40 results about "Thermobifida fusca" patented technology

The invention relates to genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and a method for constructing the same, belonging to the field of the bioengineering technology. The genetically engineered bacteria are escherichia coli BL21 (DE3) which carry two recombinant plasmids, and the recombinant plasmids are respectively plasmid pSTV28 carrying the specific genes in the Alpha-hemolysin A (hly A) pathway and plasmid pET20b(+) containing the cutinase-hly As genes. The method for constructing the genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase comprises the following steps: constructing two key recombinant plasmids and transforming the constructed recombinant plasmids in the escherichia coli BL21 (DE3) to obtain the genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase. The cutinase is produced by using the genetically engineered bacteria through culturing liquid and inducing and expressing the cutinase. The cutinase Tfu_0883 for thermophilic monospore bacteria of Thermobifida Fusca WSH03-11 is used as the report protein. The shaking flask fermentation shows that the extracellular output of the cutinase is 306U / mL which is 1.7 times of the output of the cutinase which adopts the II-type secreting pathway in the preliminary working process in the research laboratory. The cutinase is secreted and expressed efficiently.

The invention discloses a compound microbial agent for promoting fermentation of pig manure composting. The compound microbial agent is prepared from thermobifida fusca, ureibacillus thermophaericus, pantoea agglomerans, gibsonii, bacillus amyloliquefaciens, bacillus pumilus and a liquid culture medium. Compared with other treatment methods, the fermentation time of the compound microbial agent on the pig manure composting is shorter, and more obvious facilitation effect on fermentation of the pig manure composting is obtained.

The invention provides a mutant of themobifida fusca cutinase and a preparation method thereof. The mutant comprises one or more substituents adjacent to amino acid residues corresponding to the active center of cutinase of thermobifida fusca. I218A, Q132A / T101A and W195A / F249A can improve catalyzing efficiency to cotton fiber. The catalyzing efficiency of mutant I218A to keratin is improved by 50% compared with that of the original cutinase. The catalyzing efficiency of Q132A / T101A on synthetic fibre ethylene glycol terephthalate (PET) is improved by 3 times compared with the original cutinase. The three types of mutants have wide application prospect in pre-treatment of textile fiber.

The invention discloses a trehalose synthase mutant and a preparation method and application thereof and belongs to the field of genetic engineering and enzyme engineering. The 271st Gln codon near the active center of trehalose synthase of trehalose synthase of Thermobifida fusca YX is converted into the Met codon, the 220th Ile codon is converted into the Val codon, the 228th Val codon is converted into the Ile codon, the 290th Cys codon is converted into Val codon, the 294th Phe codon is converted into the Tyr codon, and the 332nd Ile codon is converted into the Phe codon, so as to respectively obtain mutants Q271M, I220V, V228I, C290V, F294T and I332F. Based on the I220V, amphimutation is carried out to obtain mutants I220V / Q271M, I220V / V228I, I220V / C290V, I220V / F294Y and I220V / I332F. The mutants can be used for improving the trehalose conversion rate prepared by trehalose synthase and have relatively high industrial value.

The invention discloses trehalose synthase with an increased maltose conversion rate, a method for preparing the trehalose synthase and application thereof, and belongs to the field of gene engineering and enzyme engineering. The method includes mutating glutamic acid at 289 sites near trehalose synthase active centers of Thermobifida fusca YX into glycine to obtain mutants E289G; mutating histidine at 295 sites into asparagine mutants H295N; mutating methionine at 344 sites into lysine mutants M344K; mutating methionine at 367 sites into leucine mutants M367L and carrying out combined mutation on the basis of the H295N to obtain mutants H295N / E289G, H295N / M344K, H295N / M367L and H295N / M344K / M367L. The trehalose synthase, the method and the application have the advantages that by the aid of the mutants, the conversion efficiency of trehalose prepared from the trehalose synthase still can be prevented from being severely affected even if substrates contain certain glucose, and accordingly the trehalose synthase, the method and the application have high industrial values.

The invention relates to high temperature cutinase and a gene sequence thereof, which belongs to the fields of enzyme gene engineering and enzyme engineering. The invention obtains a high temperaturecutinase gene SEQ ID NO:1 from thermobifida fusca WSH03-11 total DNA, the cutinase gene takes a plasmid pET 20b (+) as an expression vector and takes E. coli BL21 Rosetta (DE3)PlysS as an expression host to realize high level expression of the high temperature cutinase gene, the whole length of the cutinase gene is 783 nucleotides, the code is 261 amino acids, a prokaryotic expression plasmid is constructed, and escherichia coli expression cutinase is transformed. Recombinase has the activity of the cutinase. The most suitable temperature for the high temperature cutinase is 60DEG C, the mostsuitable pH value is 8, the high temperature cutinase has vey high thermal stability under the temperature of 60 DEG C, and the half-life is 45 hours. The high temperature cutinase is very suitable for the demands of being applied in the textile industry.

The invention provides a method for promoting the utilization of a xylan-containing substrate by thermobifida fusca. The thermobifida fusca generates xylanase to decompose xylan to produce xylo-oligosaccharide and xylose. The method comprises a step of controlling the content of xylo-oligosaccharide in a reaction system. Besides, the invention further provides a method for improving the utilization efficiency of a xylan-containing substrate by the thermobifida fusca. The method includes a step of culturing the thermobifida fusca by utilizing a substrate containing xylan; and the thermobifida fusca can produce xylanase and cellulase. And the method further comprises a step of adding thermomyces lanoginosus in the culture process.

The present invention provides a method of increasing extracellular secretion of secretory proteins by co-expressing the secretory proteins with a mature cutinase. Cutinase can improve the permeability of E. coli cell membrane without destroying the membrane, and thus facilitate cross-membrane transfer of the secretory proteins co-expressed in E. coli. Increased extracellular secretion of target proteins can shorten cell culture time, reduce the formation of inclusion bodies and increase production of target proteins.

The invention relates to heat resistant exogenous enzyme, and the coding gene and the expression formula thereof, and belongs to the biological engineering technical technology. The invention provides the bacteria exogenous enzyme different from the accurate exogenous enzyme, and the coding gene and the expression formula thereof. The exogenous enzyme comprises the abstraction of Thermobifida fusca WSH03-11 genomic DNA, the design of primer, and the gene of the heat resistant exogenous enzyme obtained by PCR augmenting, the exogenous enzyme is provided with SEQ ID NO: 1 showed nucleotide sequence, the total length is 906 nucleotides, the coding is 301 amino acids, so as to take plasmid pET20b (+) as the expression carrier, and to take E.coli BL21 Rosetta (DE3) PlysS as expression host, and the high-efficient expression of the heat resistant exogenous enzyme agene can be realized. The thermal stability of the exogenous enzyme is good, and the invention which has good hydrolytic action to cutin, triglyceride and various solubility synthesized greases can be widely used in industries such as spinning and cleaning agent.

The invention relates to genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and a method for constructing the same, belonging to the field of the bioengineering technology. The genetically engineered bacteria are escherichia coli BL21 (DE3) which carry two recombinant plasmids, and the recombinant plasmids are respectively plasmid pSTV28 carrying the specific genes in the Alpha-hemolysin A (hly A) pathway and plasmid pET20b(+) containing the cutinase-hly As genes. The method for constructing the genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase comprises the following steps: constructing two key recombinant plasmids and transforming the constructed recombinant plasmids in the escherichia coli BL21 (DE3) to obtain the genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase. The cutinase is produced by using the genetically engineered bacteria through culturing liquid and inducing and expressing the cutinase. The cutinase Tfu_0883 for thermophilic monospore bacteria of Thermobifida Fusca WSH03-11 is used as the report protein. The shaking flask fermentation shows that the extracellular output of the cutinase is 306U / mL which is 1.7 times of the output of the cutinase which adopts the II-type secreting pathway in the preliminary working process in the research laboratory. The cutinase is secreted and expressed efficiently.