567 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 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 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 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 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 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 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.

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 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.

The invention discloses a method for improving biological control efficacy of pichia caribbica to fruit postharvest diseases, belonging to the field of fruit postharvest biological control. The method comprises the steps of: activating pichia caribbica, inoculating in an NYTB culture medium added with 0.2-1% of trehalose for inducing culture, and centrifuging to obtain thalli; diluting the thalli with sterile water to prepare 1*10<8>/ml of bacterial suspension; placing fruits in the bacterial suspension, soaking for 1 minute and then immediately taking out, and naturally airing; and placing in a plastic crate, sealing with a preservative film, and storing in an environment at room temperature. The efficacy of controlling the fruit postharvest diseases by the pichia caribbica is increased by using the inducing culture of the trehalose, thus the method has the advantages of simplicity in use, convenience for operation, good effect and low cost. The pichia caribbica subjected to the inducing culture can be used for replacing a chemical bactericide to control the fruit postharvest diseases, can avoid the damage to people due to the use of the chemical bactericide, and has remarkable economic benefit and social benefit.

The invention relates to pichia pastoris for efficiently converting methanol to produce single cell protein and an application of the pichia pastoris. The classification designation is pichia pastoris HGD-01. The pichia pastoris is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: M2012342. As the pichia pastoris strain is used for methanol protein production, the whole fermentation period is 70-84h, the fermentation time is shortened, and the yield of a single fermentation tank is increased to 140kg of dry weight of the mycoprotein per ton of fermentation liquid.

The invention discloses an aroma producing yeast strain and application thereof. The aroma producing yeast strain YfenjiaZ capable of generating strong fruity flavor is separated and screened out fromfen-flavor Baijiu fresh fermented grains through smelling technology, belongs to pichia kudriavzevii and is collected under the number of CGMCC No.16197. The strain has high aroma producing ability in culture media taking cereals as a raw material, and ethyl acetate in main aroma substances accounts for 59.59%. Fen-flavor Baijiu high in ethyl acetate content and ethyl acetate/ethyl lactate ratioand strong in fruity flavor can be produced by utilizing the strain for fermentation and can serve as high-ethyl acetate/ethyl lactate-ratio flavoring liquor or fen-flavor base liquor to be used for blending of Baijiu.

The invention discloses an alpha-glucosidase genetic engineering bacterium and application thereof, and belongs to the technical field of enzyme engineering. The genetic engineering bacterium is PichapastorisGS115 carrying recombinant plasmid, wherein the recombinant plasmid is plasmid pPIC9K-aglu containing Aspergillus niger alpha-glucosidase cDNA, and the P.pastorisGS115 is utilized to express alpha-glucosidase of an A.niger source. The constructed recombinant P.pastorisGS115/pPIC9K-aglu serving as a production strain to perform high density cultivation of P.pastoris by a dissolved oxygen feedback regulated feed batching strategy to produce the recombinant alpha-glucosidase; glycerol is continuously fed after 18 to 20 hours of fermentation so as to realize high density cultivation of the P.pastoris; the methanol is continuously fed after 24 to 26 hours of fermentation to control certain residual methanol concentration; and the enzyme activity of the recombinant alpha-glucosidase is over 3.5U/mL after 120 hours of fermenting cultivation. Cheap inorganic salt and the glycerol are adopted as raw materials for production, the fermentation process is simple and practical, and the method is suitable for large-scale production.