where a and b are both integers. For example, an integer numerical range of "1-N" means 1, 2 … … N, where N is an integer.
In the present invention, unless otherwise specified, "combinations thereof" mean multicomponent mixtures of the elements described, for example two, three, four and up to the maximum possible.
The term "a" or "an" as used herein means "at least one" if not otherwise specified.
All percentages (including weight percentages) stated herein are based on the total weight of the composition, unless otherwise specified.
The "ranges" disclosed herein are in the form of lower and upper limits. There may be one or more lower limits, and one or more upper limits, respectively. The given range is defined by the selection of a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this manner are inclusive and combinable, i.e., any lower limit can be combined with any upper limit to form a range. For example, ranges of 60-120 and 80-110 are listed for particular parameters, with the understanding that ranges of 60-110 and 80-120 are also contemplated. Furthermore, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5.
Herein, unless otherwise specified, the proportions or weights of the components are referred to as dry weights.
In this context, each reaction is carried out at normal temperature and pressure unless otherwise specified.
Herein, unless otherwise specified, the individual reaction steps may or may not be performed sequentially. For example, other steps may be included between the various reaction steps, and the order may be reversed between the reaction steps. Preferably, the reaction processes herein are carried out sequentially.
The inventor of the invention finds that the enzyme activity of the strain for producing the phospholipase C can be effectively improved by adding the manganese ions, the L-arginine and/or the olive oil into the fermentation culture medium of the strain for producing the phospholipase C, and the effect of improving the enzyme activity of the strain for producing the phospholipase C is better when two or three of the manganese ions, the L-arginine and/or the olive oil are added into the fermentation culture medium of the strain for producing the phospholipase C. In the art, an increase in the enzymatic activity of a phospholipase C producing strain, particularly a wild-type strain, of lipase C also means an increase in the production of phospholipase C by the strain. Therefore, based on the findings of the present invention, it is clear to those skilled in the art that the addition of manganese ions, L-arginine and/or olive oil to the fermentation medium of a phospholipase C producing strain is effective in increasing the production of phospholipase C by the strain, and that the addition of two or three of manganese ions, L-arginine and/or olive oil to the fermentation medium of a phospholipase C producing strain is more effective in increasing the production of phospholipase C by the strain.
Accordingly, in a first aspect of the present invention, there is provided a fermentation medium for a phospholipase C producing strain.
The fermentation medium provided by the invention contains one or more of manganese ions, L-arginine and olive oil.
In a preferred embodiment of the present invention, the fermentation medium comprises two or three of manganese ions, L-arginine and olive oil.
In a preferred embodiment of the invention, the fermentation medium comprises:
manganese ion 0.59mM-17.7mM, L-arginine 0.05-2wt%, preferably L-arginine 0.1-2wt%, and/or olive oil 0.42-1.5wt%, preferably olive oil 0.42-1.32 wt%.
In a preferred embodiment of the invention, the fermentation medium comprises:
manganese ion 2.95mM-11.8mM, L-arginine 0.3-1.5wt%, and/or olive oil 0.66-1.02 wt%.
In a preferred embodiment of the invention, the manganese ions in the fermentation medium are derived from inorganic salts of manganese ions. In the present invention, the inorganic salt providing manganese ion is well known to those skilled in the art, and can be selected according to the common knowledge or need in the art when preparing the medium. In a preferred embodiment of the invention, the manganese ions in the fermentation medium are derived from one or more of manganese sulfate, manganese chloride, manganese acetate, manganese carbonate, manganese nitrate, manganese phosphate.
Fermentation media useful for phospholipase C producing strains are well known to those skilled in the art and may include, for example, but are not limited to: zwaal et al (R.F.A.Zwaal, et.al.complete Purification and protocols of yeast C from Bacillus strain Acta (BBA)1971, 474-479.), Taguchi Ryo et al (Taguchi Ryo, et.al. Purification and protocols of yeast-specific protease C of Bacillus strain Biophysical Acta (BBA) -Lipids and Lipid Metabolism,1980, 48-57.), Emanul Kambev et al (Emanniv, Kambe.purification, et.g. yeast J. biological strain Biochemical Acta, 1980, 48-57.), Emulation Kamben et al (yeast J. biological strain of yeast) culture of yeast J. biological strain Acta (BBA) -Lipids and Lipid Metabolism, 1990), Emulation Kamben et al (Emanniu.K.P.J. culture of yeast J. biological strain hydrolysate, 1980,48-57.,. fermentation of yeast J.,. yeast J., (biological strain hydrolysate J.) (Bacillus strain hydrolysate J.) -yeast J., (biological strain C. hydrolysate J.) -1990, culture of yeast strain, Bacillus strain C.S.S.S.. In one embodiment of the invention, the fermentation medium used is: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium hydrogen phosphate and pH 7.0. Other components of the fermentation medium used may be combined and adjusted by those skilled in the art in light of the present disclosure and the prior art, and the methods of combining and adjusting are conventional to those skilled in the art.
In a preferred embodiment of the present invention, the strains include, but are not limited to: bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, Bacillus atrophaeus or Bacillus simplex; in a preferred embodiment of the present invention, the strains include, but are not limited to: bacillus subtilis CGMCC No.7506, bacillus subtilis GIM1.135, bacillus subtilis GIM1.286, bacillus subtilis GIM1.129, bacillus licheniformis GIM1.182, bacillus licheniformis GIM1.362, bacillus licheniformis CGMCC1.0265, bacillus licheniformis CGMCC No.7878, bacillus licheniformis CGMCC1.0521 and bacillus licheniformis CGMCC 1.0813; bacillus pumilus CGMCC No.7879, Bacillus mojavensis CGMCC No.4961, Bacillus atrophaeus CGMCC No.7881 or Bacillus simplex CGMCC No. 7880.
In a second aspect of the invention, a method for increasing the yield of phospholipase C produced by fermentation of a strain is provided.
The method for improving the yield of the phospholipase C produced by the strain fermentation comprises the step of adding one or more of manganese ions, L-arginine and olive oil into a fermentation medium of the strain.
In a preferred embodiment of the present invention, two or three of manganese ions, L-arginine and olive oil are added to a fermentation medium of the strain.
In a preferred embodiment of the invention, the fermentation medium comprises:
manganese ion 0.59mM-17.7mM, L-arginine 0.05-2wt%, preferably L-arginine 0.1-2wt%, and/or olive oil 0.42-1.5wt%, preferably olive oil 0.42-1.32 wt%.
In a preferred embodiment of the invention, the fermentation medium comprises:
manganese ion 2.95mM-11.8mM, L-arginine 0.3-1.5wt%, and/or olive oil 0.66-1.02 wt%.
In a preferred embodiment of the invention, the manganese ions in the fermentation medium are derived from inorganic salts of manganese ions. In the present invention, the inorganic salt providing manganese ion is well known to those skilled in the art, and can be selected according to the common knowledge or need in the art when preparing the medium. In a preferred embodiment of the invention, the manganese ions in the fermentation medium are derived from one or more of manganese sulfate, manganese chloride, manganese acetate, manganese carbonate, manganese nitrate, manganese phosphate.
Fermentation media useful for phospholipase C producing strains are well known to those skilled in the art and may include, for example, but are not limited to: zwaal et al (R.F.A.Zwaal, et.al.complete Purification and protocols of yeast C from Bacillus strain Acta (BBA)1971, 474-479.), Taguchi Ryo et al (Taguchi Ryo, et.al. Purification and protocols of yeast-specific protease C of Bacillus strain Biophysical Acta (BBA) -Lipids and Lipid Metabolism,1980, 48-57.), Emanul Kambev et al (Emanniv, Kambe.purification, et.g. yeast J. biological strain Biochemical Acta, 1980, 48-57.), Emulation Kamben et al (yeast J. biological strain of yeast) culture of yeast J. biological strain Acta (BBA) -Lipids and Lipid Metabolism, 1990), Emulation Kamben et al (Emanniu.K.P.J. culture of yeast J. biological strain hydrolysate, 1980,48-57.,. fermentation of yeast J.,. yeast J., (biological strain hydrolysate J.) (Bacillus strain hydrolysate J.) -yeast J., (biological strain C. hydrolysate J.) -1990, culture of yeast strain, Bacillus strain C.S.S.S.. In one embodiment of the invention, the fermentation medium used is: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium hydrogen phosphate and pH 7.0. Other components of the fermentation medium used may be combined and adjusted by those skilled in the art in light of the present disclosure and the prior art, and the methods of combining and adjusting are conventional to those skilled in the art.
In a preferred embodiment of the present invention, the strains include, but are not limited to: bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, Bacillus atrophaeus or Bacillus simplex; in a preferred embodiment of the present invention, the strains include, but are not limited to: bacillus subtilis CGMCC No.7506, bacillus subtilis GIM1.135, bacillus subtilis GIM1.286, bacillus subtilis GIM1.129, bacillus licheniformis GIM1.182, bacillus licheniformis GIM1.362, bacillus licheniformis CGMCC1.0265, bacillus licheniformis CGMCC No.7878, bacillus licheniformis CGMCC1.0521 and bacillus licheniformis CGMCC 1.0813; bacillus pumilus CGMCC No.7879, Bacillus mojavensis CGMCC No.4961, Bacillus atrophaeus CGMCC No.7881 or Bacillus simplex CGMCC No. 7880.
In a third aspect of the invention, a method for improving the yield of phospholipase C produced by fermentation of a strain is provided, wherein the strain is a phospholipase C producing strain fermented by using the culture medium of the first aspect.
In a preferred embodiment of the present invention, the strains include, but are not limited to: bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, Bacillus atrophaeus or Bacillus simplex; in a preferred embodiment of the present invention, the strains include, but are not limited to: bacillus subtilis CGMCC No.7506, bacillus subtilis GIM1.135, bacillus subtilis GIM1.286, bacillus subtilis GIM1.129, bacillus licheniformis GIM1.182, bacillus licheniformis GIM1.362, bacillus licheniformis CGMCC1.0265, bacillus licheniformis CGMCC No.7878, bacillus licheniformis CGMCC1.0521 and bacillus licheniformis CGMCC 1.0813; bacillus pumilus CGMCC No.7879, Bacillus mojavensis CGMCC No.4961, Bacillus atrophaeus CGMCC No.7881 or Bacillus simplex CGMCC No. 7880.
In the following examples of the invention, the enzymatic activity of phospholipase C in the supernatant was determined by the P-nitrophenylphosphocholine (P-NPPC) method (J.Shiloach, et. al. Phospholipase C from Bacillus cereus: Production, purification, and properties [ J ] Biotechnology and Bioengineering,1973, 551-560).
In the following examples of the invention, the media used are as follows:
fermentation medium a: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium hydrogen phosphate and pH 7.0.
Fermentation medium B1: manganese sulfate was added to fermentation medium A to 8.85 mM.
Fermentation medium B2: manganese chloride was added to fermentation medium A to 8.85 mM.
Fermentation medium B3: manganese acetate was added to the fermentation medium A to 8.85 mM.
Fermentation medium C1: adding L-arginine to 0.5wt% in the fermentation medium A.
Fermentation medium D1: olive oil was added to the fermentation medium a to 0.5 wt%.
Fermentation medium E1: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium hydrogen phosphate, 8.85mM of manganese sulfate, 0.5wt% of L-arginine and pH 7.0.
Fermentation medium E2: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium phosphate, 8.85mM of manganese chloride, 0.5wt% of L-arginine and pH 7.0.
Fermentation medium E3: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium phosphate, 8.85mM of manganese acetate, 0.5wt% of L-arginine and pH 7.0.
Fermentation medium F1: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium hydrogen phosphate, 8.85mM of manganese sulfate, 0.5wt% of olive oil and pH 7.0.
Fermentation medium F2: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium hydrogen phosphate, 8.85mM of manganese chloride, 0.5wt% of olive oil and pH 7.0.
Fermentation medium F3: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium phosphate, 8.85mM of manganese acetate, 0.5wt% of olive oil and pH 7.0.
Fermentation medium G1: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium hydrogen phosphate, 0.5wt% of olive oil, 0.5wt% of L-arginine and pH 7.0.
Fermentation medium H1: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium phosphate, 8.85mM of manganese sulfate, 0.5wt% of L-arginine, 0.5wt% of olive oil and pH 7.0.
Fermentation medium H2: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium phosphate, 8.85mM of manganese chloride, 0.5wt% of L-arginine, 0.5wt% of olive oil and pH 7.0.
Fermentation medium H3: 1wt% of D (+) galactose, 0.5wt% of yeast extract, 0.5wt% of tryptone, 0.5wt% of dipotassium phosphate, 8.85mM of manganese acetate, 0.5wt% of L-arginine, 0.5wt% of olive oil and pH 7.0.
LB Medium (units g.L)-1): tryptone 10, yeast extract 5, sodium chloride 10, ph 7.0.
In the following examples of the present invention, Bacillus subtilis WBRD01280 with the preservation number of CGMCC No.7506 was used. Bacillus licheniformis (Bacillus licheniformis) GIM1.182, available from Guangdong province center for culture Collection of microorganisms. Bacillus pumilus (Bacillus pumilus) WBRD00240 with the preservation number of CGMCC No. 7879.
In the following examples of the invention, use is made of
A seed liquid of Bacillus subtilis WBRD01280 is prepared by inoculating Bacillus subtilis WBRD01280 to LB culture medium, and fermenting at 180rpm and 28 deg.C for 24 hr.
A seed solution of Bacillus licheniformis (GIM 1.182) was prepared by inoculating Bacillus licheniformis (GIM 1.182) in LB medium and fermenting at 28 deg.C and 180rpm for 24 hours.
A seed liquid of Bacillus pumilus (Bacillus pumilus) WBRD00240 is prepared by inoculating Bacillus pumilus (Bacillus pumilus) WBRD00240 into LB medium, and fermenting at 180rpm and 28 deg.C for 24 hr.
Example 1 Effect of manganese sulfate, L-arginine or Olive oil addition to fermentation Medium on phospholipase C production by Bacillus subtilis WBRD01280
Bacillus subtilis WBRD01280 seed liquid was inoculated into fermentation media A, B1, C1 and D1 at an inoculum size of 2wt%, respectively, and cultured at 180rpm at 28 ℃ for 24 hours. The fermentation broth was centrifuged at 12000rpm for 10 minutes, and the supernatant after centrifugation was collected to obtain a fermentation supernatant. The enzyme activities of phospholipase C in the fermentation supernatants were measured, respectively, and the results are shown in Table 1.
TABLE 1,
| Fermentation Medium A | Fermentation Medium B1 | Fermentation Medium C1 | Fermentation Medium D1 | |
| Enzyme activity (U/ml) | 0.042 | 0.81 | 0.094 | 0.071 |
According to the results in table 1, compared with the fermentation medium a, the phospholipase C obtained by fermentation in the fermentation medium B1 had the highest enzyme activity, which was 1928% of the enzyme activity of the phospholipase C obtained by fermentation in the fermentation medium a; the enzyme activities of the phospholipase C enzymes obtained by fermentation in the fermentation medium C1 and the fermentation medium D1 were 229% and 173% of the enzyme activity of the phospholipase C enzyme obtained by fermentation in the fermentation medium A, respectively.
Example 2: influence of two-by-two combination addition of manganese sulfate, L-arginine and olive oil on phospholipase C production of Bacillus subtilis WBRD01280
Bacillus subtilis WBRD01280 was inoculated into fermentation media E1, F1, and G1 at an inoculum size of 2wt%, respectively, and cultured at 180rpm at 28 ℃ for 24 hours. The fermentation broth was centrifuged at 12000rpm for 10 minutes, and the supernatant after centrifugation was collected to obtain a fermentation supernatant. The enzyme activities of phospholipase C in the fermentation supernatants were measured, respectively, and the results are shown in Table 2.
TABLE 2,
| Fermentation Medium E1 | Fermentation Medium F1 | Fermentation Medium G1 | |
| Enzyme activity (U/ml) | 1.50 | 1.34 | 0.13 |
According to the results in tables 1 and 2, compared with the case that manganese sulfate, L-arginine or olive oil is added into the fermentation medium alone, the case that another 1 of manganese sulfate, L-arginine and olive oil is added into the fermentation medium for fermentation culture is obtained, the enzyme activity of the phospholipase C in the obtained fermentation supernatant is obviously improved, wherein the enzyme activity of the phospholipase C is 185% and 1596% compared with the case that manganese sulfate or L-arginine is added into the fermentation medium alone; adding manganese sulfate monohydrate and olive oil into a fermentation medium, wherein the enzyme activity of phospholipase C is 165% and 1887% respectively compared with that of singly using manganese sulfate monohydrate or olive oil; the enzyme activity of phospholipase C is 138% and 183% compared with L-arginine or olive oil alone when L-arginine or olive oil is added into the fermentation medium.
Example 3 Effect of the amount of L-arginine added on phospholipase C production by fermentation of Bacillus subtilis
The amounts of L-arginine added to the fermentation media E1 were adjusted to obtain fermentation media E1-1 to E1-9 (see Table 3 for details).
Inoculating 2wt% of Bacillus subtilis WBRD01280 seed solution to fermentation culture medium E1-1-E1-9, respectively, and culturing at 28 deg.C and 180rpm for 24 hr. The fermentation broth was centrifuged at 12000rpm for 10 minutes, and the supernatant after centrifugation was collected to obtain a fermentation supernatant. The enzyme activities of phospholipase C in the fermentation supernatants were measured, respectively, and the relative enzyme activities of phospholipase C in the fermentation supernatants obtained by fermentation using fermentation media E1-1 to E1-9 were calculated with fermentation medium B1 as a control, and the results are shown in Table 3.
TABLE 3 influence of the amount of L-arginine added on the production of phospholipase C by fermentation
| L-arginine content | Relative enzyme activity% | |
| Fermentation Medium B1 | 0wt% | 100 |
| Fermentation Medium E1-1 | 0.1wt% | 117.08 |
| Fermentation Medium E1-2 | 0.3wt% | 125.25 |
| Fermentation Medium E1-3 | 0.5wt% | 142.08 |
| Fermentation Medium E1-4 | 0.7wt% | 143.56 |
| Fermentation Medium E1-5 | 0.9wt% | 172.03 |
| Fermentation medium E1-6 | 1.1wt% | 185.11 |
| Fermentation Medium E1-7 | 1.3wt% | 190.96 |
| Fermentation medium E1-8 | 1.5wt% | 175.53 |
| Fermentation Medium E1-9 | 2wt% | 175 |
According to the results in Table 3, the enzyme activity of phospholipase C can be improved by adding 0.1wt% -2wt% of L-arginine into fermentation medium B1, and when the content of L-arginine is more than 0.3wt%, especially more than 0.5wt%, the enzyme activity of phospholipase C is improved obviously. But the addition of the L-arginine to 1.5wt% tends to saturate, and the enzyme activity is not increased by increasing the addition amount.
Example 4 Effect of manganese sulfate addition on phospholipase C production by Bacillus subtilis fermentation
Adjusting the addition amount of manganese sulfate in fermentation medium E to obtain fermentation media E1-11 to E1-18 (see Table 4 for details).
Inoculating 2wt% of Bacillus subtilis WBRD01280 seed solution to fermentation culture medium E1-11-E1-18, respectively, and culturing at 28 deg.C and 180rpm for 24 hr. The fermentation broth was centrifuged at 12000rpm for 10 minutes, and the supernatant after centrifugation was collected to obtain a fermentation supernatant. The enzyme activities of phospholipase C enzymes in the fermentation supernatants were measured, respectively, and the relative enzyme activities of phospholipase C enzymes in the fermentation supernatants obtained by fermentation using fermentation media E1-11 to E1-18 were calculated using fermentation medium C1 as a control, and the results are shown in Table 4.
TABLE 4 influence of manganese sulfate addition on phospholipase C production by fermentation
| Manganese sulfate content | Phospholipase C enzyme activity (U/ml) | Relative enzyme activity% | |
| Fermentation Medium C1 | 0mM | 0.094 | 100 |
| Fermentation medium E1-11 | 0.59mM | 0.8358 | 889.15 |
| Fermentation medium E1-12 | 2.95mM | 1.0353 | 1101.38 |
| Fermentation medium E1-13 | 5.9mM | 1.0790 | 1147.87 |
| Fermentation medium E1-14 | 8.85mM | 1.1165 | 1187.76 |
| Fermentation medium E1-15 | 11.8mM | 1.1044 | 1174.89 |
| Fermentation medium E1-16 | 14.75mM | 0.9014 | 958.93 |
| Fermentation medium E1-17 | 17.7mM | 0.42 | 446.80 |
| Fermentation medium E1-18 | 20.65mM | 0.0581 | 61.80 |
According to the results of Table 4, the enzyme activity of phospholipase C in the fermentation supernatant was improved when manganese sulfate was added in an amount of 0.59mM-17.7mM, particularly 2.95-11.8mM, wherein the enzyme activity of phospholipase C in the fermentation supernatant was the highest when manganese sulfate was added in an amount of 8.85 mM.
Example 5 Effect of the amount of Olive oil added on the production of phospholipase C by fermentation of Bacillus subtilis
The addition amount of olive oil in the fermentation medium F1 was adjusted to obtain fermentation media F1-1 to F1-7 (see Table 5 for details).
Inoculating 2wt% of Bacillus subtilis WBRD01280 seed solution to fermentation media F1-1 to F1-7, respectively, and culturing at 28 deg.C and 180rpm for 24 hr. The fermentation broth was centrifuged at 12000rpm for 10 minutes, and the supernatant after centrifugation was collected to obtain a fermentation supernatant. The enzyme activities of phospholipase C enzymes in the fermentation supernatants were measured, and the relative enzyme activities of phospholipase C enzymes in the fermentation supernatants obtained by fermentation of fermentation media F1-1 to F-7 were calculated using fermentation medium B1 as a control, and the results are shown in Table 5.
TABLE 5 Effect of the amount of Olive oil added on the production of phospholipase C by fermentation
| Olive oil content | Relative enzyme activity% | |
| Fermentation Medium B1 | 0wt% | 100 |
| Fermentation medium F1-1 | 0.3wt% | 91.35 |
| Fermentation medium F1-2 | 0.42wt% | 109.79 |
| Fermentation medium F1-3 | 0.54wt% | 104.61 |
| Fermentation medium F1-4 | 0.66wt% | 146.97 |
| Fermentation medium F1-5 | 0.78wt% | 130.26 |
| Fermentation medium F1-6 | 1.02wt% | 129.68 |
| Fermentation medium F1-7 | 1.32wt% | 129.68 |
According to the results of Table 5, the enzyme activity of phospholipase C in the fermentation supernatant was improved when the amount of olive oil added was more than 0.42wt%, for example, 0.42wt% to 1.32wt%, and the enzyme activity of phospholipase C in the fermentation supernatant was the highest when the amount of olive oil added was 0.66 wt%. The addition of olive oil to 1.02wt% is saturated, and the increase of the addition amount does not increase the enzyme activity.
Example 6: effect of manganese sulfate, L-arginine and olive oil added together on phospholipase C production by bacillus subtilis WBRD01280
Bacillus subtilis WBRD01280 seed liquid was inoculated to the fermentation medium H1 at an inoculum size of 2wt%, and cultured at 180rpm at 28 ℃ for 24 hours. The fermentation broth was centrifuged at 12000rpm for 10 minutes, and the supernatant after centrifugation was collected to obtain a fermentation supernatant. The enzyme activity of the phospholipase C in the fermentation supernatant is detected, and the result shows that the enzyme activity of the phospholipase C in the fermentation supernatant is 1.81U/ml.
Manganese sulfate, L-arginine and olive oil are added into a fermentation medium H1 at the same time, and the enzyme activity of phospholipase C in the supernatant of fermentation liquid obtained by fermentation with the fermentation medium is obviously higher than that of the phospholipase C in the supernatant of the fermentation liquid obtained by adding any two of the manganese sulfate, the L-arginine and the olive oil into the fermentation medium. The enzyme activities in the fermentation medium H1 were 121.2%, 136.7% and 1392% of those of phospholipase C in the supernatants obtained by the culture in the fermentation media E1, F1 and G1, respectively.
Example 7:
respectively using a fermentation medium B2 and a fermentation medium B3 instead of the fermentation medium B1; the fermentation medium E2 and the fermentation medium E3 replace the fermentation medium E1; the fermentation medium F2 and the fermentation medium F3 replace the fermentation medium F1; examples 1, 2, 4 and 6 were repeated with fermentation medium H2, fermentation medium H3 in place of fermentation medium H1.
The results show that the enzyme activity of the phospholipase C in the supernatant obtained by fermentation can be obviously improved by using manganese chloride, manganese acetate or manganese sulfate, and the effect is not obviously different, so that the enzyme activity of the phospholipase C in the supernatant obtained by fermentation can be obviously improved by adding 0.59-17.7 mM, preferably 2.95-11.8mM, of manganese ions into the culture medium.
Example 8: effect of manganese ion, L-arginine and olive oil on production of phospholipase C by Bacillus licheniformis GIM1.182 shake flask fermentation
Bacillus licheniformis GIM1.182 seed solution was inoculated into the fermentation medium (see Table 6 for details) and cultured at 28 ℃ and 180rpm for 24 hours, respectively, as in examples 1 to 6. The fermentation broth was centrifuged at 12000rpm for 10 minutes, and the supernatant after centrifugation was collected to obtain a fermentation supernatant. The enzyme activities of phospholipase C in the fermentation supernatants were measured, respectively, and the results are shown in Table 6.
TABLE 6,
| Fermentation medium | Phospholipase C enzyme activity | Fermentation medium | Phospholipase C enzyme activity |
| Fermentation Medium A | 0.039U/ml | Fermentation Medium E1 | 0.83U/ml |
| Fermentation Medium B1 | 0.57U/ml | Fermentation Medium F1 | 0.91U/ml |
| Fermentation Medium C1 | 0.10U/ml | Fermentation Medium G1 | 0.24U/ml |
| Fermentation Medium D1 | 0.05U/ml | Fermentation Medium H1 | 0.96U/ml |
According to the results of Table 6, the use of fermentation media B1, C1 and D1 all increased the enzyme activity of phospholipase C in the fermentation supernatant compared to fermentation medium A, which was 1461%, 256% and 128%, respectively, of the fermentation using fermentation medium A. Therefore, the enzyme activity of the phospholipase C in the fermentation supernatant can be improved by adding manganese ions, L-arginine or olive oil into the fermentation medium.
When two of manganese ions, L-arginine and olive oil are added into a fermentation medium at the same time, the enzyme activity of phospholipase C in fermentation supernatant is obviously improved. The enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium E1 was 146% of the enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium B1, which was 830% of the enzyme activity of phospholipase C in fermentation medium C1. The enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium F1 was 160% of the enzyme activity of phospholipase C in fermentation medium B1, and 1820% of the enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium D1. The enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium G1 was 240% of the enzyme activity in the fermentation supernatant of fermentation medium C1 and 480% of the enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium D1.
Manganese ions, L-arginine and olive oil are added into a fermentation medium at the same time, and the enzyme activity of phospholipase C in the supernatant obtained by fermentation is higher than that of the phospholipase C in the supernatant obtained by adding any two of the manganese ions, the L-arginine and the olive oil into the fermentation medium. The enzyme activities in the fermentation medium H1 were 116%, 105% and 400% of the enzyme activities of phospholipase C in the supernatants obtained by the culture in the fermentation media E1, F1 and G1, respectively.
Example 9: influence of manganese ions, L-arginine and olive oil on phospholipase C production by shake flask fermentation of Bacillus pumilus (WBRD 00240)
Bacillus pumilus (Bacillus pumilus) WBRD00240 seed liquid was inoculated into fermentation medium A, B1-H1, respectively, and cultured at 28 ℃ and 180rpm for 24 hours according to the method of example 7. The fermentation broth was centrifuged at 12000rpm for 10 minutes, and the supernatant after centrifugation was collected to obtain a fermentation supernatant. The enzyme activities of phospholipase C in the fermentation supernatants were measured, respectively, and the results are shown in Table 7.
TABLE 7
| Fermentation medium | Phospholipase C enzyme activity | Fermentation medium | Phospholipase C enzyme activity |
| Fermentation Medium A | 0.059U/ml | Fermentation Medium E1 | 1.09U/ml |
| Fermentation Medium B1 | 0.069U/ml | Fermentation Medium F1 | 0.96U/ml |
| Fermentation Medium C1 | 0.10U/ml | Fermentation Medium G1 | 0.13U/ml |
| Fermentation Medium D1 | 0.07U/ml | Fermentation Medium H1 | 1.18U/ml |
According to the results of Table 6, the use of fermentation media B1, C1 and D1 all increased the enzyme activity of phospholipase C in the fermentation supernatant compared to fermentation medium A, 1169%, 169% and 117% of the fermentation using fermentation medium A, respectively. Therefore, the enzyme activity of the phospholipase C in the fermentation supernatant can be improved by adding manganese ions, L-arginine or olive oil into the fermentation medium.
When two of manganese ions, L-arginine and olive oil are added into a fermentation medium at the same time, the enzyme activity of phospholipase C in fermentation supernatant is obviously improved. The enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium E1 was 158% of the enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium B1 and 1090% of the enzyme activity of phospholipase C in fermentation medium C1. The enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium F1 was 139% of the enzyme activity of phospholipase C in fermentation medium B1 and 1371% of the enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium D1. The enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium G1 was 130% of the enzyme activity in the fermentation supernatant of fermentation medium C and 186% of the enzyme activity of phospholipase C in the fermentation supernatant of fermentation medium D1.
Manganese ions, L-arginine and olive oil are added into a fermentation medium at the same time, and the enzyme activity of phospholipase C in the supernatant obtained by fermentation is higher than that of the phospholipase C in the supernatant obtained by adding any two of the manganese ions, the L-arginine and the olive oil into the fermentation medium. The enzyme activities in the fermentation medium H1 were 108%, 123% and 908% of the enzyme activities of phospholipase C in the supernatants obtained by the culture in the fermentation media E1, F1 and G1, respectively.
Claims (8)
1. A method for improving the yield of phospholipase C produced by fermentation of a strain is characterized in that one or more of 0.59-17.7 mM manganese ions, 0.05-2wt% of L-arginine and 0.42-1.5wt% of olive oil are added into a fermentation medium of the strain, and the strain is Bacillus subtilis, Bacillus licheniformis or Bacillus pumilus.
2. The method of claim 1, wherein two or three of manganese ions, L-arginine and olive oil are added to the fermentation medium of the strain.
3. The method of claim 2, wherein the fermentation medium comprises 0.1-2wt% L-arginine, and/or 0.42-1.32wt% olive oil.
4. The method of claim 3, wherein the fermentation medium comprises manganese ions in an amount of 2.95mM-11.8mM, L-arginine in an amount of 0.3-1.5wt%, and/or olive oil in an amount of 0.66-1.02 wt%.
5. The method according to any one of claims 1 to 4, wherein the strain is: bacillus subtilis CGMCC No.7506, Bacillus subtilis GIM1.135, Bacillus subtilis GIM1.129, Bacillus licheniformis GIM1.182, Bacillus licheniformis GIM1.362, and Bacillus licheniformis CGMCC No. 7878; bacillus pumilus CGMCC No. 7879.
6. The method of any one of claims 1-4, wherein the manganese ions in the fermentation medium are derived from inorganic salts of manganese ions or organic salts of manganese ions.
7. The method of claim 6, wherein the manganese ions in the fermentation medium are derived from one or more of manganese sulfate, manganese chloride, manganese acetate, manganese carbonate, manganese nitrate, and manganese phosphate.
8. The method of claim 6, wherein the strain is:
bacillus subtilis CGMCC number 7506, bacillus subtilis GIM1.135, bacillus subtilis GIM1.129, bacillus licheniformis GIM1.182, bacillus licheniformis GIM1.362 and bacillus licheniformis CGMCC No. 7878; bacillus pumilus CGMCC No. 7879.
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