1586 results about "Pichia pastoris" patented technology

The invention discloses a recombinant humanized collagen. Its amino acid sequence is as shown in SEQNO.3 and has 599 amino acids. The molecular weight is 55.0kDa. A preparation method of the recombinant humanized collagen comprises the following steps of: constructing a gene engineering strain for the expression of the recombinant humanized collagen to obtain a Pichia pastoris engineering strain; carrying out fermentation culture on the engineering strain, and realizing inducible expression of the recombinant humanized collagen to obtain a recombinant humanized collagen fermentation broth; and purifying the fermentation broth to obtain the recombinant humanized collagen. The humanized collagen gene Ge1 designed in the invention is a brand-new sequence which is greatly shorter than a natural humanized collagen gene (Kbp) in length. By the preparation method, operation simplification at the molecular level is realized, and the gene engineering strain fermentation production of the collagen is more easily realized. The humanized collagen gene Ge1 contains characteristics of a natural humanized collagen gene. And simultaneously, its expressed protein has excellent characteristics of a natural humanized collagen.

Use of mixed mode chromatography for purification of an antibody from an antibody mixture, for example) a Pichia pastoris fermentation mixture containing impurities such as host cell proteins and DNA is described Mixed mode chromatography is used instead of protein A chromatography and presents certain advantages over protein A chromatography Furthermore, the integration of such a method into a multi-step procedure with other fractionation methods for purification of antibodies suitable for in vivo applications is provided.

Functionally assembled antigen-specific intact recombinant monoclonal antibody produced by transformation of the methylotropic yeast, P. pastoris with mouse / human immunoglobulin genes encoding heavy and light chains. A method for production of the intact monoclonal antibodies, a recombinant yeast expression vector and the antibody-specific MRNA synthesis. A process for a large-scale production of the functionally assembled intact recombinant antibody.

The invention relates to candida Antarctica lipase B gene and applications thereof in yeast display. Coded protein of improved candida Antarctica B and wild type candida Antarctica lipase protein have the same function on the amino acid level; the heat resistance capacity of the enzyme is 50-80 DEG C, and the half-lift is 3-24 hours; a nucleotide sequence is hybridized with SEQ.ID.NO2 from 1st to 978th of nucleotide under the moderate precise condition; and a preservation number of colon bacillus DH5Alpha / Puc57-CALB (Escherichia coliDH5Alpha / pUC57-CALB) which carries the plasmids is CCTCC M 209081. The candida Antarctica lipase B gene is transferred into pichia pastoris host bacteria so as to realize the high-efficient display expression of the candida Antarctica lipase B in pichia pastoris; and the provided pichia pastoris bacteria can effectively display candida Antarctica lipase B, can be widely applied to the synthesis of ethyl caproate, has different melting points and does not contain triglyceride of various fatty acids, a plurality of structured lipids, and the like.

The invention provides a preparing method of reconstructing immunoregulatory protein of a ganoderma lucidum. The method includes the following steps: construction of engineering bacteria, wherein, LZ-8 protein gene is designed and coded, and is connected with a leading peptide coding sequence of saccharomyces cervisiae alpha-factor into fusion gene, which is transferred to a Pichia Pastoris expression system GS115 for reconstructing expression, high expressed rLZ-8 protein bacterial strain is acquired; the fermentation under the scale of 100L fermentation cylinder, wherein, improved seed culture medium and fermentation medium are used, additions of glycerol and methanol are regulated, 0.5M(NH4)2SO4 is added at the late stage of the fermentation; the purification on a column via centrifugal separation, wherein, salt is hyperfiltrated, powder is prepared after being frozen and dried. The method has large scale of fermentation, optimized technological condition and high production rate.

A novel gene encoding P. pastoris orotate-phosphoribosyl transferase (URA5) is disclosed. Methods for producing and selecting yeast strains capable of stable genetic integration of heterologous sequences into the host genome are also provided.

The invention discloses a method for producing recombinant human-like collagen by expression by Pichia pastoris, which adopts Pichia pastoris C13 with a collection number CGMCC No.4437 as a strain and comprises: culturing primary seeds, culturing secondary seeds, culturing in a fermentation tank and inducing expression; and centrifuging fermentation liquor obtained after induced expression, collecting supernate after centrifugation, and performing concentration, gel column chromatography, precipitate decolorization, ion-exchange column chromatography and ultrafiltration for desalination in turn to obtain the recombinant human-like collagen. Compared with other purification methods, the method is simpler in process and higher in target protein yield, realizes a recovery rate of over 60 percent and a purity of over 95 percent, can realize complete decolorization and is suitable for industrial large-scale production of the recombinant human-like collagen.

The invention discloses acid beta-mannase, genes, engineering bacteria and a structure thereof. A coding gene manAsp is from Aspergillus niger CBS 513.88, the coding gene has a nucleotide sequence expressed as SEQ ID NO.3 or 4, and the acid beta-mannase obtained by coding has an amino acid sequence expressed as SEQ ID NO.1 or 2. The structure of the engineering bacteria is obtained by guiding a pichia pastoris constitutive expression vector (pGAPZa-man) containing the coding gene sequence of the beta-mannase into pichia pastoris. The acid beta-mannase of the invention has the following properties: at the optimal pH of 5.0 and the optimal temperature of 40 DEG C, the beta-mannase has better stability when the pH is between 3.0 and 7.0, and the residual enzyme activity is 66.39 percent when the beta-mannase is treated for 1 hour at the temperature of 90 DEG C; the engineering bacteria achieve efficient expression of the acid beta-mannase; and the fermentation process is simple, has low extracting cost, is suitable for large-scale industrialized production, and has broad application prospect in the industries of feed, food, medicine, energy and the like.

The invention provides site-specific mutagenesis high temperature resistant phytase gene TP and an expression vector and application thereof. Amino acid in a specific area in a phytase amino acid sequence is mutated into praline and arginine so as to enable the phytase to have a special stable protein structure. Simultaneously, a gene sequence is optimized to synthesize a phytase gene according to pichia pastoris codon preference and GC content, recombinant plasmids are built and transferred to pichia pastoris, and positive converters are obtained through screening to conduct induction expression to obtain the high temperature resistant phytase. Experiments prove that the phytase obtained through expression of the pichia pastoris can resist high temperature plasmids with the temperature higher than 85 DEG C, and survival rate reaches 85%. Popularization and application of the high temperature resistant phytase have substantial economical benefit and social benefit on development of feed animal husbandry of our country, and simultaneously great ecological benefit can be obtained.

The invention discloses a bacillus subtilis chitosanase as well as a preparation method and application thereof. The invention optimizes an encoding gene of bacillus subtilis chitosanase according to the preference of pichia pastoris codon. The optimized nucleotide sequence is shown as SEQ ID NO.2. A pichia pastoris expression system is further utilized to perform efficient secretory expression on the optimized chitosanase encoding gene, so as to obtain the bacillus subtilis chitosanase with the amino acid sequence as SEQ ID NO.1. The bacillus subtilis chitosanase obtained according to the invention has higher hydrolytic activity to chitosan substrates at different degrees of deacetylation; the crude enzyme generated through shake-flask fermentation has the hydrolysis capacity of degrading 5g of chitosan by 1mL crude enzyme (0.3mg of protein), about 150mg of non-specific commercial enzyme is required for degrading the same amount of chitosan, and the efficiency is theoretically increased by 500 times; and the bacillus subtilis chitosanase has excellent industrial application prospects.

The invention discloses a recombinant Ganoderma Lucidum immunomodulatory protein (rLZ-8) which has anti-tumor effect and is expressed by Pichia pastoris, and a pharmaceutical preparation thereof. The rLZ-8 has the effect of directly killing or damaging NB4 or K562 or HL-60 or S180 or H22 cells and not affecting normal cells; and the rLZ-8 can quickly and efficiently induce a plurality of tumor cells to apoptosize in vitro, can also effectively kill the tumor cells in a body of a mouse tumor model, has no obvious toxic side effect, and can keep or raise the level of leucocytes. In addition, the invention also provides an anti-tumor pharmaceutical preparation type taking the rLZ-8 as a core composition.

The invention discloses a microbial compound preparation, a preparation method and a method for treating kitchen garbage. The method for treating kitchen garbage is to separate out mixed bacteria of bacteria and fungi with a certain ratio from the natural world and fermented food, and then prepare the mixed bacteria preparation to effectively treat the kitchen garbage. The preparation comprises the mixed bacteria including various bacteria, actinomycetes, yeast and mycetes. The preparation is characterized in that the mixed bacteria further comprise bacillus subtilis, bacillus licheniformis, high-temperature monad, streptomycete, lactobacillus plantarum, lactobacillus acidophilus, brewer's yeast, pichia pastoris, aroma-producing yeast, rhizopus chinensis, rhizopus oryzae and mucor indicus. The preparation is good for treating the kitchen garbage, non-toxic, harmless, and of genetic stability.

The invention discloses Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent and the establishing method, which belongs to the biological engineering field. The collection number of the Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent is CGMCC No.1853. The invention has the advantages that the Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent built by the invention can prevent the generation of excessive manna saccharify, when expressing extrinsic glucoprotein, reduce the immunogenicity of the pressed protein, and therefore, the invention has important application value in the biomedical field, and other fields; simultaneously, the strain can also be applied to the further gene knockout or the metabolic engineering reconstruction. The invention also builds a method for knocking out the alpha-1, 6-mannosyl transferase gene through secondary homologous recombination, mutant strain can be relatively easily obtained through the method, therefore the sieving workload is greatly reduced, and the success ratio is improved.

The invention discloses multi-strain combined fermentation red date wine and a brewing method thereof, which belongs to the technical field of brewing. The red date wine is produced by taking red date as raw materials through such processes as pretreatment, digestion, enzymolysis, sterilization, fermentation, aging and clarification. The adopted Candida, Kloeckera, Hanseniaspora and Pichia pastoris move at the early stage of fruit wine fermentation generally, so that a great deal of aromatic substances and special flavor components can be generated, the quality of fruit wine is improved, and positive impact is generated on the total flavour of the red date wine. Non-brewing yeast and brewing yeast are accessed at different fermentation stages, the species and content of the flavor substances in the date wine can be increased through the combined fermentation of multiple strains, thereby improving the mouthfeel of the date wine and the quality of the date wine; the date wine has unique style, rich wine body, sweet and pure bouquet, fresh mouthfeel and typicality.

The invention provides a modified coding gene of xylanase that has high activity in the condition of high temperature and strong alkali, a recombinant plasmid thereof, a yeast recombination genetic engineering strain containing the gene and a preparation method of the gene. Compared with the original xylanase, the modified xylanase is mutated into cysteine from glycin of the 201 site of amino acid sequence of the original xylanase; and the modified xylanase has higher heat stability, after being processed for 10 minutes at the temperature of 75 DEG C, the residual activity of the modified xylanase reaches 80 percent, while that of the original xylanase is only 15 percent. The activity of the modified xylanase is 1.5 to 2 times of that of the original xylanase at the environment with high temperature and strong alkali and can decompose xylan for a long time at high temperature. The invention also provides the Pichia pastoris yeast recombination genetic engineering strain containing the modified gene. The modified xylanase can be widely applied to industries of paper making, feedstuff and food.

The invention provides a method for efficiently expressing an antibacterial peptide NZ2114 in recombinant pichia pastoris. An expression vector comprising a DNA (Deoxyribose Nucleic Acid) sequence of the antibacterial peptide NZ2114 which is subjected to optimization coding by yeast preferred codons is constructed, pichia pastoris is transformed, the obtained recombinant pichia pastoris secretes to generate the antibacterial peptide NZ2114 by fermentation cultivation. According to the invention, by optimizing the genic sequence of the antibacterial peptide NZ2114 and constructing the special expression vector, efficient expression of the antibacterial peptide NZ2114 in the pichia pastoris is realized for the first time; yield breaks through the gram-grade level; the problem of excessively low yield or excessively high chemical synthesis cost in the large-scale production is solved; a perfect purification system is established; scale production can be realized; the method can be applied to the fields of development of antibacterial agents, development of feed additives and the like and has high application values and broad market prospects.

The invention discloses a method for producing ethanol fuel from kitchen waste, belonging to the technical field of biology. The invention aims to reduce the environmental pollution of kitchen waste, and change wastes into valuable substances, thereby recycling the kitchen waste. According to the method, the kitchen waste, which contains rich starch, cellulose, fat, protein and the like, is comprehensively utilized with a biological bacterial liquid and an enzyme preparation. The biological bacterial liquid contains Bacillus licheniformis, Bacillus megaterium, Bacillus natto, Bacillus subtilis, Lactobacillus plantarum, Lactobacillus delbrueckii subspecies bulgaricus, Saccharomyces cerevisiae and Pichia pastoris. The enzyme preparation comprises amylase, cellulase, lipase, protease, pectinase and saccharifying enzyme. The method comprises the following steps: fermenting the kitchen waste, biological bacterial liquid and enzyme preparation in a fermentation tank at the constant temperature of 25-32 DEG C for 48-80 hours, distilling the fermentation broth, and rectifying to obtain the ethanol fuel.

The invention discloses a microorganism bacterium agent and a preparation method thereof, and relates to a composite bacterium agent and a preparation method thereof. The composite bacterium agent can be used for efficiently removing a large amount of COD (chemical oxygen demand), ammonia and nitrogen in leather wastewater. The composite bacterium agent is prepared from mycobacterium, microbacterium, acinetobacter, pichia pastoris, lactobacillus, klebsiella oxytoca and a liquid culture medium. The preparation method comprises the following steps of 1, activating the bacteria strains of raw materials; 2, enriching and culturing; 3, mixing the bacterium strains according to a weight ratio, and adding into the liquid culture medium, so as to obtain the composite bacterium agent. The composite bacterium agent has the advantages that the biological activity is high, the growth speed is high, the leather wastewater can be treated under the low temperature condition, the removal rate of organic matter can be obviously increased, the contents of COD, ammonia and nitrogen in the leather wastewater are effectively decreased, and the effect is obvious.

The invention discloses an optimized gene of recombinant glucose oxidase and an expression vector and application of the optimized gene. On the premise that the amino acid sequence of the glucose oxidase is not changed, the gene sequence of the glucose oxidase is optimized according to pichia pastoris preferred codons by comprehensively considering the influencing factors such as use frequency ofthe codons, adjustment of GC content, deletion of instable sequences, secondary mRNA structure and the like; and the nucleotide sequence of the optimized glucose oxidase gene is shown as SEQ ID NO.1.The invention further provides the expression vector and a recombinant host strain containing the optimized gene of the glucose oxidase. The optimized gene is transferred to the pichia pastoris for expression, and the test results show that: compared with the gene before optimization, the secreting expression quantity of the optimized gene in the pichia pastoris is remarkably improved. The application effect tests of the glucose oxidase show that the expressed recombinant glucose oxidase has the same using effect as a commercial enzyme preparation.

The present invention relates to genes of the Pichia pastoris expression codon-optimized main capsid protein L1 of human papilloma virus types 52,31 and 45, a vector containing the genes, a strain, a preparation method, and an expression method thereof.

A process for purifying the genetically recombined insulin precursor features that the activated carbon as adsorbent and cationic chromatography are used to remove the pigment from fermented liquid and purify the active protein. Its recovery rate is more than 85% and its purity is more than 95%.

The invention discloses a feed fermentation agent and a fermented feed, the feed fermentation agent includes the following components by mass: 20-25% of pichia pastoris, 15-20% of lactobacillus, 10-15% of enterococcus faecalis, 3-10% lactobacillus plantarum, 10-15% of cellobiose lactobacillus and 15%-25% a compound enzyme agent. The fermented feed is a fermented product of soybean meal, corn and wheat bran, mulberry-rich plants and a mixture of mulberry-rich plants and soybean meal by use of the feed fermentation agent, after fermentation of the soybean meal or corn or wheat bran and other traditional feed by use of the feed fermentation agent, harmful materials such as accumulated stool and the like cumulated in pig intestines can be decomposed and removed by cooperation of crude fiber in the traditional feed and microorganism, so that the intestinal absorption efficiency of nutrients can be provided.

The invention relates to a compound biological deodorizer as well as a preparation method and application thereof. The compound biological deodorizer comprises a plant extract, pichia pastoris, pediococcus pentosaceus, pediococcus acidilactici, candida mycoderma and bacillus megatherium. Through the utilization of compound biological deodorizer to treat a waste transfer station, a livestock and poultry farm and a composting site, the NH3 removal rate can respectively reach 65.2-71.6%, 63.4-73.7% and 53.42-75.77%, and the H2S removal rate can reach 53.5-60.1%, 35.5-45.9% and 47.2-53.8%, so that the odor concentration is obviously reduced, the environment of the periphery of the waste transfer station, the livestock and poultry farm and the composting site is effectively improved, and the improvement of both economic benefits and environment-friendly benefits is facilitated.

The invention relates to a preparation method and application of a compound leavening agent. The preparation method comprises the steps of: separating lactobacillus plantarum, lactobacillus bulgaricus, streptococcus thermophilus, saccharomyces cerevisiae, candida rugosa, candida tropicalis and fermented pichia pastoris, as a leavening agent, from naturally fermented panada to produce a strain so as to prepare freeze-dried strain powder; weighing 1-2 parts of lactobacillus plantarum, 1-2 parts of lactobacillus bulgaricus, 1-2 parts of streptococcus thermophilus, 3-5 parts of saccharomyces cerevisiae, 3-5 parts of candida rugosa, 3-5 parts of candida tropicalis and 3-5 parts of fermented pichia pastoris based on parts by weight, and uniformly mixing to obtain the compound leavening agent. When the compound leavening agent is applied to the cooked wheaten food, bacterial contamination can be effectively prevented, the sanitation of the cooked wheaten food is guaranteed, the quality of the cooked wheaten food is improved, the production cycle is shortened, a production process is controllable, and the preparation method of the compound leavening agent lays a foundation for industrial production.

The invention discloses a recombinant vector and recombinant bacterium of Trichoderma reesei beta-glucosaccharase gene BGL1, and expression of the Trichoderma reesei beta-glucosaccharase gene BGL1 in the recombinant bacterium. The recombinant vector containing the Trichoderma reesei beta-glucosaccharase gene BGL1 is constructed by the following steps: (1) carrying out PCR (Polymerase Chain Reaction) amplification on the Trichoderma reesei beta-glucosaccharase gene BGL1, and connecting to a TA vector; and (2) constructing the Trichoderma reesei beta-glucosaccharase gene to a pichia pastoris expression vector. The recombinant bacterium disclosed by the invention can effectively express the target protein Trichoderma reesei beta-glucosaccharase BGL1. The invention overcomes the defect that a great deal of beta-glucosaccharase can not be purified from Trichoderma reesei in the prior art. Lots of waste lignocellulose exists in the natural world. The method provided by the invention can increase the yield of beta-glucosaccharase, and has very important meanings in enhancing degradation efficiency of cellulose, efficiently producing energy from waste cellulose and solving the problem of energy crisis at present.

The invention discloses recombinant pichia pastoris for heterogenous high level secretory expression of a rhizomucor miehei lipase. The recombinant pichia pastoris is obtained by introducing a self leader peptide gene for encoding the rhizomucor miehei lipase by employing a molecular biology technology, then optimizing a copy number of an objective gene, and constructing a vector which can be expressed in pasteur pichia pastoris. Researches show that: the introduction of the self leader peptide greatly increases the secretion amount of the objective lipase, a pichia pastoris recombinant of a lipase gene (pro-rml) containing two copies has highest enzyme activity, and the lipase Pro-RML has higher activity of hydrolysis of tri-acylglycerol.

The invention relates to the field of gene engineering, and in particular relates to a lipase, gene and application thereof. The lipase has an amino acid sequence shown as a SEQ ID NO.1. The invention aims to solve the defects of an existing technology, and clones a lipase gene from Aspergillus niger. High expression of the lipase in Pichia pastoris reaches a level higher than that of the existing technique. Therefore, the recombinant lipase provided by the invention can greatly reduce production cost in industrial production, so as to show great application potentials in industries such as washing, papermaking and food.

The invention discloses a biosynthesis method of glucuronic acid and glucuric acid, belonging to the field of metabolic engineering. The biosynthesis method is characterized by expressing myo inositol oxygenase (MIOX) from pichia pastoris in Escherichia coli to convert myo inositol into glucuronic acid, and expressing aldehyde acid dehydrogenase (Udh) from pseudomonas putida to convert produced glucuronic acid into glucuric acid, thereby successfully establishing a synthesis path from myo inositol to glucuric acid. Recombinant Escherichia coli established by the method has very great application prospect, and a new idea is provided for generating glucuronic acid and glucuric acid through conversion by the biological method.

The invention discloses a method fermentable producing adenosylmethionine utilizing genetic engineering bacteria, which is characterized in that the engineering bacteria is inoculated to a seed culture medium after activated in the plate culture medium, and then the material is inoculated to the fermentation culture medium for not less than one step after inoculated in the seed culture medium; glycerol is renewed as carbon source, obtaining the wet cells with a high density of 300 to 350g / L; then methanol and L-methionine are renewed for induction and transformation cultivation, obtaining the adenosylmethionine. The engineering bacteria is pichia pastoris GS115-sam II-208, and the book reservation number is CCTCC NO: M206106. The invention has the advantages of simple method, short cycle, high yield and low cost.

The invention discloses a carrier and engineering bacteria to express the human serum albumin. The carrier is the plasmid which inserts the encoding human serum albumin sequence into the multi clone locus of the Pichia pastoris expression carrier. The 5'end of the encoding sequence of the serum albumin is connected to the encoding sequence of the secretory signal peptide and the leading peptide which the 5' end is connected to the sequence GAAACG of the AOX1 gene for the Pichia pastoris. The bacteria including the expression carrier especially the Pichia pastoris HSA75-10 CGMCCNo.1360 can be cultured to get the high yield of the human serum albumin (10g / L of the supernate) which is 80% of the secretory protein.