75 results about "Pichia stipitis" 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.

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'.

Genetic recombinant yeast expressing xylose reductase (XR), (wild-type or mutant) xylitol dehydrogenase (XDH), and xylulokinase (XK) and a method for highly efficiently producing ethanol from xylose using the yeast are provided. Pichia stipitis-derived XR and (wild-type or modified-type) XDH genes and Saccharomyces cerevisiae-derived XK gene were introduced via chromosomal integration. Thus, a genetic recombinant yeast having a high xylose fermentation rate, being capable of producing ethanol from xylose in high yields, and having high xylose fermentability in the presence of glucose, as well as a method using the recombinant yeast for highly efficiently producing ethanol from xylose or a saccharified solution from lignocellulose-based biomass are provided. Furthermore, a method for improving the xylose fermentability of the genetic recombinant yeast of the present invention via acclimatization treatment is also provided herein.

Strains of the yeast Pichia stipitis NPw9 (ATCC PTA-3717) useful for the production of ethanol using oxygen for growth while fermenting normally toxic lignocellulosic prehydrolysates.

The invention discloses clone and expression of an Alpha-glucosidase (abbreviated as AGLU enzyme) gene and belongs to the fields of enzyme gene engineering and enzyme engineering. The invention obtains the aglu gene with SEQ ID NO of 1 through an Aspergillus niger (A.niger) WX-07 total DNA, and aglu cDNA uses an expression vector of plasmid pPIC9K and an expression parasitifer of pichia stipitis (P.pastoris), thus realizing extracellular dissolubility expression of the aglu gene; and the aglu cDNA totally has 2880 nucleic acid and encoding of 960 amino acid, constructs eukaryotic expression plasmid and transforms Pichia pastoris KM71 for AGLU enzyme expression. The recombinant enzyme has transnucleosidation activity, can generate isomalto oligosaccharide through maltose transnucleosidation, is applicable to the requirements of industrial application including food, feeding stuffs and medicaments and the like, and can be applied into the industrial production of isomalto oligosaccharide.

A novel isolated Pichia stipitis strain is provided. The strain is capable of fermenting at least a pentose sugar in the presence of one or more inhibitory substances to produce ethanol. A method of utilizing the strain to produce ethanol is also provided.

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 a method for producing the mixture of isoamylase, alpha-amylase and glucamylase and belongs to the field of biological technology. The method comprises: firstly, amplifying an isoamylase gene out of the genome of bacillus amyloliquefaciens, an alpha-amylase gene out of the genome of bacillus licheniformis, an alpha-amylase gene out of the genome of barley and a glucamylase gene out of the genome of aspergillus niger; secondly, constructing a bacillus subtilis expression vector containing the isoamylase gene, a pichia stipitis expression vector containing two alpha-amylase genes and a saccharomyces cerevisiae expression vector containing the glucamylase gene; thirdly, constructing engineering bacteria, namely transforming the genome of bacillus subtilis through the bacillus subtilis expression vector, transforming the genome of pichia stipitis through the pichia stipitis expression vector, and transforming the genome of the saccharomyces cerevisiae through the saccharomyces cerevisiae expression vector; and finally, allowing the fermentation engineering bacteria to produce the mixture of recombinant isoamylase, alpha-amylase and glucamylase. The mixed use of the enzymes can obviously improve the action under which starch can be hydrolyzed into glucose. The production of the mixed enzymes has the advantage that the change from the production of isoamylase, alpha-amylase and glucamylase by several steps regularly into production by one step obviously reduces energy consumption and lowers production cost.

The invention relates to a composite microorganism deodorizing agent and a preparation method and applications thereof. The microorganism agent includes bacillus cereus 2-2, pichia manshurica 2-6 andlactobacillus zeae 2-20, wherein the total bacteria count of the composite microorganism deodorizing agent is 2x10<9> cells / mL. By combining the three strains of the bacillus cereus 2-2, the pichia pastoris 2-6 and the lactobacillus cornicula 2-20, the method finds that the three strains not only have no antagonistic reaction, but also can significantly improve the effect of removing NH3 and H2S.Actual test indicates that the ammonia removal rate of the microorganism deodorizing agent compounded of the three strains reaches 73.04%, and the removal rate of hydrogen sulfide reaches 71.19%.

The invention discloses a method for carrying out alkaline pretreatment on plant fiber raw materials for preparing ethanol through enzymolysis and fermentation. According to the method, firstly, green liquid is used for carrying out alkaline treatment on the air dried plant fiber raw materials; then, under the condition that the substrate w / v concentration being 5 to 15 percent, the cellulose is adopted for batch type hydrolysis for 48 to 72 hours; and after the hydrolysis completion, hydrolysate is subjected to solid-liquid separation, the obtained clear liquid is concentrated to a state that the glucose concentration in the sugar liquid is 100 to 200g / L, hexose in the concentrated sugar liquid is converted into the ethanol through brewer's yeast, the ethanol mash is removed through distillation, and then, the pichia stipitis is used for converting pentose into the ethanol. The method realizes the goals that the mature process and equipment of the paper pulp process are utilized for recovering chemical medicine and heat energy, the consumption of chemicals and the loss of heat energy are reduced, the environment pollution is reduced, the clean production of the ethanol is realized, the efficient proceeding of the enzymolysis and the hexose and pentose fermentation are respectively ensured through the low-substrate-concentration enzyme hydrolysis and the sequential fermentation, particularly, through the pentose fermentation, the ethanol yield of each ton of plant fiber raw materials is improved, and the raw material cost of the ton ethanol is reduced.

Genetic recombinant yeast expressing xylose reductase (XR), (wild-type or mutant) xylitol dehydrogenase (XDH), and xylulokinase (XK) and a method for highly efficiently producing ethanol from xylose using the yeast are provided. Pichia stipitis-derived XR and (wild-type or modified-type) XDH genes and Saccharomyces cerevisiae-derived XK gene were introduced via chromosomal integration. Thus, a genetic recombinant yeast having a high xylose fermentation rate, being capable of producing ethanol from xylose in high yields, and having high xylose fermentability in the presence of glucose, as well as a method using the recombinant yeast for highly efficiently producing ethanol from xylose or a saccharified solution from lignocellulose-based biomass are provided. Furthermore, a method for improving the xylose fermentability of the genetic recombinant yeast of the present invention via acclimatization treatment is also provided herein.

The invention discloses a brewing method of purple sweet potato Huangguan pear composite fruit wine. The brewing method comprises following steps: material selection and raw material pretreatment; enzymatic saccharification; SO2 sterilization; sugar blending and acid blending; preparation of a saccharomycetes seed solution; inoculation and fermentation; aging and fermentation; bottle changing and clarification; and finished product blending and diatomite filtering. According to the brewing method, purple sweet potato is subjected to enzymatic hydrolysis with amylase, and then is mixed with pear juice; an obtained mixture is subjected to saccharification with pectase and glucoamylase, so that enzymatic hydrolysis juice yield of mashed purple sweet potato is increased to be 68% or higher, and purple sweet potato Huangguan pear juice saccharification juice yield is increased to be 70% or higher via appropriate enzymatic hydrolysis and saccharification; Pichia kudriavzevii fermentation is adopted, and appropriate fermentation technical conditions are adopted, so that the alcohol by volume of the purple sweet potato Huangguan pear composite fruit wine is 12%vol or higher. The color of the purple sweet potato Huangguan pear composite fruit wine is clear; the purple sweet potato Huangguan pear composite fruit wine is transparent, the texture is fine and stable, the purple sweet potato Huangguan pear composite fruit wine possesses the fruit flavor and health care effect of both purple sweet potato and Huangguan pear, the flavor is unique, mellow and full, and fresh, and the purple sweet potato Huangguan pear composite fruit wine is abundant in nutrients.

The invention discloses a novel compound microbial agent. The novel compound microbial agent is prepared from an active seed fluid and a producing culture medium by volume ratio of 2 to 10. The active seed fluid is prepared from bacillus velezensis, camellia flower lactobacillus, Pichia kudriavzevii, brewer's yeast, acetobacter pasteurianus, lactobacillus plantarum and lactobacillus casei by volume ratio of (10-20) to (3-9) to (2-4) to (1-5) to (1-5) to (1-3) to (1-3). The producing culture medium is prepared from 200-300 kg of tomatoes, 200-300 kg of bean sprouts, 100-200 kg of wheat bran, 300-400 kg of brown sugar, 250-350 kg of molasses, 2-6 kg of peptone, 2-7 kg of yeast extract and 3-9 kg of glacial acetic acid which are contained in every 10 tons of water. The production and fermentation process is simple, the deodorization efficiency is high, microbial flora are stable, and he applicability is high.

The invention relates to a pichia stipitis gene expression system as well as construction and an application thereof. The pichia stipitis gene expression system comprises a novel expression vector which is of an annular structure; the following elements are operably connected from 5' to 3' sequentially: a pMD19-T simple plasmid skeleton, an rDNA homologous recombination sequence, an exogenous gene expression cassette and a selective marker gene expression cassette; from upstream side to downstream side, the exogenous gene expression cassette sequentially comprises a promoter, an exogenous gene insertion enzyme cutting site and a transcription terminator; the selective marker gene expression cassette comprises a promoter, an antibiotics resistance gene and a transcription terminator; and the yeast capable of making use of xylose is pichia stipitis. The expression vector disclosed by the invention can be used for achieving the integrated stable expression in the pichia stipitis; and the expression vector is of great significance for the basic theory research and product development of the pichia stipitis.

The present invention relates to a polypeptide which has a novel specific arabinose transporter function as well as to nucleic acids coding therefore. The invention further relates to host cells, in particular modified yeast strains which contain the coding nucleic acids and express the polypeptide and functionally integrate it into the plasma membrane and are thus able to absorb L-arabinose. When using modified host cells which express additional proteins of the arabinose metabolic pathway, arabinose can be fermented by these cells, in particular into ethanol. The present invention is therefore relevant, inter alia, in connection with the production of biochemicals from biomass, such as bioethanol for example.

The invention discloses a gene sequence of encoding an antibacterial peptide-chitin peptide cloned from procambarus clarkia, and provides a recombinant expression and purification method thereof. The invention further discloses an amino acid sequence of the antibacterial peptide-chitin peptide cloned from the procambarus clarkia. The invention further provides the uses of the gene sequence and the chitin peptide. In the invention, a yeast expression vector can be prepared by virtue of the procambarus clarkia chitin peptide gene of the invention; an engineering strain can be obtained by transfecting Pichia stipitis; and recombinant protein with antibacterial activity can be obtained by induction expression. The recombinant chitin peptide obtained by the invention can be applied to an antibacterial feed additive, food preservation, animal and plant gene transformation and drug development.

Strains of the yeast Pichia stipitis NPw9 useful for the production of ethanol using oxygen for growth while fermenting normally toxic lignocellulosic prehydrolysates.

The invention provides a method for preparing ethanol by fermenting pentose and hexose mixed sugar by using pichia stipitis. According to the method, pichia stipitis is used an origin strain, the pentose and hexose mixed sugar is used as a carbon source, and the conditions required by high-sugar concentration fermentation of pichia stipitis are determined.

The invention relates to a preparation method for mannanase by induction of a substrate batch fed into a carbon source of glycerol, in particular to a fermentation production process for genetically engineered strain of the mannanase with Pichia stipitis as a carrier. The invention is characterized in that a seed culture solution is used for culture in a first-stage seed culture fermentor; a basic culture solution is used for culture in a second-stage seed fermentor; part of the basic culture solution is poured into a production fermentor for culture at initial time, and the glycerol, methanol and mannan substrate are respectively fed into a fermentation solution to finish the fermentation at a certain proportion and speed in the fermentation process. The yield of the mannanase can be obviously increased by 15 to 24 percent.

The present invention describes an integrated genome-scale metabolic network model construction and analysis method with Pichia stipitis as an example, belonging to the system biology field. Based on the method, a constructed and obtained Pichia stipitis genome-scale metabolic network model is subjected to cell growth simulation of a system level and identification of a minimal gene and reaction set, and an important platform is provided for comprehensive understanding of metabolic characteristics and mechanism of the Pichia stipitis is provided.

The invention provides a method for adopting single strains to carry out fermentation to in-situ virus-eliminated alcohol of hydrolyzate of lignocellulose; therefore, the invention firstly provides a new strain of pichia stipitis of trunks and the preservation number is CGMCC No.2661; the experiment proves that the strain can carry out detoxification in situ to lignocellulose diluted acid hydrolyzate, can transform glucose and xylose in lignocellulose diluted acid hydrolysate into ethanol effectively and reach 92.4 percent of the highest theoretical value of the ethanol. The use of the strain can simplify the technique that the lignocellulose is used as materials to produce the ethanol, reduces the production cost of the ethanol and has significant theoretical and practical significance to the commercialization of the production of lignocellulose ethanol.

The invention provides grape vinegar brewed by using saccharomycetes and a preparation method thereof. The grape vinegar is prepared from raw material grape wine and acetic acid bacteria seed liquid. The preparation method comprises the following steps: culturing the acetic acid bacteria seed liquid with Pichia pastoris; mixing the seed liquid with a filling material and then filling into a double-layer spraying tower; and pasteurizing, insulating heat, cooling, processing in the double-layer spraying tower, fermenting and sterilizing so as to obtain a finished product. The invention relates to a production method and device for producing the grape wine into the grape vinegar by using Pichia pastoris. In the whole brewing process, no additive is needed; the produced grape vinegar is high-quality grape vinegar which maintains the original nutrition value of grape and also has pure and mild mouth feel and high acetic acid content (the content of total acid) up to 6%; and production process is simple, sanitary condition is good, productive labor intensity is low, and product quality is high.

The invention is a process for producing ethanol from sugarcane bagasse, the principal steps of which are mild sulphuric acid hydrolysis of the hemicellulose fraction of the sugarcane bagasse, followed by extraction of the hydrolysate and then fermentation thereof with the yeast Pichia stipitis. The process can be carried out with different solid : liquid ratios, and provides a step of acclimatizing the Pichia stipitis yeast, which results in a greater rate of ethanol production. The process takes place in a press reactor specially designed for this purpose, which allows more efficient extraction of the hydrolysate and, as a consequence, better process performance.

The invention discloses a method of producing stalk ethanol by means of mixed fermentation of transgenetic yeast. The transgenetic yeast can respectively degrade cellulose and xylan of stalks to glucose and xylose and ferment glucose and xylose to ethanol without additionally providing cellulase and xylanase. Degradation of cellulose and xylan and fermentation are synchronously carried out. The method disclosed by the invention has the advantages that by means of transgenetic saccharomyces cerevisiae, transgenetic pichia stipitis and transgenetic candida shehatae, synchronous enzyme hydrolysis-fermentation of a stalk cellulose raw material is realized to produce ethanol without additionally adding cellulase and xylanase, thereby greatly lowering the production cost of stalk cellulose ethanol and promoting the progress of replacing petroleum fuel by the stalk cellulose ethanol.

The invention provides an ester-producing yeast strain and application thereof. The invention provides a pichia kudriavzevii strain, the pichia kudriavzevii strain is a pichia kudriavzevii strain C4.12, the pichia kudriavzevii strain C4.12 is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the pichia kudriavzevii strain C4.12 is CCTCC NO: M 2021124. The pichia kudriavzevii strain is a pichia kudriavzevii strain, and the pichia kudriavzevii strain is a pichia kudriavzevii strain. The yeast strain provided by the invention has the characteristics of multi-environment tolerance, broad-spectrum substrate applicability and ester production, and can improve the sensory flavor of a traditional brewed product, improve the product quality and enrich the product taste.

The invention discloses clone and expression of an Alpha-glucosidase (abbreviated as AGLU enzyme) gene and belongs to the fields of enzyme gene engineering and enzyme engineering. The invention obtains the aglu gene with SEQ ID NO of 1 through an Aspergillus niger (A.niger) WX-07 total DNA, and aglu cDNA uses an expression vector of plasmid pPIC9K and an expression parasitifer of pichia stipitis (P.pastoris), thus realizing extracellular dissolubility expression of the aglu gene; and the aglu cDNA totally has 2880 nucleic acid and encoding of 960 amino acid, constructs eukaryotic expression plasmid and transforms Pichia pastoris KM71 for AGLU enzyme expression. The recombinant enzyme has transnucleosidation activity, can generate isomalto oligosaccharide through maltose transnucleosidation, is applicable to the requirements of industrial application including food, feeding stuffs and medicaments and the like, and can be applied into the industrial production of isomalto oligosaccharide.

The invention provides a method for preparing functional recombinant human hemoglobin. The method comprises the following steps of culturing yeast Pichia in a culture medium and recovering human hemoglobin from an obtained culture, wherein the yeast Pichia is transformed from an expression vector of encoded human hemoglobin alpha chain DNA which is contained in the yeast Pichia under the regulation of a promoter capable of playing a role, as well as an expression vector of encoded human hemoglobin beta chain DNA which is contained in the yeast Pichia under the regulation of another promoter capable of playing a role. The invention also provides an expression vector. From the upstream side in a transcription direction, the expression vector successively comprises one ethanol oxidase 1 promoter, the encoded human hemoglobin beta chain DNA, one terminator capable of playing a role in the yeast Pichia, one ethanol oxidase 1 promoter, the encoded human hemoglobin alpha chain DNA and one terminator capable of playing a role in the yeast Pichia.

The invention provides a method for fermenting ethanol by immobilized cells of Pichia stipitis, wherein the method uses the Pichia stipitis as a starting strain and replaces calcium alginate by manganese alginate gel, and the service life of immobilized yeast is obviously prolonged.

Disclosed are nucleic acid constructs comprising coding sequences operably linked to a promoter not natively associated with the coding sequence. The coding sequences encode Pichia stipitis proteins that allow recombinant strains of Saccharomyces cerevisiae expressing the protein to grow on xylose, and allow or increase uptake of xylose by Pichia stipitis or Saccharomyces cerevisiae expressing the coding sequences. Expression of the coding sequences enhances uptake of xylose and / or glucose, allowing increased ethanol or xylitol production.

The invention discloses a selective culture medium for quantitative detection of pichia kudriavzevii, and a preparation method and application thereof. Each liter of the selective culture medium comprises the following components: 20-40g of lactic acid; 20-40g of ethanol; 10-20g of peptone, 5-15g of yeast extract; 1-5g of MgSO4;0.5-1.5g of NaCl; 0.1-1g of FeSO4; 15-20g of agar, the balance being distilled water, and the natural pH is 3.0-0.5, as the selective medium is for the characteristics of the pichia kudriavzevii; wherein a high concentration of lactic acid and an amount of ethanol are added so that the pH of the selective medium is relatively low; the selective culture medium can effectively inhibit the growth of other microorganisms in the brewing process of the white spirit; the purpose of fast and accurate quantitative detection of the pichia kudriavzevii can be achieved by the selective culture medium.