599 results about "Pichia membranaefaciens" patented technology

The present invention relates to the elimination of mannosylphosphorylation on the glycans of glycoproteins in the yeast genus Pichia. The elimination of mannosylphosphorylated glycoproteins results from the disruption of the PNO1 gene and the newly isolated P. pastoris MNN4B gene. The present invention further relates to methods for producing modified glycan structures in host cells that are free of glycan mannosylphosphorylation.

The invention relates to a saline-alkali soil improving agent and its preparation method. The preparation method comprises the following steps: mixing Rhodopseudomonas palustris, Pichia membranefaciens Hansen bacterium, lactobacillus casei, Staestomyces microflavus and seaweed slurry, organically combining a plurality of beneficial microorganisms and seaweed through multi-time fermentation, culturing in a compound mode to obtain a novel microorganism agent-seaweed bio-fungus. adding a proper amount of humic acid, organic matter, active zeolite powder and molasses, fermenting and drying the fermented product. The saline-alkali soil improving agent uses a plurality of microorganisms and employs strong effects of a unique ionic adsorbent, an organic neutralization balancing agent, an ion-exchange agent and a beneficial bacteria conversion starter to achieve the purposes of improving the soil environment, regulating acid-base balance, reducing the salt content, effectively raising the soil fertility and permeability, loosening the soil, eliminating the soil hardening, degrading the harmful substance and promoting the accumulation and conversion of the organic substance.

The invention provides a composite microbial flora and an enzyme product thereof, and a method of applying the composite microbial flora and the enzyme product thereof in soil restoration. The composite microbial flora is composed of a microbial flora and a bioactive enzyme, wherein the microbial flora is composed of bacillus subtilis, bacillus mucilaginosus, bacillus amyloliquefaciens, pediococcus pentosaceus, pichia farinose and pediococcus acidilactici, and the bioactive enzyme is composed of phytase, protein hydrolase, cellulose hydrolase and urease. Compared with the prior art, an effective microbial community and a microecological environment are constructed to realize biological catalysis and improve the content of organic matters and beneficial microbial flora in soil, and thus, the fertility of the soil is improved; biochemical degradation rate of soil pollutant can be substantially accelerated, digestion and biodegradation of the pollutant can be accelerated, and final inertial products like carbon dioxide, water and nitrogen are obtained; and toxic intermediate produced in the process of biochemical degradation can be rapidly removed, odor is eliminated, and emission of organic volatile matters (VOC) is reduced.

The invention aims to provide a method for improving the expression amount of secretory foreign protein in pichia pastoris. Byintracellular co-expression of double genes of HAC1 and ERO1 or three genes of HAC1, ERO1 and BIP in pichia pastoris, the expression amount of secretory foreign protein in pichia pastoris is effectively improved. According to the invention, double genes of HAC1 and ERO1 or three genes of HAC1, ERO1 and BIP are transformed into pichia pastoris secreting and expressing exogenous xylanase to obtain recombinant pichia pastoris strains, thereby significantly increasing the expression amount of the exogenous xylanase. During co-expression of double genes of HAC1 and ERO1 in pichia pastoris, the secretion expression amount of exogenous xylanase is generally increased by 15%-25% compared with the initial level; and during the co-expression of three genes HAC1, ERO1 and BIP, the secretion expression amount of xylanase is increased by 45%-57% compared with the initial level.

The invention discloses recombinant pichia pastoris engineering bacteria. The recombinant pichia pastoris engineering bacteria comprises a codon-optimized Richter trichoderma xylanase gene, and the nucleotide sequence of the gene is shown in SEQ:1 in a sequence table. The invention also discloses a production method of the recombinant pichia pastoris engineering bacteria. The production method comprises the following steps: (1) performing synonymous mutation on the Richter trichoderma xylanase gene according to pichia pastoris flavored codons so that the Richter trichoderma xylanase gene is completely mutated into the pichia pastoris favored codons; (2) synthesizing an optimized xylanase gene by utilizing a total-gene synthesis technology, and connecting the optimized xylanase gene with a pMD20-T vector to build a cloning vector pMD20T-Xynopti; (3) building a recombinant expression vector pPICZalpha-Xynopti; (4) transforming a pichia pastoris host; (5) performing induction culture of a positive converter; (6) producing xylanase by small-scale fermentation by adopting the recombinant pichia pastoris engineering bacteria. The recombinant pichia pastoris engineering bacteria disclosed by the invention can be used for secreting a great amount of xylanase.

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 relates to an improved rhizomucor miehei lipase gene and an application to yeast display. The sequence of the improved rhizomucor miehei lipase gene is SEQ.ID.No2, with respect to a recombinant vector pMD18-T-RML containing the gene, RML means lipase gene; and the collection number of a bacterial strain Escherichia coli TOP10 / pMD18-T-RML carrying the plasmid is CCTCC M 208136. In the invention, the gene is transferred into pichia stipitis host strain, so that the rhizomucor miehei lipase is displayed and expressed in the pichia stipitis. The provided pichia stipitis can effectively display the rhizomucor miehei lipase. The lipase can be widely applicable for producing fatty acid methyl ester, ethyl caproate, triglycerides which have different melting points but does not contain various types of fatty acid and a few 'reconstructed esters'.

The present invention is related to methods and for producing higher titers of recombinant protein in a modified yeast host cell, for example Pichia pastoris, wherein the modified yeast cell lacks vacuolar sorting activity or has decreased vacuolar sorting activity relative to an unmodified yeast host cell of the same species. In particular embodiments vacuolar sorting activity is reduced or eliminated by deletion or disruption of a gene encoding Vps10 or a Vps10 homolog. The invention is also related to the modified yeast cells which are modified in accordance with the methods disclosed herein.

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 relates to alginate lyases, and in particular, relates to coding genes and applications of three algin-degrading alginate lyases. The alginate lyases comprise alginate lyases AlgAT0, AlgAT1 and AlgAT5 respectively. The coding genes of the alginate lyases have the base sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively. The alginate lyase genes are cloned into escherichia coli and pichia pastoris by a genetic engineering method. The AlgAT0, AlgAT1 and AlgAT5 are cloned into an expression vector of pichia pastoris; after fermentation condition optimization, fermentation is performed for 120 h, the concentrations of extracellular proteins are 0.312 g / L, 1 g / L and 9.39 g / L respectively, and the enzyme activities are 64666.67 U / mL, 126666.67 U / mL and 136025.6 U / mLrespectively. An escherichia coli recombinant strain and a pichia pastoris strain capable of producing alginase are obtained. The recombinant enzymes have stable properties and can be used for algin conversion with high added value, the enzyme activities of the three enzymes are much higher than values those reported so far, and the three enzymes have quite good potential for industrial application.

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 provides a Pichia guilliermondii X25 which is enriched and screened from soil, has high diastereoselectivity and is a carbonyl reductase active microbial new strain, and application of the Pichia guilliermondii X25 in preparation of 6-cyano-(3R, 5R)-dyhydroxyl hexanoic acid tert-butyl ester by biological asymmetric reduction (R)-6-cyano-5-hydroxyl-3-carbonyl hexanoic acid tert-butylester. The strain is preserved in China Center for Type Culture Collection (CCTCC), the address is Wuhan university, Wuhan, China, the post code is 430072, the CCTCC No. is M 2011386, and the preservation date is November11th,2011. The optical pure 6-cyano-(3R, 5R)-dyhydroxyl hexanoic acid tert-butyl ester prepared by using carbonyl reductase generated by the Pichia guilliermondii X25 to convert (R)-6-cyano-5-hydroxyl-3-carbonyl hexanoic acid tert-butyl ester is high in stereoselectivity, mild in reaction condition and environment-friendly.

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 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 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 discloses a preparation method of multi-copy high expressed recombined plectasin by pichia pastoris. The method comprises the following steps: a plectasin expressing gene sequence is designed according to preference performance to codon translated by pichia pastoris; the optimized plectasin gene is fused on an alpha-factor signal peptide C terminus of an expression vector pPICZalphaA to construct a single-copy expression vector, the vector comprises a plectasin expression cassette containing a start signal element alcohol oxygen dehydrogenase strong promoter (AOX), alpha-factor signal peptide gene and a plectasin gene fused in C terminus, a stop signal element AOX (TT) and the like. A complementation principle of restriction endonuclease Bg1II and BamHI cohesive end is used to obtain plectasin gene-containing recombinant plasmid of different copy cascade expression cassettes, pichia pastoris is electrotransformed and secreted and expressed plectasin with high efficiency under the methanol induction. The expression level and plectasin gene copy number exist a linear relation. The constructed multi-copy high expressed yeast cells can be used for raising the output and reducing the cost, and is adapted to large scale production of plectasin.

The invention relates to a field of environmental biology, and discloses a laccase gene, an engineering bacteria of the laccase gene, and an application thereof in treatment of papermaking waste water by microbial hydrolysis. The laccase gene has a nucleotide sequence as shown in a SEQIDNO:2, and codes a protein having an amino acid sequence as shown in a SEQIDNO:1. According to the invention, the laccase gene optimized according to preferred codons of pichia yeast can be constitutively expressed in high efficiency in yeast, and an expression vitality of the laccase can be improved, thereby being of great significance for promoting the application of the laccase in biological treatment of the papermaking waste water.

The invention provides novel antimicrobial peptide MP1106 (Seq ID No.1) designed based on Plectasin, and provides a high-efficiency expression of the novel antimicrobial peptide MP1106 (Seq ID No.1) in pichia pastoris. Through the preference of yeast codon for optimization of antibacterial peptide MP1106 encoding gene and construction of recombinant plasmid, the recombinant plasmid is transformed into the pichia pastoris, secretory expression of high density fermentation protein in a 5 L fermentation tank can be achieved, the total protein level reaches 2.134g / L, and purified recombinant MP1106 yield reaches 831mg / L. In addition, recombinant MP1106 cation-exchange chromatography one-step purification method is established, and novel antimicrobial peptide MP1106 in-vitro properties show that the novel antimicrobial peptide MP1106 has strong inhibitory activity against staphylococcus aureus, is free of cell toxicity and stable in high temperature, alkaline environment, serum incubation conditions, and is resistant to papain, pepsase and the like. The novel antimicrobial peptide MP1106 is superior in properties, can realize large-scale industrial production, and is potential to be developed into a novel anti staphylococcus aureus preparation.

Belonging to the field of microbial agents and fertilizers, the invention specifically relates to an Enteromorpha microbial agent suitable for the demands of planting, livestock and poultry, aquaculture, sewage treatment and other fields, and further discloses a preparation method of the microbial agent. The Enteromorpha microbial agent provided by the invention employs Enteromorpha to prepare a slurry, and is compounded with Streptomyces microflavus, Lactobacillus casei, bacillus subtilis, rhodopseudomonas palustris, Pichia membranaefaciens and other main beneficial microbial floras, and has the multiple functions of effectively improving agricultural product quality, effectively lowering the cost of animal and plant growth period, increasing agricultural product output and the like. The microbial agent also has the functions of purifying water, removing hydrogen sulfide and other odors from sewage, garbage and livestock raising housings, and improving the environment, etc.

The invention discloses a lactic acid-tolerant ester-producing pichia pastoris, and belongs to the technical field of a bioengineering technology and a brewing biotechnology. The strain is preserved at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on April 24, 2017, the classification is named pichia kudriavzevii (Pichia kudriavzevii) and the preservation number is CGMCC No.14068. The pichia pastoris is high in environmental tolerance, is capable of tolerating 0-15% lactic acid, is capable of metabolizing to generate multiple ethyl ester volatile flavor substances, such as ethyl acetate, ethyl propionate, ethyl caprylate, phenethyl acetate and ethyl phenylacetate in the environment of 0-12% lactic acid, and is a brewing functional strain with excellent performance.

The invention provides and discloses an optimized nucleotide sequence of alkaline pectinase pel168s and a high-level expression method thereof. According to the method, a pel168 gene sequence (wherein the gene is Bacillus subtilis 168 the accession number of which in a GenBank is AL009126) is optimized by DNA works software, restriction enzyme cutting sites SalI and PmeI are shielded, a restriction enzyme cutting site EcoRI is added at the 3' end of a primer, and a restriction enzyme cutting site NotI is introduced at the 5' end of the primer. After the procedures of PCR (Polymerase Chain Reaction) amplification, connection transformation and sequencing verification, the optimized gene sequence of the alkaline pectinase pel168s is obtained. Recombinant plasmid pel168s-9k is constructed according to the sequence, and then is transformed into pichia yeast GS115, thereby obtaining a positive recombinant strain GS115 / pel168s-9k. According to the invention, when alkaline pectinase is produced by adopting the optimized nucleotide sequence of the alkaline pectinase pel168s and utilizing the pichia yeast, the target protein expression index is high, the purge process is simple, the production cost of the alkaline pectinase is reduced greatly, and the utilization rate of an enterprise on the alkaline pectinase is enhanced.

The invention discloses recombinant pichia pastoris engineering bacteria and metabolic recombinant xylanase as well as preparation of the metabolic recombinant xylanase. A recombinant yeast expression vector pPIC9K-Xyn11Bm is constructed by a nucleotide sequence Xyn11Bm which is optimized by a xylanase gene Xyn11B of synthetically coded Neocallimastix frontalis; a host cell pichia pastoris GS115 is transformed to obtain recombinant genetically engineered bacteria Pichia pastoris GS115 / pPIC9K-Xyn11Bm with the preservation number of CGMCC No.9398; the recombinant Xyn11Bm gene is expressed in the yeast cell, and further the purified and metabolized high-enzyme-activity expressed recombinant xylanase has a wide application value when applied to non-therapy targeted xylanase decomposition.

The invention relates to a genetic-engineering antibacterial peptide, as well as a preparation method and application thereof. The preparation method comprises the following steps: according to the characteristics of the amino acid sequence of Magainin and Cecropin P and LL-37 which are antibacterial peptide, a novel antibacterial peptide gene is designed; the sequence of the gene is synthesized and transformed to be expressed in pichia to form antibacterial-peptide gene transformation pichia engineering bacteria, and to be expressed in a fermentor to achieve the effects of high-density cultivation and efficient expression. Fermentation broth is separated and purified to be an antibacterial peptide product which can be applied to feed and food additives and be used as injection. The antibacterial peptide has the advantages of good antibacterial activity to most gram-negative bacteria and gram-positive bacteria, in particular better effects of inhibiting and killing ampicillin-resistant bacteria and kanamycin-resistant bacteria.

This invention discloses the expression method and engineering bacterium for a beta-mannase. The bate-mannase is a protein that can degrade mannan, containing one of the following amino acid residue sequences: (1) SEQ ID No.1in the sequence list; and (2) protein obtained by substituting, deleting or adding 1-10 amino acid residues to the amino acid residue sequence in SEQ ID No.1. The engineering bacterium for the mannase is obtained by introducing yeast Pichia pastoris expression carrier containing beta-mannase mature protein coding sequence into yeast Pichia pastoris. The engineering bacterium can realize high-efficiency expression of beta-mannase, the fermentation technique is simple and the extraction cost is low, thus is suitable for mass production. The beta-mannase and its engineering bacterium will have important applications in industrial manufacture and animal feed additives.

The invention discloses Pichia kudriavzevii ZJPH0802 and an application thereof in preparation of curcumin derivatives. The strain is collected in China Center for Type Culture Collection (CCTCC), the address is Wuhan University, Wuhan, China, the collection data is September 24, 2012, and the collection number is CCTCC M 2012373. The invention provides a new strain, namely the Pichia kudriavzevii ZJPH0802 for preparing the curcumin derivatives by biotransformation, and structural modification can be performed on curcumin by utilizing a strain resting cell transformation method for obtaining the corresponding derivatives; the pharmacological or biological activity of structurally modified matters of the curcumin can be improved to different extents relative to a curcumin substrate before modification so as to be conductive to development of new pharmaceutical preparations. The technology for obtaining the curcumin derivatives by adopting the biotransformation method, disclosed by the invention, is simpler and environment-friendly, a biological catalyst is a microbial thallus, and the Pichia kudriavzevii ZJPH0802 further has the advantages of self-fermentation and production, stable quality and low cost.

The invention discloses a genetically engineered bacterium for producing glucose oxidase as well as a construction method and an application thereof, and belongs to the technical field of genetic engineering. A recombinant DNA (Deoxyribonucleic Acid) technology is used for cloning and connecting an Aspergillus niger glucose oxidase (GOD) gene to a Pichia pastoris expression carrier pPIC9K and converting Pichia pastoris GS115; screening and identifying to obtain recombinant Pichia pastoris GS115-pPIC9K-GOD which can produce the glucose oxidase with the higher activity and has the preservation number of CCTCC (China Center For Type Culture Collection) NO: M2012266. The glucose oxidase expressed by the bacterial strain has the enzyme activity of 52 U / mL in a shaking bottle and the enzyme activity is improved by about 24 times as compared with the enzyme activity of wild funguses, so as to lay a good foundation for the large-scale production of the glucose oxidase.

The invention discloses a preparation method of an EV71 vaccine and the vaccine prepared by the preparation method, relating to the preparation method of taking a Pichia yeast as an expression system for co-expression of P1 and 3CD proteins of the EV71 after codon optimization by utilizing different promoters, and obtaining the viral like particle vaccine with immunogenicity, and also relating to the vaccine prepared by the preparation method.

The invention discloses preparation and application of recombinant plectasin. The method comprises designing plectasin gene according to preferred codons of Pichia pastoris, wherein possible nucleotide sequences of the plectasin gene are expressed in SEQ ID NO. 1, constructing recombinant expression vectors pPICPlectasin and recombinant genetic engineering bacteria Pichia pastoris X33pPICPlectasin (CGMCC NO. 3564), carrying out a high density fermentation process on the recombinant genetic engineering bacteria Pichia pastoris having a high expression level, wherein a total protein concentration of supernate from the high density fermentation process is 729 microgrammes per milliliter, dialyzing and freeze-drying the supernate, and orderly carrying out a gel filtration chromatography treatment and a reversed phase high performance liquid chromatogram treatment on the freeze-dried supernate to obtain high purity recombinant plectasin. The high purity recombinant plectasin is not hemolytic, has favorable PH stability, heat stability and anti-pepsin activity, and can inhibit effectively the growth of gram-positive pathogen Streptococcus pneumonia, staphylococcus aureus and staphylococcus epidermidis. Therefore, the high purity recombinant plectasin can be utilized for treating and preventing gram-positive bacterium and especially streptococcus and has potential antimicrobial drug development values.

The invention relates to the field of genetic engineering, in particular to mutated glucose oxidase with the increased expression quantity and an encoding gene and application thereof. The amino acid sequence of the mutated glucose oxidase is shown as SEQ ID NO.1. The site-specific mutagenesis technology is adopted for carrying out site-specific mutagenesis on a gene (Genebank:FJ979866.1) of glucose oxidase (GOD) of aspergillus niger GIM 3.452(CICC 2377) to enable mutation sites of the amino acid sequence of the mutated glucose oxidase to be Y76C and Q279K; the mutant gene is cloned and connected to a pichia pastoris expression vector pPICZalphaA, and pichia pastoris X33 is converted, and screened to obtain a glucose oxidase pichia pastoris strain P.pastoris X33-pPICZalphaA-GODmut with the increased expression quantity. Enzymatic property determination shows that the mutated glucose oxidase gene can be expressed and inherited in pichia pastoris stably and efficiently, and the enzymatic activity and the stability of glucose oxidase expressed by the strain are remarkably higher than those of an original strain, which lays a good foundation for large-scale production of glucose oxidase.