142 results about "Hansenula polymorpha" patented technology

The invention provides a compound microbial agent for urban sewage treatment. The compound microbial agent comprises a compound thallus which is compounded by bacillus subtilis, bacillus licheniformis, lactobacillus plantarum, hansenula polymorpha and saccharomyces cerevisiae. The compound microbial agent provided by the invention can be used for reasonably combining various strains capable of forming a dominant bacterial community, has a good degrading effect to macromolecules and degradation-resistant organic matters by being added into a sewage treatment system in a certain proportion, and can be used for effectively improving the biodegradability of sewage, reducing CODcr and BOD5 of urban sewage with poor biodegradability and purifying the water quality.

The invention relates to a human papilloma virus 18 L1 (HPV18L1) polynucleotide sequence and its expression vector, host cell and use and especially relates to an amino acid sequence of an HPV L1 capsid protein, a synthetic nucleotide sequence for coding the amino acid sequence, a recombinant expression vector containing the synthetic nucleotide sequence, and a hansenula polymorpha expression host strain containing the synthetic nucleotide sequence. The invention also relates to a use of an HPV18L1 protein composed of the amino acid sequence and derivatives of the amino acid sequence in preparation of vaccines. Through modification of a nucleotide sequence of a gene of an HPV18L1 wide-type virus, a recombinant HPV18L1 capsid protein can be highly expressed in a hansenula polymorpha expression system and thus hansenula polymorpha expression system-based industrial production of an HPV18L1 capsid protein is realized. Compared with the existing eukaryotic expression systems, the HPV18L1 polynucleotide sequence and its expression vector and host cell have advantages of higher yield and lower cost.

The invention belongs to the biotechnology field, and relates to a vaccine for controlling the persistent infection of hepatitis B virus. According to the invention, a Hansenula polymorpha cell that is heat inactivated and expresses hepatitis B surface antigen is adopted as the hepatitis B vaccine, wherein the HBsAg is an antigen part of the vaccine and the Hansenula polymorpha cell is an adjuvant part of the vaccine. Animal immunity experiment shows that: the vaccine can induce the aggregation of immune cells to the spleen, promote the maturation of DCs, and both induce Th1 immune response and enhance Th2 immune response in mice, including total IgG, IgG1, IgG2a, specific CTL activity, and the production ability of antigen-specific IFN-gama. The invention can make up for the deficiency of the impossibility of inducing Th1 immune response for traditional vaccines that take aluminium hydroxide as an adjuvant, and is helpful to the control of the persistent infection of hepatitis B virus.

The invention relates to the technical field of wine production, in particular to hansenula polymorpha and application thereof in wine making. The invention provides the hansenula polymorpha with the preservation number being CGMCC (China General Microbiological Culture Collection Center) NO.13712 and the application thereof in the wine making. The strain of bacteria and saccharomyces cerevisiae are jointly applied in wine fermentation, so that in obtained wine, the content of aroma substances are greatly increased, the aroma quality of the wine is improved, the problem that the aroma is single due to pure fermentation of the saccharomyces cerevisiae is solved, and the wine aroma is more complex and layered.

The invention relates to an HPV16L1 polynucleotide sequence, and an expression vector, a host cell and application of the HPV16L1 polynucleotide sequence. The HPV16L1 polynucleotide sequence comprises an amino acid sequence of recombinant human papillomavirus (HPV) L1 capsid protein, a synthetic nucleotide sequence coding the amino acid sequence, and a recombinant expression vector and a hansenula polymorpha expression host strain comprising the nucleotide sequence. The invention also relates to the application of HPV16L1 protein consisting of the amino acid sequence and derivatives of the HPV16L1 polynucleotide sequence in preparing vaccine. According to the invention, by transforming the nucleotide sequence of HPV16L1 wild type virogene, the recombinant HPV16L1 capsid protein in a hansenula polymorpha system is efficiently expressed, and the HPV16L1 capsid protein can be industrially produced by using the hansenula polymorpha expression system; and compared with the conventional other eukaryotic expression systems, the hansenula polymorpha expression system has the advantages of high yield, low cost and the like.

The invention provides a recombinant DNA sequence, hansenula polymorpha, a preparation method for hepatitis B surface antigen, and a hepatitis B vaccine. The recombinant DNA sequence is obtained by codon optimization of coding genes of the hepatitis B virus surface antigen according to codon usage frequency of the hansenula polymorpha. The invention also provides the hansenula polymorpha comprising the recombinant DNA sequence, a method for preparing adr sub-type hepatitis B surface antigen by using the recombinant DNA sequence, and the hepatitis B vaccine. The adr sub-type hepatitis B surface antigen has high expression level of the recombinant DNA sequence. The recombinant hansenula polymorpha is fast in growth speed, has high HBsAg yield, can be fermented in high cell density by using a cheap chemically synthetic medium, has low fermentation contamination rate and is beneficial to large-scale production; and HBsAg adr vaccine provided by the invention has high trend of Th1 and Th2 type cellullar immunologic response.

The invention discloses a composite aroma-producing microorganism as well as a preparation method and application thereof. The composite aroma-producing microorganism is formed by compounding bacillus subtilis, trichoderma reesei, aspergillus niger and hansenula polymorpha at a wet thallus mass ratio of 5:2:1:1. The composite aroma-producing microorganism disclosed by the invention can be used for preparing a filter material for a cigarette filter and greatly improving the aroma and taste of the cigarettes, and has good application value in the cigarette processing production.

The invention discloses a composite flour product fermenting agent and a preparation method thereof, and relates to the fermenting agent and the preparation method thereof, which solve the problems that the fermentation speed and production efficiency are low in the preparation of the flour product by the conventional fermenting agent and the nutritive value and flavor of the conventional fermented flour product fermented by using an active dry yeast fermenting agent are not as good as those of the flour product obtained by the traditional fermentation. The composite flour product fermenting agent is prepared from lactobacillus acidophilus, lactobacillus paracasei, beer yeast and abnormal hansenula polymorpha yeast. The method comprises the following steps of: 1, preparing seed solution; 2, expanding culture solution; 3, centrifugally collecting strains and washing; and 4, mixing the four strains, adding a cryoprotective agent for pre-freezing and drying to obtain the fermenting agent. The composite flour product fermenting agent prepared by the method can restore the conventional flavor and nutritive value of the flour products, has certain fermenting power and high fermenting speed, is obviously superior to the conventional fermenting agent, improves the production efficiency and can improve the situation of single strain content of the conventional fermenting agent.

The invention relates to a compound microorganism deodorization fungicide used for waste compression stations, waste transfer stations, public toilets and livestock and poultry farms. The microorganism deodorization fungicide contains abnormal Wacker hansenula polymorpha of which the biological preservation number is ATCC (American Type Culture Collection) 18205, Guilliermond candida of which thebiological preservation number is ACCC (Agricultural Culture Collection of China) 20232, plant lactobacillus of which the biological preservation number is ACCC 10241 and lactobacillus acidophilus ofwhich the biological preservation number is ACCC 00159. The microorganism deodorization fungicide can inhibit a situation that organic matters are subjected to putrefactive fermentation to generate malodorous gas, meanwhile, malodorous substances including NH3, ammonium sulfate, methyl mercaptan and the like can be adsorbed and utilized, and the compound microorganism deodorization fungicide has an obvious deodorization effect. The invention also discloses a preparation method for the microorganism deodorization fungicide, and the application of the microorganism deodorization fungicide.

The invention provides a hansenula polymorpha expression system, a hansenula polymorpha construction method and application of hansenula polymorpha. The expression system contains uracil auxotroph hansenula polymorpha AU-0501, of which the preservation number is CGMCC NO.7013. The uracil auxotroph hansenula polymorpha AU-0501 provided by the invention has the advantages of definite mutation site, low reverse mutation frequency, good hereditary stability, high biological expression quantity and the like, plays a significant role in researching and producing gene engineering vaccine, has the advantages of higher yield and low cost as compared with other eukaryotic expression systems adopted at present. The invention further provides an expression vector applied to the hansenula polymorpha expression system and a construction method of the expression vector. Two or more genes can be expressed simultaneously through the expression vector. Two target genes can be expressed in a hansenula polymorpha auxotroph cell at a high level without interference.

The invention discloses aroma-enhancing puffed tobacco stem particles and a preparation method and application thereof. The preparation method of the aroma-enhancing puffed tobacco stem particles comprises the steps that a micropore structure is formed in each tobacco stem, and the tobacco stems are dried and smashed after being fixed and formed to form particles; the tobacco stem particles are inoculated with a compound aroma-generating microorganism, fermental cultivation and drying are carried out on the tobacco stem particles and the compound aroma-generating microorganism, and then the aroma-enhancing puffed tobacco stem particles can be obtained. The compound aroma-generating microorganism is formed by compounding bacillus subtilis, Trichoderma reesei, Aspergillus niger and Hansenula polymorpha according to the wet-cell mass ratio of 5:2:1:1. According to the aroma-enhancing puffed tobacco stem particles and the preparation method and application thereof, the tobacco stems serve as raw materials to be processed to obtain the puffed tobacco stem particles, then the compound aroma-generating microorganism is added, and serving as a filter material of a filter tip, the finally-obtained aroma-enhancing puffed tobacco stem particles have the advantages of being high in filter efficiency, strong in adsorption capacity, free of offensive odors and consistent with tobacco in aroma and have good application prospect.

The invention belongs to the technical field of wastewater environmental-protection treatment, and particularly relates to a microorganism water treatment agent for heavy metal organic wastewater. The microorganism water treatment agent is prepared from the following raw materials: oyster shells, potassium feldspar, olivine, talc, serpentine, chlorite, attapulgite, ammonium molybdate modified bentonite, diatomaceous earth, aluminium polychlorid, magnesium hydrate, gram-negative bacteria, Bacillus mojavensis, polyacrylamide, tartaric acid, malic acid, citric acid, chitosan, ethylene glycol diglycidyl ether, epoxy chloropropane, thiosemicarbazide cellulose, corn starch, calcium alginate, hansenula polymorpha, candida, trailing plant, fagus longipetiolata sawdust, pinus sylvestris sawdust, walnut shell, lupulus, dehalogenase, horseradish peroxidase, urease and propionibacterium pentosaceum. A precipitate (alum floc) produced after treatment with the microorganism water treatment agent has good strippability, no solid particle is released into water basically, and the microorganism water treatment agent has rapid action speed and stable treatment effect, so that the adsorption time is usually not required to be prolonged during use.

This invention relates to a method for separating and purifying recombinant hepatitis B virus surface antigen expressed in Hansenula polymorpha. The method comprises: (1) crushing culture solution of recombinant B virus surface antigen expressed in Hansenula polymorpha till 50-90% cells are crushed; (2) centrifuging to remove cell debris, adjusting pH value and electrical conductivity, performing anion exchange chromatography, and collecting the eluates with activity higher than 20%; (3) incorporating the eluates, adjusting the pH value and electrical conductivity, and performing hydrophobic chromatography; (4) concentrating the eluate with ultrafiltration membrane; (5) separating by a gel filtration column to obtain recombinant B virus surface antigen expressed in Hansenula polymorpha with purity higher than 99%. The method has such advantages as high recovery rate, high product purity, few steps, and short time.

The invention discloses a semi-fermented low-salt fish leisure food, which is prepared by the following steps: (1) inoculating the cultured hansenula polymorpha and aspergillus oryzae into fresh fish sections for primary fermentation, and standing for 6-10hours; (2) placing the primarily fermented fish sections into a chain type drying oven for segmented temperature-variable drying, wherein the drying time is controlled for 30-60 minutes, and the moisture content of fish is controlled at 15-18%; and (3) adding the activated lactobacillus acidophilus and streptococcus lactis fermentation liquor into the dried fish sections for secondary fermentation, and fermenting for 30-50 hours at 10-15 DEG C; discharging liquid, standing and clarifying. No edible salt is added, primary fermentation is combined with the segmented temperature-variable drying, and water in fish sections can be evenly evaporated through the temperature change so as to form a porous cavity, so that the fish has a loosened structure, the texture of the fish can be improved and the secondary fermentation is facilitated, and the fermentation efficiency can be increased. Unique taste and mouth feeling can be generated through secondary fermentation, and the nutritional value and flavor of the fish products can be enhanced.

The invention provides a polymorphic hansenula polymorpha mutant strain and application of the polymorphic hansenula polymorpha mutant strain in glutathione biosynthesis. Firstly, low-energy ions N+ serve as a mutagenesis source, mutagenesis treatment for Hansenula polymorpha DL-1 is achieved, a glutathione (GSH) high-producing strain (DL-18) is obtained, the temperature remains 37 DEG C, the speed remains 180 r / min, shake flask fermentation remains for 42 hours, the dry cell weight (DCW) in end products is 7.04 g / L, the total quantity of the GSH is 420.46 mg / L, and the DCW in the end products and the total quantity of the GSH are respectively improved by 1.73 times and 1.94 times compared with the original mutant strain. Hereditary stability of the DL-18 is detected through subculture, yatsushiro is cultivated, content changes of the GSH are not significant, and therefore, the hereditary stability of the mutant strain is high.

The invention aims to provide a deodorized fermentation liquor of excrements of livestocks and a preparation method thereof. The deodorized fermentation liquor of excrements of livestocks comprises the following components in percentage by weight as follows: 10-18% of cane molasses, 0.5-1.8% of milt paste, 10-20% of starch, 0.6-1.5% of coprophilous lactobacillus, thermophilic streptococcus, saccharomyces cerevisiae, abnormal Hansenula polymorpha and anaerobic cellulose decomposition bacteria seed liquor, and the balance of purified water. The deodorized fermentation liquor of excrements of livestocks provided by the invention has the beneficial effects that under the high temperature anaerobic condition, cellulose is converted into organic acid and alcohol and then into methane and carbon dioxide by means of the anaerobic cellulose decomposition bacteria, so that odor of excrements of livestocks are thoroughly overcome; strains are skillfully combined with special process fermentation to increase organic matters in soil and indispensable nutrients for crops, so that the quality of crops is improved, and both production and income are increased.

The invention provides a hansenula polymorpha recombination strain and application thereof in the biosynthesis of gentiopicroside. A recombination saccharomycete used for producing gentiopicroside is obtained by a method for conversion of low energy ion implantation mediated gentiana macrophylla genome total DNA in the saccharomycete, and is named as hansenula polymorpha of which the preservation number is CGMCC.No.8998. The hansenula polymorpha recombination strain and the application realize the technical breakthrough in the aspect of using the recombination saccharomycete to be subjected to biological fermentation to produce gentiopicroside, solves the source shortage of gentiopicroside, protects the gentianaceae plant resources of our country, and provides a new way for sustainable development of the gentianaceae plant resources.

The present invention provides EV71 virus-like particles and a preparation method and application thereof. The method comprises: connecting a P1 protein gene and a 3CD protease gene of an EV71 virus with a PMV plasmid to construct a PMV-P1-3CD recombinant expression plasmid; then transforming a Hansenula polymorpha AU-0501 expression strain with the PMV-P1-3CD recombinant expression plasmid to obtain an AU-PMV-P1-3CD recombinant expression strain; fermenting and culturing the recombinant expression strain, and inducing the recombinant expression strain to express the EV71 virus-like particle protein with methanol; centrifuging and collecting mycelia for homogeneous breakage at a high pressure; and purifying the supernatant through ion-exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography, so as to obtain EV71 virus-like particles.

The invention relates to a double auxotrophic yeast, wherein the yeast is Hansenula polymorpha, and the orotic glycoside-5-phosphate decarboxylase gene and the beta-isopropyl malate dehydrogenase gene of the Hansenula polymorpha are blocked. The invention also relates to a preparation method of the double auxotrophic yeast. In addition, the invention also provides a DNA sequence and a recombinant vector used to prepared the double auxotrophic yeast. The double auxotrophic yeast of the invention has the advantages of low reverse mutation, high genetic stability, high biomass, and the like, and plays an important role in genetic engineering vaccine production; for example, HPV16-type L1 and 58L2 protein have double expression with high efficiency in the yeast.

The invention discloses hansenula polymorpha with a double selection marker and an application thereof. The hansenula polymorpha with a double selection marker provided by the invention is uracil-synthesized and tryptophan-synthesized double-deficient hansenula polymorpha HUT-31 with a preservation number of CGMCC No.4778. The strain has a uracil and tryptophan double-auxotrophic selection marker, maintains the physiological biochemical characteristics of wild-type strains, facilitates the culture of recombinant strains and the high-level expression of heterologous protein; not only recombinant strains can be selected easily and rapidly, but also the double selection marker can increase the copy number of target genes integrated on a chromosome, can increase the expression level of targetprotein, and has important industrial application value. Meanwhile, the double selection marker is also applicable to the polygene genetic modification of hansenula polymorpha in functional genomics,synthetic biology, and metabolic engineering.

The invention relates to a method for purifying recombined hansenula polymorpha hepatitis E virus-like particles and application. The method comprises the following steps: cultivating and fermenting working seed lot recombined hepatitis E vaccine engineering strains, and collecting a fermented culture; sequentially performing cell disruption, clarification, ultra-filtration, silica gel adsorption, ultra-filtration and concentration liquid change, chromatographic purification, desalination, disinfection and filtration on the culture to obtain the purified recombined hansenula polymorpha hepatitis E virus-like particles. The size of the purified recombined hansenula polymorpha hepatitis E virus-like particles is in accordance with the theoretical size of natural hepatitis E virus particles and is 27-34nm, a gene sequence is in accordance with a theoretical gene sequence, the expressed foreign protein molecular weight is in accordance with the expected target protein size, and the recombined hansenula polymorpha hepatitis E virus-like particles are good in immunogenicity. The invention also discloses the application of the recombined hansenula polymorpha hepatitis E virus-like particles in preparation of vaccine. The recombined hansenula polymorpha hepatitis E virus-like particles are mixed with an aluminum adjuvant to form the vaccine. An animal immunogenicity detection result shows that the vaccine prepared from the recombined hansenula polymorpha hepatitis E virus-like particles is good in immunogenicity.

The invention discloses multi-strain compound fermentation type jujube wine and a brewing method thereof. The jujube wine obtained by carrying out compound fermentation on candida, hansenula polymorpha, pichia pastoris, kloeckera and saccharomyces cerevisiae is obviously superior to jujube wine prepared by fermenting single saccharomyces cerevisiae in flavor and taste. In the invention, non-saccharomyces generate aromatic matters, i.e. higher alcohol, lower fatty acids, esters, and the like, and special flavor components so that the jujube wine is obviously improved in flavor characteristics and has smoother taste and stronger flavor, thereby overcoming the defects of single saccharomyces fermentation and generating positive effects on the total flavor of the jujube wine; and in addition,the jujube wine has the advantages of full wine body, rose color, clarification, brightness, mellow and smooth taste and pure and elegant jujube aroma and bouquet.

The invention relates to the field of genetic engineering, particularly to a method for realizing high expression of CP4 (cysteine proteinase 4)-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) fusion protein gene in a constitutive manner in Hansenula polymorpha cells by using a composite enhancer. The method comprises the following steps: (1) improving the yield of codon-optimized CP4-EPSPS fusion protein gene in Hansenula polymorpha by using the composite enhancer; (2) regulating and controlling CP4-EPSPS fusion protein gene to express in a constitutive manner in polytype Hansenula by adopting Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter; (3) fermenting optimized Hansenula polymorpha; and (4) rapidly purifying the recombinant heterologous protein by employing nanofiltration and nickel column affinity chromatography. The CP4-EPSPS recombinant fusion protein prepared by the method can serve as an alternative protein standard substance in the field of study on the origin of protein and the like.

The invention discloses a method for efficiently expressing and producing recombinant protein of T4 lysozyme in a recombinant hansenula polymorpha cell in a constitutive mode. The method comprises the following steps: 1) increasing the biological output of T4 lysozyme genes in a eukaryotic expression system, namely hansenula polymorpha cells, by using the T4 lysozyme gene with optimized codon; 2) taking a coded sequence of a plasmalemma ATP enzyme nucleotide derived from the hansenula polymorpha as a homologous sequence for completely integrating exogenous plasmid into the hansenula polymorpha genome; 3) adjusting and controlling the high-efficiency expression of the T4 lysozyme gene in the hansenula polymorpha in the constitutive mode by using pichiapastoris glyceraldehyde-3-phosphate dehydrogenase promoter; and 4) providing a specific hansenula polymorpha engineering bacteria fermentation culture and growth condition so as to improve the biological output of recombinant protein of T4 lysozyme and quickly extract the recombinant exogenous protein. The recombinant protein of T4 lysozyme finally prepared by the method has biological activity and can be widely applied in fields such as medicinal treatment, foods, feeds, scientific research and the like.

The invention discloses a composite microbial deodorizer and a preparation method thereof. According to the invention, thiobacillus ferrooxidans, rhizobium sludge, bacillus amyloliquefaciens, rhodopseudomonas palustris, pseudomonas stutzeri, bacillus coagulans and hansenula polymorpha are selected for use; liquid fermentation is performed respectively, and then compounding is performed in proportion; then linalool is added in proportion and the substances are fully mixed to prepare a composite microbial deodorizer capable of effectively expelling flies and insects, inhibiting growth and propagation of harmful flora and reducing and removing foul gas in the environment. The thiobacillus ferrooxidans (CFU / g) in the product is greater than or equal to 2.95*10<8>; the rhizobium sludge (CFU / g)is greater than or equal to 1.05*10<8>; the bacillus amyloliquefaciens (CFU / g) is greater than or equal to 1.73*10<8>; the rhodopseudomonas palustris (CFU / g) is larger than or equal to 2.55*10<8>; thepseudomonas stutzeri (CFU / g) is larger than or equal to 1.15*10<8>; the bacillus coagulans (CFU / g) is larger than or equal to 2.86*10<8>; the hansenula anomala (CFU / g) is larger than or equal to 2.97*10<8>; and the total amount of organic acids is larger than or equal to 25.8 g / L. The product provided by the invention has an obvious deodorization effect, the NH3 removal rate is greater than or equal to 78.14%, the H2S removal rate is greater than or equal to 71.64%, the odor concentration elimination rate is greater than or equal to 84.95%, and the mosquito and fly expelling rate is greater than or equal to 74.16%.

The invention discloses a method for preparing a flavored product from yellow water and liquor-making microbes, and belongs to the field of pickled and fermented foods. The method comprises the following steps that processed loaches, barley flour, the yellow water, edible alcohol and salt are mixed to be uniform, and loach and flour mixed liquor is obtained; saccharomycopsis fibuligera, saccharomyces cerevisiae and zygosaccharomyces rouxii are inoculated into the loach and flour mixed liquor for fermentation, and first-time fermented liquor is obtained; torulopsis mogii, hansenula polymorpha and candida albicans are inoculated into the first-time fermented liquor for fermentation, and second-time fermented liquor is obtained; corn flour and pepper paste are added into the second-time fermented liquor, bacillus licheniformis is inoculated into the mixture for fermentation, and third-time fermented liquor is obtained; the third-time fermented liquor is put into a pottery jar for 12-16 months, and the flavored product which is slightly sweet, delicious and harmonious in fragrance is obtained. According to the method for preparing the flavored product from the yellow water and the liquor-making microbes, the use value of the loaches and the yellow water is improved, and the delicious flavor of the product is improved through addition of auxiliaries and fermentation of various microbes.

The invention provides a municipal refuse treatment method. As the high grease decomposing capability, lipase can provide nutrient substances for various microorganisms. When alcaligenes xylosoxidans subsp.denitrificans, bacillus amyloliquefaciens, depubyomyces hansenii hansenula polymorpha variants, diamond issatchenkia, keratinophilic aleurisma carnis and Pasteur nocardia are used in combination, nutrient substances can be fully and reasonably utilized; especially, after lipase is transferred into yeast, the decomposition capability on grease is greatly improved, and thus the capability and efficiency of treating municipal refuse are greatly improved; besides, the whole treatment cycle is shortened, and high application and popularization value is achieved.