133 results about "Phospholipases C" patented technology

The present invention relates to a method for screening therapeutic drugs of schizophrenia using an animal model of the disease. More specifically, this invention relates to a screening method based on the phospholipase C β1 (PLCβ1) knockout mouse as an animal model of schizophrenia with all the major symptoms of the human disease. This knockout mouse exhibits symptoms similar to human schizophrenia such as locomotor hyperactivity, impaired prepulse inhibition of the startle response, lack of barbering and nesting behaviors, socially subordinate status, impaired learning, and lack of type II theta rhythm which has been implicated in working memory. Thus, the knockout mouse of the present invention can be useful as an animal for screening therapeutic drugs against schizophrenia.

A method for degumming an oil composition comprises the steps of (a) providing an oil composition containing a quantity of phospholipids, (b) contacting said oil composition simultaneously with one or more phospholipase A enzymes and one or more phospholipase C enzymes, under conditions sufficient for the enzymes to react with the phospholipids to create phospholipid reaction products, and (c) separating the phospholipids reaction products from the oil composition, the remaining oil composition after the separation being a degummed oil composition, whereby during step (b) the reaction of said one or more phospholipase A enzymes proceeds at a faster rate than it would in the absence of said one or more phospholipase C enzymes.

In alternative embodiments, the invention provides phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes, nucleic acids encoding them, antibodies that bind specifically to them, and methods for making and using them. Industrial methods and products comprising use of these phospholipases are also provided. In certain embodiments, provided herein are methods for hydration of non hydratable phospholipids (NHPs) within a lipid matrix. The methods enable migration of NHPs to an oil-water interface thereby allowing the NHPs to be reacted and / or removed from the lipids. In certain embodiments, provided is a method for removing NHPs, hydratable phospholipids, and lecithins from vegetable oils to produce a degummed oil or fat product that can be used for food production and / or non-food applications. In certain embodiments, provided herein are methods for hydration of NHPs followed by enzymatic treatment and removal of various phospholipids and lecithins. The methods provided herein can be practiced on either crude or water-degummed oils.

In alternative embodiments, the invention provides phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes, nucleic acids encoding them, antibodies that bind specifically to them, and methods for making and using them. Industrial methods and products comprising use of these phospholipases are also provided. In certain embodiments, provided herein are methods for hydration of non hydratable phospholipids (NHPs) within a lipid matrix. The methods enable migration of NHPs to an oil-water interface thereby allowing the NHPs to be reacted and / or removed from the lipids. In certain embodiments, provided is a method for removing NHPs, hydratable phospholipids, and lecithins from vegetable oils to produce a degummed oil or fat product that can be used for food production and / or non-food applications. In certain embodiments, provided herein are methods for hydration of NHPs followed by enzymatic treatment and removal of various phospholipids and lecithins. The methods provided herein can be practiced on either crude or water-degummed oils.

The invention discloses a method for degumming soybean oil by phospholipase catalysis, which belongs to the field of vegetable oil degumming and includes the steps of preheating, adjusting pH, adding enzyme prior to mixing, allowing for enzymolysis, deactivating the enzyme and separating. The method is technically characterized by including: using phospholipase C as the phospholipase for catalysis, and preheating crude soybean oil prior to acid treatment; cooling oil-water mixture subjected to acid treatment to 37-60 DEG C, adding NaOH solution to adjust pH to 3-8, adding distilled water accounting for 1%-5% of the weight of the oil, adding the phospholipase C according to an oil proportion of 100-2000U / g, and mixing well; setting the mixing speed at 100-600r / min, and allowing for enzyme reaction for 0.5-2 hours; and deactivating the enzyme at high temperature for a reaction system, and performing high-speed centrifugal separation to complete degumming. The degumming time of the method is shortened by 1-2 times as compared with that of using phospholipase A1, phosphorus content is reduced, diglyceride content of the oil is increased slightly, and economic benefit is increased for oil production industries.

The invention discloses a rapeseed oil processing method. The rapeseed oil processing method comprises the following steps: 1) preparing crude rapeseed oil; 2) performing ultrasonic assisted phospholipase C degumming treatment on the crude rapeseed oil, and performing centrifugal separation after degumming to obtain degummed rapeseed oil; 3) performing anhydrous long-mixing deacidification treatment on the degummed rapeseed oil to obtain deacidified rapeseed oil; 4) adding spent earth into the deacidified rapeseed oil to perform pre-decolorizing treatment to obtain pre-decolorized rapeseed oil, and adding attapulgite and activated carbon into the pre-decolorized rapeseed oil to perform re-decolorizing treatment to obtain re-decolorized rapeseed oil; 5) performing deodorizing treatment on the re-decolorized rapeseed oil to obtain finished product rapeseed oil. The rapeseed oil processing method has the advantages of good safety and environmental-friendliness, simple process, convenience in operation, low cost and high refining rate, and is applicable to industrial production of rapeseed oil.

The invention relates to an efficient zinc ion independent phospholipase C mutant. Specifically, the invention provides a separated amino acid sequence, wherein the amino acid sequence includes (1) an amino acid sequence shown as SEQ ID NO:7, or (2) polypeptide which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of (1), reserves phospholipase C activity of the SEQ ID NO:7 and is derived from (1). The invention also relates to a polynucleotide sequence for coding the amino acid sequence, a nucleic acid construct and a host cell containing the polynucleotide sequence, a composition containing the amino acid sequence and an application of the amino acid sequence.

Plant stresses such as pest drought, and excessive temperatures can lead to significant losses of crops each year. There is a continuing need to develop novel plant varieties that are less susceptible to damage or loss by such stresses. The present invention provides a system and method for generating plants having an increased resistance to plant stresses such as drought or adverse temperatures. Transgenic plants expressing a phosphoinositide-specific phospholipase C (PI-PLC) gene can show an unexpected and dramatic improvement in their capacity to tolerate a variety of stress conditions. Moreover, increased PI-PLC expression can further lead to marked increase in plant growth rates, maturation, and lipid content. The present invention encompasses transgenic plants having modifications with regard to PI-PLC pathways, such as altered PI-PLC levels, and plant products thereof.

Compounds of formula (I)in which R1, R2, R3, R4 and R5 are hydrogen atoms or chromogenic substituents and X is hydroxyl, OR6 wherein R6 is selected from the group consisting of C1-C4 alkyl, or O-Me+ wherein Me+ is a cation derived from an organic or inorganic base; these compounds do not exhibit significant fluorescence but are capable of being cleaved by phosphatidyl-inositol-specific phospholipase C, an enzyme which is indicative of bacterial activity; the umbelliferyl moity resulting from such cleavage is a strong fluorogen thus providing effective test methods for various pathogenic bacteria, such as Listeria, Staphylococcus and Clostridium species. Also disclosed are plating media for detection of microorganisms that are capable of metabolic generation of a phosphatidyl inositol-specific phospholipase C (PI-PLC). The plating medium can be in a dry, liquid, or semi-liquid form, depending upon its water content, and comprise at least one compound capable of forming an aqueous gel when in contact with water; at least one nutrient capable of supporting growth of said microorganism; and at least one indicator compound of formula I and / or IV, notably 4-methylumbelliferyl myo-inositol-1-phosphate or salts thereof and 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate or salts thereof. PI-PLC generated by the microorganisms of interest leads to cleavage of the indicator compounds causing formation of fluorescence and / or color suitable for identification of type and count of such hygienically and pathologically important microorganisms as Listeria species.

A process of water degumming an edible oil (preferably a crude edible oil) comprising the steps of: a) admixing approximately 0.1-5% w / w water with an edible oil (preferably a crude edible oil) and a lipid acyltransferase, b) agitating the admixture for between about 10 minutes and 180 minutes at about 45 to about 900 C, and c) separating the oil phase and the gum phase. Preferably said lipid acyltransferase is a polypeptide having lipid acyltransferase activity which polypeptide is obtained by expression of the nucleotide sequence shown as SEQ ID No. 49 or a nucleotide sequence which as has 70% or more identity therewith; and / or is obtained by expression of a nucleic acid which hybridises under medium stringency conditions to a nucleic probe comprising the nucleotide sequence shown as SEQ No. 49; and / or is a polypeptide having lipid acyltransferase activity which polypeptide comprises the amino acid sequence shown as SEQ ID No. 68 or an amino acid sequence which as has 70% or more identity therewith. In one embodiment the lipid acyltransferase is preferably used in combination with a phospholipase C enzyme. A process for modifying the gum phase of a degummed oil using a lipid acyltransferase is also taught herein.

The invention discloses a method of using phospholipase C to extracellularly express intracellular protein and belongs to the technical field of bioengineering. Through co-expression phospholipase C, intracellular positioning protein is released to the outsides of cells by means of nonspecific leaking, and high-performance phospholipase C and co-expression recombinant bacteria high in target protein extracellular enzyme activity are obtained by screening. Enzyme activity of recombinant bacteria extracellular sucrose phosporylase co-expressing sucrose phosphorylase and phospholipase C is 1445.1U mL-1, and enzyme activity of recombinant bacteria extracellular trehalase co-expressing trehalase and phospholipase C is 322.3U mL-1. The method can realize extracellular expression of intracellular positioning protein, improve production efficiency and simplify post-extraction process, has wide application value in food, cosmetics and pharmaceuticals industry and has great demand in market at home and abroad.

The present application provides a wild type phosphatidylcholine specificity phospholipase C (PLC) mutant of Bacillus cereus, the related mutations comprising asparagine at position 63 mutating to another amino acid; also comprising mutation of arginine to histidine at position 20 and of alanine to aspartic acid at position 83. The present application also provides a nucleic acid molecule encoding said mutant, a vector containing said nucleic acid molecule, and a cell containing said nucleic acid molecule or vector. The present application also provides uses of said mutant, nucleic acid molecule vector, and cell.

In alternative embodiments, the invention provides phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes, nucleic acids encoding them, antibodies that bind specifically to them, and methods for making and using them. Industrial methods and products comprising use of these phospholipases are also provided. In certain embodiments, provided herein are methods for hydration of non hydratable phospholipids (NHPs) within a lipid matrix. The methods enable migration of NHPs to an oil-water interface thereby allowing the NHPs to be reacted and / or removed from the lipids. In certain embodiments, provided is a method for removing NHPs, hydratable phospholipids, and lecithins from vegetable oils to produce a degummed oil or fat product that can be used for food production and / or non-food applications. In certain embodiments, provided herein are methods for hydration of NHPs followed by enzymatic treatment and removal of various phospholipids and lecithins. The methods provided herein can be practiced on either crude or water-degummed oils.

The invention belongs to the field of antibody drug, and discloses a full human-derived anti-human VEGFR2 monoclonal antibody screened by a phage antibody base technology and prepared by a gene engineering method, and further discloses a carrier including polynucleotide for encoding the monoclonal antibody, a host cell and an application. Through combining with VEGFR2, the monoclonal antibody stops the combination of VEGF with VEGR2, and does not induce receptor dimerization, cause the following tyrosine residue phosphorylation in intracellular tyrosine kinase structure and activate downstream signal access including activation of phospholipase C and increase of calcium ion concentration in the cell and others; the antibody drug triggers endothelial proliferation, survival, cytoskeleton rearrangement, cell migration, gene expression and others, and finally stops the vascular proliferation caused by combination of VEGF and VEGFR2 and induction of dimerization. The monoclonal antibody can be used for treating diseases caused by tumor angiogenesis.

The invention provides a method for degumming soybean crude oil by using immobilized phospholipase C (PLC). According to the method, free PLC can hydrolyze phosphorus-oxygen bonds between phosphate groups and glyceryl specifically to generate diglyceride, and the diglyceride is dissolved in oil to form one part of edible oil, so that the yield of degummed oil can be improved; but a miscible natural enzyme is not easy to separate and recycle in a reaction system and is high in cost, so the miscible natural enzyme cannot be used repeatedly or continuously. The immobilized PLC is added into the soybean crude oil to perform degumming, and the phosphorus content and the yield of grease of the degummed soybean crude oil and the repeated using time of the immobilized PLC are determined, so that after the soybean crude oil is degummed, the minimum phosphorus content is 3.8 mg / kg, the yield of the grease is improved by 1.2 times, and after the immobilized PLC is used for 6 batches continuously, the relative vigor of the immobilized PLC is still kept about 80 percent. Therefore, degumming is performed by using the immobilized PLC, so that the refining rate can be improved, and the production cost can also be reduced.

The present invention relates to a method for producing cheese comprising treating the cheese milk, or a fraction of the cheese milk, with phospholipase C and / or phospholipase D.

The invention discloses a method for degumming fragrant rap oil through an enzymic process. The method is characterized by comprising the following steps: heating the squeezed fragrant crude rap oil without impurities to about 40 DEG C, adding a 50% citric acid aqueous solution according to 0.1% by weight of the oil, mixing the 50% citric acid aqueous solution with the fragrant crude rap oil, mixing and stirring at a rate of 50-60r / min, and stirring for one hour; adding soft water according to 1.8% by weight of the oil, adding a phospholipase A, phospholipase B or phospholipase C solution diluted to 100 times according to 0.13% of the total mass of oil and water, simultaneously stirring and reacting for 60 minutes at a rate of 40-50r / min under the temperature of 30-50 DEG C, filtering or centrifugally separating, and drying to obtain the finished product of the fragrant rap oil.

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.

The invention provides a method for degumming soybean crude oil by virtue of combined application of immobilized phospholipase A1 and immobilized phospholipase C. The method comprises the following steps: developing an immobilized phospholipase A1-immobilized phospholipase C two-enzyme degumming technology according to respective characateristics of the phospholipase A1 and phospholipase C, namely, performing conventional enzymic method degumming by adopting a small amount of immobilized phospholipase A1, forming an emulsifying system favorable for the subsequent immobilized phospholipase C effects by utilizing lysophosphatide, and adding a proper amount of the immobilized phospholipase C so as to realize degumming. The lowest obtained degummed phosphorus content is 2.8mg / kg, and after the phospholipase C is continuously used in six batches, the relative activity of the immobilized enzyme is still maintained to be about 80 percent. Therefore, a combined degumming method is adopted, so that the refining rate can be improved, the production cost can be reduced, the degumming reaction time can be reduced by 2 hours, and the oil yield is improved by 1.3 times.

The present invention relates to inositol phosphate derivatives, in which the inositol phosphate is substituted with one or two reactive groups G or one or two conjugated substances or molecules M, said reactive group(s) G or said substance(s) or molecule(s) M being linked to IP1 via a linkage group L, M being chosen from the following group: a tracer, an immunogen, a member of a binding partner pair, a solid support.Application: tools allowing the study of the inositol phosphate cycle and therefore, indirectly, the study of seven transmembrane domain receptors coupled to phospholipase C, receptors having a tyrosine kinase activity, and in general enzymes involved in the variations of the intracellular concentration of IP1.

The invention provides compounds of formula (I) in which R1, R2, R3, R4 and R5 are independently selected from hydrogen and chromogenic substituents, and X is selected from the group consisting of hydroxyl; OR6 wherein R6 is selected from the group consisting of C1-C4 alkyl; and O-Me+ wherein Me+ is a cation derived from an organic or inorganic base. A preferred species of formula (I) is 4-Methylumbelliferyl myo-inositol-1-phosphate and salts thereof with an organic or inorganic base. The invention also provides fluorogenic methods for detecting various pathogenic bacteria, e.g., Listeria, Staphylococcus and Clostridium species, using substrates containing at least one formula (I) compound. A kit for detecting a phosphatidyl-inositol-specific phospholipase C enzyme as an indication of bacterial activity is disclosed.

The invention relates to phospholipase C, a food enzyme agent comprising the same and a processing method for food raw materials, providing the phospholipase C. The phospholipase C has the capability of efficiently hydrolyzing various phospholipids close to an acidic pH region or a neutral pH region, also has activity in a citric acid buffer solution, has thermal stability to a certain extent and also has the property of not hydrolyzing organic phosphate not containing a lipid part. The phospholipase C shows activity in the region with pH from acidic pH to neutral pH and does not substantially hydrolyze the organic phosphate except for the phospholipids.

The invention discloses a method for preparing a chicken infectious bronchitis virus HA antigen. The chicken infectious bronchitis virus HA antigen is prepared by the steps of virus multiplication, virus solution concentration and phospholipase C treatment, and antigen inactivation and stabilization. The stability of the prepared HA antigen at a temperature of 4 DEG C is prolonged to more than 6 months from the original 24 hours; besides, the valence of antigen is stable so as to solve the difficult problems which are not solved for a long time that the antigen is temporarily prepared for the test of each time and needs to be detected before use, and reduce the workload of the HI test. The prepared IBV HA antigen can be successfully applied to detecting the valence of antibody of serum HI after the chicken IBV vaccine immunization, and solves the problem that the detection of the IBV vaccine immunization effect is realized through neutralization tests in China for a long time, thereby wasting time and energy and having the danger of poison diffusion.

Compounds of formula (I) in which R1,R2,R3,R4 and R5 are hydrogen atoms or chromogenic substituents and X is hydroxyl, OR6 wherein R6 is selected from the group consisting of C1-C4 alkyl, or O-Me+ wherein Me+ is a cation derived from an organic or inorganic base; these compounds do not exhibit significant fluorescence but are capable of being cleaved by phosphatidyl-inositol-specific phospholipase C, an enzyme which is indicative of bacterial activity; the umbelliferyl moity resulting from such cleavage is a strong fluorogen thus providing effective test methods for various pathogenic bacteria, such as Listeria, Staphylococcus and Clostridium species. Also disclosed are plating media for detection of microorganisms that are capable of metabolic generation of a phosphatidyl inositol-specific phospholipase C (PI-PLC). The plating medium can be in a dry, liquid, or semi-liquid form, depending upon its water content, and comprise at least one compound capable of forming an aqueous gel when in contact with water; at least one nutrient capable of supporting growth of said microorganism; and at least one indicator compound of formula I and / or IV, notably 4-methylumbelliferyl myo-inositol-1-phosphate or salts thereof and 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate or salts thereof. PI-PLC generated by the microorganisms of interest leads to cleavage of the indicator compounds causing formation of fluorescence and / or color suitable for identification of type and count of such hygienically and pathologically important microorganisms as Listeria species.

The invention discloses recombinant escherichia coli, a method for preparing phospholipase C and application. The recombinant escherichia coli BL21(DE-3)-pET-28a-L.m-phospholipase C (PLC) of which the target gene is from listeria monocyogenes (L.monocytogenes) is utilized as a fermentation strain to carry out liquid fermentation, so as to prepare phospholipase C. The method disclosed by the invention is simple in enzyme production process, short in production cycle and low in cost. A glycerophosphate bond on glyceryl phosphatide C3 can be specifically hydrolyzed to produce diglyceride (DAG) by using recombinant phospholipase C; plant crude oil (soybean oil, colleseed oil, rice bran oil and the like) is degummed by phospholipase C.

The present invention provides a phospholipase C enzyme(s) having ability to hydrolyze phospholipid in both acidic and around neutral ranges and the activity in a citrate buffer solution as well as having some degree of heat stability, and having a property not to hydrolyze phosphate esters not containing lipid moieties. The phospholipase C enzyme(s) shows the activity at from acidic to neutral pH and does not substantially hydrolyze any phosphate esters except for phospholipids.

The invention discloses a mutton fat degumming method which comprises the following steps that a citric acid solution is added to a mutton fat raw material, acidizing is conducted under assistance of ultrasonic waves, and acidized mutton fat is obtained; a certain quantity of distilled water is added to the acidized mutton fat, the pH and the temperature of the acidized mutton fat are adjusted, phospholipase A1 is added for conducting ultrasound-assisted enzymolysis, and the mutton fat obtained after preliminary enzymolysis is obtained; the pH and the temperature of the mutton fat obtained after preliminary enzymolysis are adjusted, phospholipase B is added for conducting ultrasound-assisted enzymolysis, and mutton fat obtained after secondary enzymolysis is obtained; the pH and the temperature of the mutton fat obtained after secondary enzymolysis are adjusted, phospholipase C is added for conducting ultrasound-assisted enzymolysis, and mutton fat obtained after third enzymolysis is obtained, enzyme deactivation processing is conducted on the mutton fat obtained after third enzymolysis, centrifugation is conducted to take fat samples on the upper layer, and mutton fat obtained after enzyme deactivation is conducted is obtained; the mutton fat obtained after enzyme deactivation is conducted is subjected to vacuum drying processing, and degummed mutton fat is obtained. The mutton fat degumming method is safe in process and convenient and fast to operate.

A method for degumming an oil composition comprises the steps of (a) providing an oil composition containing a quantity of phospholipids, (b) contacting said oil composition simultaneously with one or more phospholipase A enzymes and one or more phospholipase C enzymes, under conditions sufficient for the enzymes to react with the phospholipids to create phospholipid reaction products, and (c) separating the phospholipids reaction products from the oil composition, the remaining oil composition after the separation being a degummed oil composition, whereby during step (b) the reaction of said one or more phospholipase A enzymes proceeds at a faster rate than it would in the absence of said one or more phospholipase C enzymes, and wherein the reaction of step (b) continues for a duration of less than about one hour.

The present invention relates to a TGF-β1 binding protein called r150. This protein has a GPI-anchor contained in r150 itself and not on a tightly associated protein and that it binds TGF-β1 with an affinity comparable to those of the signaling receptors. Furthermore, the released (soluble) form of this protein binds TGF-β1 independent of the types I and II receptors. Also, the soluble form inhibits the binding of TGF-β to its receptor. In addition, evidence that r150 is released from the cell surface by an endogenous phospholipase C is provided. Also, the creation of a mutant human keratinocyte cell line with a defect in GPI synthesis which displays reduced expression of r150 is described. Our results using these mutant keratinocytes suggest that the membrane anchored form of r150 is a negative modulator of TGF-beta responses. These findings, taken together with the observation that r150 forms a heteromeric complex with the signaling receptors, suggest that this accessory receptor in either its membrane anchored or soluble form may antagonize TGF-β responses in human keratinocytes. Experiments with mutants confirmed that TGFβ1 activity can be modulated when the expression of the accessory receptor r150 is silenced. The complete nucleic acid and deduced amino acid sequences are now provided. The r150 cloned nucleic acid was used to study overexpression of r150. When r150 gene is overexpressed, TGFβ responses are increased. r150 and its derivatives or precursors (fragments, variants and nucleic acids encoding the same) will find a broad clinical utility, knowing that TGFβ1 is an important cytokine.