36 results about "Pichia garciniae" patented technology

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

The invention discloses a recombinant bacterium capable of high efficiently secreting and expressing natto kinase, and the recombinant bacterium can prominently increases the output of natto kinase. The recombinant bacterium is a recombinant pichia pastoris containing at least two copies of a natto kinase Pro-NK gene and a Haclp regulatory factor gene. After the recombinant bacteria carry out fermentation for 96 hours in a shake flask, the enzyme activity of the natto kinase can reach 470 IU/mL.

The invention discloses a gene sequence of Lygus lucorum polygalacturonase (PG) and a method for preparing the Lygus lucorum polygalacturonase. The method comprises the following steps of: extracting total RNA (ribonucleic acid) of Lygus lucorum; designing a primer according to the conserved sequences of the Lygus lucorum polygalacturonase; amplifying by utilizing an RT-PCR (reverse transcriptase-polymerase chain reaction) method to obtain homologous gene sequences of three PGs; obtaining 5' and 3' unknown sequences by utilizing an RACE experiment; and finally, respectively carrying out the recombinant expression, purification and property analysis of the Lygus lucorum polygalacturonase by utilizing an Escherichia coli expression system and a Pichia pastoris expression system. By using the method, the obtaining problem of the gene sequence of the polygalacturonase of the Lygus lucorum is solved firstly, and the recombinant expression problem of the polygalacturonase derived from insects is also solved for the first time. The gene disclosed by the invention has the application prospect in the aspects of food (fruit juice squeezing), oil material extraction, traditional Chinese medicinal material treatment, paper-making industry, oligo-pectin health care products and the like. In addition, an inhibitor aiming at the Lygus lucorum polygalacturonase can be used as a policy for preventing and controlling a piercing-sucking type pest, thereby achieving the prevention and control on the target pest.

The invention provides a recombined pichia pastoris strain for commonly expressing glucamylase and alpha-amylase and a construction method of the recombined pichia pastoris strain as well as a mixed enzyme preparation prepared from the recombined pichia pastoris strain. The recombined pichia pastoris strain contains glucamylase genes from tiny mold funli and alpha-amylase genes at the same time; and gene sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO.2. According to the mixed enzyme preparation prepared from the recombined strain, the saccharifying enzyme activity is improved by 79% when being compared with that of a strain which only carries the glucamylase genes, and the starch liquefacation enzyme activity is improved by 183% when being compared with that of a strain which only carries the alpha-amylase genes. The optimum operation temperatures and the optimum operation pH (Potential of Hydrogen) of the two enzymes are close; and the two enzymes have the synergistic promoting effect in a starch hydrolytic process, so that the application potential and advantages are great.

The invention relates to a Pichia pastoris engineering strain GS-TA-LGH expressing a thermoascus aurantiacus var. levisporus thermostable lipase gene lgh. According to the invention, the lipase gene lgh is obtained from thermoascus aurantiacus var. levisporus through RT-PCR (reverse transcription-polymerase chain reaction), RACE (rapid-amplification of cDNA (complementary deoxyribonucleic acid) ends) and other methods, an expression vector pPIC9K/lgh is constructed and introduced into Pichia pastoris GS115, and then the Pichia pastoris engineering strain GS-TA-LGH expressing the lipase is screened out from the Pichia pastoris GS115. The enzyme activity of the lipase of the engineering strain can achieve 19.92U/mg, when heat insulation is performed on the enzyme at the temperature of 50 DEG C for 60 minutes, the loss of the activity can be avoided; and when the heat insulation is performed at the temperature of 60 DEG C for 60 minutes, the enzyme activity can still achieve 66%. The Pichia pastoris engineering strain GS-TA-LGH has higher thermal stability and has economical value and social value as the strain for producing the thermostable lipase.

The invention discloses pichia membranifaciens Y4 for controlling diseases after peach harvesting and belongs to the field of biological prevention and control of diseases after fruit harvesting. Theyeast is identified as pichia membranifaciens, and the preservation number is CCTCC NO:M 2018110. The use method comprises the following steps: during the use, cultivating the pichia membranifaciens Y4 in an NYDA culture medium at 28 DEG C for 2 to 3 days, activating, performing fermentation cultivation in the NYDB culture medium at 28 DEG C for 20 to 24 hours, repeating the activation process fortwice, centrifuging to obtain thallus, washing for twice and diluting with sterile water to prepare bacterial suspension; perforating the peaches, adding the bacterial suspension and inoculating rhizopus stolonifer spore suspension after 2 hours; and airing at a room temperature, putting into a disinfected plastic basket, sealing with a preservative film, putting into a constant-temperature culture box and storing. Soft rot morbidity and decayed diameter of juicy peaches treated by the yeast are obviously reduced. The pichia membranifaciens Y4 can be applied to prevention and control of the juicy fruit soft rot, can reduce the use of chemical sterilizers and avoids danger to people and environmental pollution caused by the use of the chemical sterilizers.

The invention discloses a VZV (varicella-zoster virus)glycoprotein E gene eukaryotic expression vector as well as a recombinant yeast strain and application thereof. The vector is a connector of alpha-gE fused gene and pPink-HC, and the alpha-gE fused gene is a gene complete sequence of VZV glycoprotein E and a fused gene alpha signal peptide. The glycoprotein E capable of successfully expressingthe VZV in an expression system of pichia pastoris discloses by the invention sets the foundation for the detection of the protein immunogenicity as well as the efficient expression of the protein inthe pichia pastoris as well as the research of the VZV vaccine.

The invention discloses a trichoderma reesei chitinase and a preparation method and applications thereof. The encoding gene of chitinase in the Trichoderma reesei chitinase is optimized according to the preference of pichia pastoris, and the optimized nucleotide sequence is shown as SEQ ID NO.2. The optimized encoding gene of chitinase is subjected to further high-efficiency secretory expression by utilizing a pichia pastoris expression system to obtain trichoderma reesei chitinase, the amino acid sequence of which is shown as SEQ ID NO.1. The obtained trichoderma reesei chitinase has higher hydrolysis activity to low-deacetylation-degree chitosan substrate, the crude enzyme liquid generated by shaking and fermentation has the hydrolysis capability of degrading 0.5g by weight of chitosan with 1mL (the content of protein is about 0.17mg), degrading of the same amount of chitosan needs about 50mg of non-specific commercial enzyme, so that the efficiency is theoretically increased by 300 times; and the trichoderma reesei chitinase and the preparation method and applications thereof have good industrial application prospects.

The invention belongs to the field of artificial synthetic gene, and particularly relates to a lipase gene COLIP, a lipase encoded by the same and gene engineering bacteria for expressing the lipase gene COLIP. The invention discloses the lipase gene COLIP having the nucleotide sequence shown in SEQIDNO.1, and the lipase encoded by the lipase gene has the amino acid sequence shown in SEQIDNO.2. Compared with the prior art, the obtained lipase gene COLIP can be ultra-efficiently expressed in pichia pastoris, and the constructed pichia pastoris gene engineering bacteria GS115 (COLIP) can be used for mass production of the lipase. Fermentation results show that under a condition of a 10 L fermentation tank, fermentation is carried out for 120 hours, the enzyme activity of the lipase in a fermentation broth can be stabilized at 45000 U/mL or more, and the protein content in the fermentation broth is stabilized at 3.2 g/L or more.

The invention discloses a pichia guilliermondii bacterium liquid biocontrol microbial agent and a method for preparing the same, and belongs to the technical field of biocontrol microbial agent preparation. The pichia guilliermondii bacterium liquid biocontrol microbial agent in a formula comprises 1*10<6>-1*10<9> CFU/mL of pichia guilliermondii, 2.5-7.5 wt% of trehalose, 0.01-0.03 wt% of glutathione, 0.01-0.03 wt% of ascorbic acid and 0.02-0.05 mol/L of phosphoric acid buffer solution with the pH (potential of hydrogen) of 6.5. The pichia guilliermondii bacterium liquid biocontrol microbial agent and the method have the advantages that fruit biocontrol effects of the pichia guilliermondii bacterium liquid biocontrol microbial agent are further verified, and as shown by results, the fruitincidence and the diameters of fruit disease speckles of nectarine treated by the Y35-1 pichia guilliermondii bacterium liquid biocontrol microbial agent are obviously reduced as compared with the fruit incidence and the diameters of fruit disease speckles of comparison groups, and are significantly different from the fruit incidence and the diameters of fruit disease speckles of the comparison groups, and occurrence and development of anthranose in nectarine and loquat fruits can be effectively inhibited by the pichia guilliermondii bacterium liquid biocontrol microbial agent which is a liquid preparation prepared by the aid of the method.

The invention discloses a synthesized duck beta-defensins-2 gene, a recombinant plasmid containing the gene as well as production methods for a recombinant yeast transformant and a recombinant duck beta-defensins-2 protein. The recombinant duck beta-defensins-2 gene refers to the cDNA (complementary Deoxyribonucleic Acid) gene sequence of a duck AvBD2 gene registered in GenBank; meanwhile, the nucleotide sequence in a mature peptide segment of the duck AvBD2 gene is transformed according to the preference of a yeast codon; the duck beta-defensins-2 gene is synthesized by using a gene synthetic method; the construction process of the recombinant plasmid containing the gene sequentially comprises the following steps of: firstly, setting an upstream primer and a downstream primer of the duckbeta-defensins-2 gene; secondly, carrying out PCR (Polymerase Chain Reaction) amplification on a duck beta-defensins-2 gene segment; and thirdly, constructing a recombinant plasmid pPICZalpha-A-AvBD2. The production method for the recombinant duck beta-defensins-2 protein comprises the following steps of: firstly, preparing and linearizing the recombinant plasmid; secondly, preparing a competent cell obtained by electrotransformation of pichia pastoris; and thirdly, electroporating the pichia pastoris. The production method for the recombinant duck beta-defensins-2 protein comprises the step of induced expression of the positive yeast transformant. Compared with the prior art, synthesized duck beta-defensins-2 gene, the recombinant plasmid and the production methods disclosed by the invention have the advantages of activities of resisting bacteria and promoting the growth, suitability for application to the livestock production, low production cost and high production efficiency.

The invention discloses a preparation method of konjaku mannan oligosaccharide and a special beta-mannase mutant adopted by the same. The beta-mannase mutant provided by the invention is from rhizomucor miehei, has the advantages of high stability, high enzymatic specific activity and the like and has very high application value in the industries of foods, feeds and the like. Engineering bacteriaformed by importing the beta-mannase mutant into pichia pastoris are fermented under a high density in a 5L fermentation tank, and the enzymatic activity of fermented liquid can reach 72,600U/mL (protein content is 9.1mg/mL). The beta-mannase mutant is used for hydrolyzing konjaku flour to prepare the konjaku mannan oligosaccharide; mainly mannan oligosaccharide with a polymerization degree beingbetween 2 and 6 is in hydrolysate; a konjaku hydrolysis rate is 90.2%; reducing sugar yield is 69.9%. The invention provides important bases for preparation of the konjaku mannan oligosaccharide by utilizing the konjaku flour.

A method of fermenting coffee beans with a Pichia yeast strain.

The invention discloses a Pichia pastoris CH01, a fermenting cultivating method thereof and an application. The preservation number of the Pichia pastoris CH01 is CGMCC No.4273. The fermenting cultivating method of the Pichia pastoris CH01 comprises the steps of: 1) inoculating activated Pichia pastoris CH01 into the frequently used fermenting culture solution of the Pichia pastoris CH01 so as tocultivate seed solution and 2) inoculating the seed solution obtained in the step 1) to the frequently used fermentation medium of the Pichia pastoris CH01 so as to aerobically culture and obtain thePichia pastoris CH01 solution. The Pichia pastoris CH01 has short fermentation period, is simple in preparation process, and has reliable industrial production. The invention has wide application prospect in preventing and treating potato late blight and controlling the postharvest diseases of fresh fruit and vegetables.

The invention discloses a method for catalyzing and synthesizing sucrose stearate through yeast show lipase, comprising the following steps of: dissolving sucrose and stearic acid in an organic solvent; adding the yeast show lipase; reacting at 50-60 degrees centigrade for 10-14 h; separating and purifying to obtain the sucrose stearate; transforming linearly treated recombinant plasmids into pichia pastoris (Pichia pastoris) GS115; inoculating the obtained transformant into a BMMY (buffered methanol-complex medium) culture medium; after inducing and culturing for 72-144 h, centrifugally collecting thallus; and washing, biologically imprinting, freezing and drying the thallus so as to obtain the yeast show lipase. By showing the lipase outside cells, the sucrose stearate is catalyzed and synthesized through the lipase preparation. By means of the method disclosed by the invention, the transformation efficiency is improved; furthermore, the reaction time is shortened; and the production cost is reduced.

The invention relates to a delta 4-fatty acid desaturase gene originated from marine microalgae isochrysis sphaerica and a cloning method thereof. The full-length delta 4-fatty acid desaturase gene isfad 4 is obtained by the following steps of: taking the isochrysis sphaerica as a material; designing degenerate primers and amplifying a gene core sequence; and amplifying the full-length delta 4-fatty acid desaturase gene isfad 4 by using a genome walking method. Heterologous expression of the isfad 4 in pichia pastoris proves the 4-fatty acid desaturase function of IsFAD 4.

The invention relates to a fermentation control process for Mut<s> type recombinant Pichia pastoris. Specifically, the invention relates to a method for determining whether methanol is excess or insufficient in the process of fermentation of Mut<s> type Pichia pastoris. The method comprises the following steps: adding methanol into fermentation broth every 3 to 18 h in the fermentation process when feeding at a basic methanol supplementation rate of 0.8 to 1.8 g/L/h is carried out, wherein 1.0 to 2.0 g of methanol is added for each L of the fermentation broth; and detecting the changes of DO and OUR of the fermentation broth in a designated period of time after addition of the methanol relative to the DO and OUR of the fermentation broth before addition of the methanol so as to determine whether the methanol in the fermentation broth is excess or insufficient. Compared with control with conventional processes, the fermentation control process provided by the invention enables the output of heterologous protein to be increased by 2 to 5 times.

The invention discloses expression and application of thymosin-repeated protein Cq-TRP1 resisting WSSV (White Spot Syndrome Virus) infection in pichia pastoris, relating to cheraxquadricarinatus thymosin-repeated protein. Cheraxquadricarinatus thymosin is named Cq-TRP1. According to multiple clone sites of a pPICZaA carrier, a specific upstream primer F1 and a downstream primer R1 for amplifying and encoding a Cq-TRP1 gene ORF are designed, and an EcoR I restriction enzyme cutting site is added at a 5 end of the upstream primer F1; an XbaI restriction enzyme cutting site, a termination codon and a basic group encoding His-tag are added at a 5 end of the downstream primer R1. The Cq-TRP1 gene is connected to an eukaryotic expression vector pPICZaA, so as to build a pPICZaA-Cq-TRP1 eukaryotic expression vector; the obtained eukaryotic recombined expression vector is imported into the pichia pastoris for induction expression, so as to acquire an expression product; and then dialysis and affinity chromatography are performed on the obtained expression product in sequence.