60 results about "Echinocandin B" patented technology

The invention discloses a method for converting echinocandin B into an echinocandin B parent nucleus through microbial fermentation. The method is characterized by comprising the following step of: adding an echinocandin B substrate in a fermentation process to carry out conversion, wherein the echinocandin B substrate is added in batches or added in a flowing manner.

The invention provides a method for separating echinocandin B from fermentation broth and a use of echinocandin B. The method comprises the following steps that through fermentation broth acidification, extraction and concentration, concentrated liquid is obtained; and the concentrated liquid is fed into a reverse phase silica gel column and then is subjected to separation and concentration so that echinocandin B is obtained. The echinocandin B separated by the method has high purity and is a good raw material for preparation of echinocandin cyclopeptide parent nucleus and antifungal drugs. The method has a high finished product yield, utilizes safe and cheap solvent and instruments, has a low production cost and simple processes and is suitable for industrial production.

The invention relates to a method for preparing echinocandin B parent nucleus. The method comprises the steps that: (1) when echinocandin B conversion is finished, pump filtration is carried out; a filtrate is obtained, and the filtrate is processed by using an acidic alumina column; (2) the pH of the liquid collected in the step (1) is regulated to 4-5; rotary evaporation concentration is carried out; the material is processed by using macroporous non-polar resin; salt is removed by water washing; and a methanol aqueous solution is used for gradient elution; (3) the pH of the liquid collected in the step (3) is regulated to 6-7; the material is processed by using reversed-phase filler chromatographic column; and a methanol aqueous solution is used for elution; and (4) the pH of the liquid collected in the step (3) is regulated to 4-5; rotary evaporation concentration is carried out; and lyophilization is carried out, such that echinocandin B parent nucleus is obtained. With the method, a final purity of echinocandin B parent nucleus can be improved to higher than 95%.

The invention discloses a recombinant ECB (echinocandin B) deacylase mutant and an application in synthesis of ECB parent nucleus from the ECB by biocatalysis. The deacylase mutant is obtained throughsingle mutation or double mutation of the 287th and 527th sites of an amino acid sequence shown in SEQ ID No.2. AuEBDA primary structure and enzyme conformation are changed through mutation of an auebda gene sequence, and the catalytic performance of AuEBDA is improved. Influences of G287Q and R527V substitution on activity of ECB deacylase are systemically analyzed. The enzyme activity of constructed double mutant AuEBDA-G287Q/R527V is improved by 2.5 times than that of parent wild AuEBDA, and the catalytic efficiency kcat/Km is increased by 2.9 times.

The invention establishes a screening method of a high-producing strain of a producing strain of echinocandins B, and a strain obtained by screened is preserved. The screening method comprises the following steps: firstly, according to the characteristic of feedback inhibition of a product of the producing strain of the echinocandins B, adding an appropriate amount of echinocandins B into a screening plate for primary screening, so as to screen out strains with the characteristic of feedback inhibition of the product removed; next, soaking mycelia, inducing the strains into an organic solvent,extracting the echinocandins B in the mycelia, and rescreening the high-producing strain of the echinocandins B from the primarily screened strains according to the size of an inhibition zone by adopting the inhibition of the echinocandins B on candida albicans, so as to achieve the purpose of rescreening; finally, investigating the production capacity of the screened out high-producing strain with a fermentation shake flask. The screening method is simple, convenient and efficient, so that the screening period is greatly shortened, and the screening efficient of the strain is improved.

The invention discloses a microbial enzymatic conversion method for echinocandin B, and particularly relates to a method of converting the echinocandin B into an echinocandin B nucleus. The method includes performing microbial fermentation to produce echinocandin B deacylase, extracting to obtain crude enzyme, and converting through adding the crude enzyme and the echinocandin B in a batch manner. The method is high in mole conversion ratio, high in product concentration, and beneficial to separation and purification. An echinocandin B substrate can be recovered and reutilized, thus reducing a production cost.

The invention discloses a method for fermenting high-yield echinocandins B through aspergillus nidulans. The method includes the steps that a fermentation culture medium is inoculated with the aspergillus nidulans, culture is carried out at the temperature of 25 DEG C, fermentation liquor containing echinocandins B is obtained, the fermentation liquor is separated and purified, and echinocandins Bare obtained; by optimizing the varieties of carbon sources, adding particles and the like, the forms of mycelia in the fermentation process of the aspergillus nidulans are controlled, the mycelia are coccoid, and the problems that as the mycelia are too long during fermentation, the viscosity is high, dissolved oxygen is low, and mass transfer is insufficient are solved; by analyzing key metabolism components in the ECB synthesis process and optimizing a precursor adding strategy in the fermentation process, the yield of fermentation products is further increased by one time or above, and broad application prospects are achieved.

The invention discloses a gene engineering bacterium for efficiently converting Echinocandin B and a preparation method thereof. The gene engineering bacterium is the engineering bacterium with an expression cassette of Echinocandin B acylase removal genes integrated in a genome of a wild strain of Streptomyces lividans. Through the conversion verification of the Echinocandin B, compared with that of the wild strain of Actinoplanes utahensis, the conversion speed of the gene engineering bacterium is improved by 1.5 times.

The invention provides a hydrate of echinocandin B mother nucleus or salt thereof. When mass percentage of water in the hydrate is 7.0-31.0%, the hydrate has excellent stability. In addition, the invention also discloses a preparation method and an application of the hydrate of echinocandin B mother nucleus or the salt thereof.

The invention discloses a method for purifying echinocandin B parent nucleus. The method comprises the following steps: 1) performing solid-liquid separation on a conversion solution; 2) concentrating, and removing the moisture from the liquid obtained in the step 1); 3) crystallizing; and 4) separating crystals. The crystallizing method used in the method disclosed by the invention is simple andconvenient to operate, time-saving, low in cost and suitable for continuous production on various scales; the product obtained by crystallization is high in purity; and the adopted non-polar solvent is easily recycled, and the cost is reduced.

The invention discloses genetically engineered bacteria for producing echinocandin B as well as a construction method and application thereof. The genetically engineered bacteria are characterized inthat a polyketide synthase gene associated with the synthesis of sterigmatomycin in a genome of the genetically engineered bacteria is inactivated or is genetically modified so as not to express the sterigmatomycin. The genetically engineered bacteria for producing echinocandin B as well as the construction method and the application thereof disclosed by the invention have the beneficial effects that the mononuclear treatment is not required to be performed on a strain in the construction process of the genetically engineered bacteria, so that the problems such as single exchange occurring through homologous recombination, poor genetic stability and easy reversion mutation are solved; the genetically engineered bacteria does not produce the sterigmatomycin, and the yield of the echinocandin B can be improved by 44 percent compared with that of a wild strain; through multiple passages, the fermentation property still remains stable.

The invention discloses a method for determining binding constants of an echinocandins medicine and protein. Aiming at a hotspot echinocandins medicine developed from an existing antifungal medicine, the pressure-driven affinity capillary electrophoresis method is used for rapidly and accurately determining the binding constants of a receptor and a ligand; the binding of ECB (Echinocandins B) and BSA (Bull Serum Albumin) is determined, effective electrophoresis mobility of ECB in protein buffering solutions with different concentration can be accurately determined, and then the binding constants are calculated. The method is used for determining the binding of ECB and BSA so as to determine that ECB belongs to a strong binding type medicine; the method provides data support on knowing medicine effect expression of the echinocandins medicine and provides optimization evidences to subsequent data screening.

The invention discloses a novel method of converting echinocandin B into echinocandin B parent nucleus. A crosslinked enzyme aggregation technique is applied to the process of conversion of echinocandin B into echinocandin B parent nucleus. The method comprises: preparing deacylase crosslinked enzyme aggregate with deacylase; allowing the crosslinked enzyme aggregate to convert echinocandin B intoechinocandin B parent nucleus. The preparation method enables the molar conversion rate to reach 85% to 90%; the production process is simplified; the cost is lowered.

The invention discloses a method for converting echinomycin B by an immobilized microbial enzyme, and belongs to the field of bioengineering. The method comprises the following steps: (1), fermentingactinoplanesutahensis, collecting mycelia, disrupting cells through ultrasonic disruption by using a cell disrupting device, centrifuging, taking a supernatant to obtain a crude enzyme solution of echinomycin B deacylase; (2), slowly adding an ammonium sulfate solution into the crude enzyme solution in the step (1) to obtain a precipitate; (3), dissolving the precipitate in the step (2) in a buffered salt solution; (4), adding odium alginate into the solution in the step (3), stirring uniformly, then dropwise adding a pre-cooled aqueous calcium chloride solution, placing in a refrigerator overnight, and filtering to obtain the immobilized microbial enzyme; and (5), dissolving echinomycin B in ethanol, and adding the immobilized microbial enzyme in the step (4), converting, and collecting afiltrate to obtain an echinocandin B mother nucleus solution. By the method, the conversion rate is high, the conversion time is short, a product is easy to separate, a conversion solution contains less pigments, and the immobilized enzyme is easy to store and can be reused.

The invention provides a method for preparing antifungal cyclic lipopeptide echinocandin B. The method comprises the following steps: 1) performing solid-liquid separation on an ECB (Echinocandin) fermentation broth; 2) enriching with a macroporous adsorption resin ADS-17 or X-5; 3) eluting the macroporous adsorption resin with hot absolute methyl alcohol, and combining eluants; 4) concentrating the eluants, adding a sodium chloride solution, stirring, cooling for crystal separation, and drying crude ECB filter cakes; 5) filtering the crude ECB filter cakes with methanol, and treating filtratewith a reverse phase resin column XT30; 6) concentrating the reverse phase resin column XT30 eluant, adding the sodium chloride solution, stirring, cooling for crystal separation, and drying ECB filter cakes; 7) collecting a dried ECB product. By adopting the method, the problems that byproducts are hard to elute and solvents are wasted are simply and effectively solved. Solid-liquid separation of water-containing ECB can be effectively completed, and product degradation is remarkably reduced. The method is simple and feasible and is applicable to industrial production, and the final purity of the echinocandin B is improved to 99% or greater.