Mutant thermophilic organisms that consume a variety of biomass derived substrates are disclosed herein. Strains of Thermoanaerobacterium saccharolyticum with acetate kinase and phosphotransacetylase expression eliminated are disclosed herein. Further, strain ALK1 has been engineered by site directed homologous recombination to knockout both acetic acid and lactic acid production. Continuous culture involving a substrate concentration challenge lead to evolution of ALK1, and formation of a more robust strain designated ALK2. The organisms may be utilized for example in thermophilic SSF and SSCF reactions performed at temperatures that are optimal for cellulase activity to produce near theoretical ethanol yields without expressing pyruvate decarboxylase.
A Clostridium thermocellum thermostable cellulaseenzyme with both endocellulase activity and exocellulase activity that is able to degrade cellulose in the absence of scaffolding and other cellulosomic proteins is provided. The use of the enzyme to degrade cellulosic materials to soluble sugars is also provided.
The invention discloses a straw-decomposing inoculant which comprises the following active components: bacillus subtilis, aspergillus niger, trichoderma viride, phanerochaete chrysosporimn and geobacillus stearothermophilus. In the straw-decomposing inoculant, the total content of effective viable bacteria is (1-2)* 10<8> / ml. With adoption of the straw-decomposing inoculant, the fertilizer prepared from straws can be rapidly degraded, and the normal compostfermentation is performed in advance for at least 15 days. When the straw organic fertilizer prepared from the straw-decomposing inoculant is used as the base fertilizer, alkali-hydrolyzale nitrogen, rapid available phosphorus, potassium and organic contents in the soil are higher than that in the soil in which straws are directly utilized and no fertilizer is applied, and the urease activity and cellulase activity in the soil are obviously higher than that in the soil in which straws are directly utilized and no fertilizer is applied.
We have discovered a new method to treat biomass with alkali, for example lime. The lime and lignin was sufficiently removed from the treated biomass b> squeezing with a high pressure device to remove alkali and other potential inhibitors of the cellulase enzymes added for sacchaπfication. The resulting fibrous material was rapidly solubilzed by cellulases, even at solid loads ranging from 10 to 30% (w / w) without inhibitory effects on the cellulase activity. The lime pretreatment removed lignin effectively and left the cellulose and hemicellulose almost intact. The method yielded a biomass with structure capable of being enzyme solubilzed and fermented readily at a solids loading of 10-30% for a production of ethanol.
The invention discloses a preparation method of a strawdecomposition agent. The strawdecomposition agent is prepared by using bacillus subtilis, aspergillus oryzae, lactobacillus plantarum, trichoderma reesei and saccharomyces cerevisiae as preparation strains, carrying out activation and primary and secondary culture on the strains respectively to obtain various strain solutions with bacterial count of 106-108 / ml and mixing the strain solutions according to a certain proportion. The strawdecomposition agent has the beneficial effects that the straw decomposition agent can be used for quickly degrading straws to prepare fertilizers and ensures that the straw decomposition time is at least 15 days ahead of the normal composting fermentation time; organic straw fertilizers prepared by the preparation method are used as base fertilizers, so that the contents of available nitrogen, rapidly available phosphorus, potassium and organic matters in soil are higher than those of available nitrogen, rapidly available phosphorus, potassium and organic matters in the soil in which straws are directly used and fertilizers are not applied; the urease activity and cellulase activity of the soil are obviously improved compared with those of the soil in which straws are directly used and fertilizers are not applied.
The invention discloses a method for breeding a high-producing strain of cellulase by gene knockout, and belongs to the field of enzymeengineering. The method for breeding the high-producing strain of cellulase by gene knockout comprises the step of knocking out an amylasegene of the cellulase producing strain. A high-producing strain of cellulase is aspergillus niger without the amylase gene. An amylasegene knockoutplasmid is obtained by connecting the front half part of the recombinant segment amylase gene-hygromycin B-an expression unit of a resistant gene- the rear half part of the amylase gene to a pCAMBIA1300 plasmid. Then, the plasmid is converted into agrobacterium, and the amylase gene is knocked out by agrobacterium-mediated conversion of the aspergillus niger. By using the gene knockout technology, the amylase gene of the aspergillus niger is knocked out. After the amylase gene is knocked out, the cell load is reduced, thereby facilitating production of a lot of cellulase. The cellulase activity of the high-producing strain of cellulase obtained can reach 21U / g.
Method for extracting oils, proteins and fermentable sugars from vegetable material in an aqueous medium, includes:a) adding water to the vegetable material;b) adding an enzyme mixture containing at least one cellulase, at least one hemicellulase, and at least one pectinase, the ratio between the pectinase activity and the cellulase activity being at least 0.14, and the ratio between the pectinase activity and the hemicellulase activity being at least 7.10−3, the pectinase activity being less than 120 μmol / min / mL;c) incubating the vegetable material and the enzyme mixture with stirring to release oils, proteins and fermentable sugars in the reaction medium;d) separating the reaction medium to obtain free oil, an aqueous phase containing proteins and fermentable sugars, and a solid phase;e) optionally separating and recycling an emulsion of free oil or aqueous phase, to the medium;f) separating the proteins and fermentable sugars from the aqueous phase.
The invention relates to a complex inoculant and an application thereof in degrading cellulose in poultry and livestockmanure. The complex inoculant consists of a bacterium strain which is cultivated in a beef extract-peptone medium and a fungus strain which is cultivated in a PDA culture medium at the volume ratio of a culture solution of 1:1. According to the complex inoculant provided by the invention, optimum enzyme-production temperatures of the various strains correspond to temperatures in different stages of composting fermentation, and changing curves of the two parts (the optimum enzyme-production temperatures of the various trains and the temperatures in different stages of the composting fermentation) fit and are matched; the various strains grow in a coordinated mode and continuously produce enzyme, so that a relatively stable cellulase producing environment is formed, and stable enzyme activity is guaranteed; the enzyme can be continuously produced in different fermentation temperature stages of a composting process, so that decomposition of the cellulose in the poultry and livestockmanure is promoted; and since a reaction substrate is continuously utilized by the various strains, a level and a peak of cellulase activity of composting fermentation of the poultry and livestockmanurecellulase can be also improved in advance and the fermentation time of a compost can be shortened, so that adaptability and a decomposing effect of the compost are enhanced.
A marine streptomyces viridochromogenes strain for producing alkali-tolerant and salt-tolerant xylanase and application of the marine streptomyces viridochromogenes strain belong to the field of biotechnology. The strain is collected in the China General Microbiological Culture Collection Center, and the collection number is 5565. The strain can normally grow under the extreme alkaline environment of pH11, shows a good oligotrophy-tolerant characteristic, can degrade a variety of hemicellulose-containing biomasses including corn straws, rice straws, Jerusalem artichoke straws, kelp fibers andthe like, and has a broad application prospect in utilizing cheap biomass resources to produce bio-based chemicals. The strain can produce xylanase under optimum conditions, and the enzymatic activity of the xylanase can be as high as 68.9U / ml. The produced xylanase has the following properties: the optimum pH value is 6.0, the optimum temperature is 70 DEG C, the optimum substrate is beechxylan, and the produced xylanase does not have cellulase activity, has high pH stability and thermal stability, and can tolerate high-concentration NaCl. As an enzyme preparation coming from the ocean, thexylanase can be widely used in papermaking, food, feed, wine making, energy and other industries.
The invention relates to the technical field of microbes, and specifically relates to trichoderma koningii, and a compound microbial agent composition and an application thereof. The invention discloses trichoderma koningii HK43-5 (CGMCC No7422), a compound microbial agent composition composed of a glycosylated bacterial preparation and a forageyeast preparation which are packaged separately, wherein the glycosylated bacterial preparation comprises the trichoderma koningii HK43-5 (CGMCC No7422). Cellulose produced by the trichoderma koningii HK43-5 (CGMCC No7422) has high activity, can be used as an efficient glycosylated bacterial preparation in production of industrial and agricultural residual proteinforage. The compound microbial agent composition can efficiently utilize industrial and agricultural residues, particularly potato residues, to produce single cell protein. Crude protein content in fermented mycoprotein is 50.49% which is far superior to that reported in a conventional technology. The industrial and agricultural residues are changed to wealth; and the produced bacterial protein can be applied in the forage to partly substitute soybean meal or fish meal. At the same time, the environment pollution problems resulted from the industrial and agricultural residues are solved.
The invention discloses a new fungusAcremonium sp. DPZ-SYz-2-3 for the high efficiency cellulose degradation and an application thereof. Acremonium sp. DPZ-SYz-2-3 is preserved in the China general microbiological culture collection center (CGMCC) on 05 / 16 / 2011 and has a preservation number of CGMCC No: 4810, wherein the address of the CGMCC is No.3 of No.1 Beichen Road West, Chaoyang District, Beijing, Institute of Microbiology Chinese Academy of Sciences. The fungus of the invention, which has a cellulase production activity, can be used for producing cellulase, so the fungus has important values to the production and the utilization of cellulase; and the fungus can be further used for making a biological organic matter degradation bacterial fertilizer.
The invention discloses a method for simultaneous high-yield production of cellulase and [beta]-glucosidase. The operating method for simultaneous high-yield production of the [beta]-glucosidase and the cellulase is implemented by the following steps: expressing polypeptide, which has a cellobiase activity, in trichoderma reesei so as to obtain a recombinant trichoderma reesei strain, and conducting fed-batch fermentation by taking a soluble inducer of a glycose transglycosylation product as a carbon source. According to the method provided by the invention, cultivation is conducted in a fermentation tank by virtue of a fed-batch fermentation technology for 156h, so that the activity of the [beta]-glucosidase in a recombinant strain fermentation broth exceeds 300CBU / mL, which is more than 60 times above that of an original strain, and meanwhile, the activity of the cellulase exceeds 50FPU / mL. With the application of the method provided by the invention, shortcoming in the prior art that the [beta]-glucosidase of a trichoderma reesei cellulase system is too low in enzymatic activity is overcome, the compounding cost of the cellulase is reduced and the enzymolysis efficiency of lignocellulose is improved.
The invention discloses a natural plant feedstuff additive for fattening mutton sheep, which relates to a mutton sheep feedstuff additive. The mutton sheep feedstuff additive solves the problems that the prior antibiotic feedstuff additive makes pathogenic microorganisms invading into the bodies of the mutton sheep develop drug resistance after long-term feeding and generates drug residues and toxic side effects on mutton products and the surrounding environment so that the weight increasing rate of the mutton sheep is low. The natural plant feedstuff additive for fattening the mutton sheep consists of gizzardskin, charred triplet, elecampane, dried orange peel, radish seed, betel nut and radix codonopsitis. The natural plant feedstuff additive can improve the daily gain of the mutton sheep, improve the utilization efficiency of protein, the energy and fiber of daily rations and improve the function of the cellulose activity in rumen juice of the mutton sheep, does not influence livers of the mutton sheep, and is a safe and efficient feedstuff.
Owner:NORTHEAST INST OF GEOGRAPHY & AGRIECOLOGY C A S
Aluminum coated glass slides provide a novel glycan array platform. Specifically, aluminum coated glass slides increase sensitivity of fluorescent based assay methods. Additionally, aluminum coated glass slides allows for mass spectroscopic analysis of carbohydrates and provide a platform for examining activity of cellulases. The unique properties of ACG slides include: 1) the metaloxide layer on the surface can be activated for grafting organic compounds such as modified oligosaccharides; 2) the surface remains electrically conductive, and the grafted oligosaccharides can be simultaneously characterized by mass spectrometry and carbohydrate-binding assay; and 3) the slides are more sensitive than transparent glass slides in binding analysis.
The invention relates to a new method of co-fermenting kitchen waste raw materials with mixed bacteria for producing fuel ethanol, which is characterized by comprising the following steps: 1, separating and screening a rhizopus oryzae strain which simultaneously has amylase activity and cellulase activity; and 2, taking the kitchen waste as a raw material only, and mixing the rhizopus oryzae and saccharomyces cerevisiae in a certain proportion for co-fermenting to produce the fuel ethanol. The method of the invention avoids the problems that in the process of fermenting the kitchen waste for producing the fuel ethanol, the raw material can not hydrolyze because of enzymic inactivation, glucose is lacked in the fermentation liquor, and ethanol fermentation can not be completely carried out. No catalyst and nutrient need to be added in the fermentation process, the availability of large molecule organic substances of starch, cellulose and the like in the waste is high, thus the invention reduces the production cost of ethanol by the kitchen waste, has the advantages of energy saving, simple required equipment and low investment cost, and is a new way with good practical value for recycling the kitchen waste.
Method for extracting oils, proteins and fermentable sugars from vegetable material in an aqueous medium, includes:a) adding water to the vegetable material;b) adding an enzyme mixture containing at least one cellulase, at least one hemicellulase, and at least one pectinase, the ratio between the pectinase activity and the cellulase activity being at least 0.14, and the ratio between the pectinase activity and the hemicellulase activity being at least 7.10−3, the pectinase activity being less than 120 μmol / min / mL;c) incubating the vegetable material and the enzyme mixture with stirring to release oils, proteins and fermentable sugars in the reaction medium;d) separating the reaction medium to obtain free oil, an aqueous phase containing proteins and fermentable sugars, and a solid phase;e) optionally separating and recycling an emulsion of free oil or aqueous phase, to the medium;f) separating the proteins and fermentable sugars from the aqueous phase.
The invention discloses a soil conditioner for tobacco field acidic soil, wherein the soil conditioner comprises, by weight, 40-80 parts of shells, 20-60 parts of potassiumfeldspar, 10-50 parts of limestone, and 15-40 parts of dolomite. According to the present invention, the soil conditioner can neutralize the active acid and the exchangeable acid in the soil, obviously increase the pH value ofthe soil, increase the soil plough layer exchangeable Ca<2+> and Mg<2+> concentrations, improve the soil microbial environment, increase the numbers of soil bacteria, actinomycetes and aerobic cellulosedecompositionbacteria, improve the activities of urease, acid phosphatase, catalase and cellulase, passivate the heavy metals in the soil, reduce the heavy metals in tobacco, reduce the occurrenceof tobacco root diseases, improve the aroma quality, the aroma amount and the concentration of tobacco leaves, and reduce the irritation, the miscellaneous gas and the like of tobacco leaves.
The invention discloses a Trichoderma reesei recombinant strain containing artificial zinc finger transcription factor and application of the Trichoderma reesei recombinant strain, and belongs to the field of microbial biotechnology. The Trichoderma reesei recombinant strain is stored in China General Microbiological Culture Collection Center on April 16, 2015, and the accession number is CGMCC No. 10649. The Trichoderma reesei recombinant strain is named Trichoderma reesei U5, which is a high-yield cellulose strain obtained by transferring an artificial zinc finger proteintranscription factor into the starting strain of Trichoderma reesei C30, and then being subjected to cellulase activity screening. Under the condition of bran inducing, the cellulase activity of the high-yield cellulose strain is increased by about 35% as compared with that of a wild type Trichoderma reesei C30, reaching 8.4 enzyme-activity units per milliliter; beta-glucosidase of the Trichoderma reesei U5 is increased by 3.5 times as compared with that of the wild type Trichoderma reesei C30, reaching 8.8 enzyme-activity units per milliliter. The increase in cellulasesecretion activity of the Trichoderma reesei U5 shows the potential application value of the Trichoderma reesei U5 in the field of renewable energy, and new microbial resources are provided for the efficient degradation of biomass.