270results about "Carbon-nitrogen lyases" patented technology

The present invention is directed to phenylalanine ammonia lyase (PAL) produced by prokaryotes, wherein such prokaryotic PAL wherein the PAL variant has a greater phenylalanine-converting activity as compared to a wild-type PAL. The invention thus provides compositions of bacterial PAL and biologically active fragments, mutants, variants and analogs thereof, as well as methods for the production, purification, and use of such compositions for therapeutic and industrial purposes.

Provided herein are phenylalanine ammonia-lyase (PAL) variants produced by prokaryotes, wherein such prokaryotic PAL variant has a greater phenylalanine-converting activity and/or a reduced immunogenicity as compared to a wild-type PAL. Further provided are compositions of prokaryotic PAL and biologically active fragments, mutants, variants or analogs thereof, as well as methods for the production, purification, formulation, and use of such compositions for industrial and therapeutic purposes, e.g., treating hyperphenylalaninemia, including phenylketonuria, and other disorders, including cancer.

A composition and method for treating bacterial infections by the use of an effective amount of at least one lytic specific for the bacteria causing specific. The lytic enzyme is genetically coded for by a bacteriophage which may be specific for said bacteria. The enzyme may be at least one lytic protein or peptides in a natural or modified form.

Yeast cells with reduced activity of certain enzymes involved in branched chain amino acid biosynthesis in yeast mitochondria are described. Target enzymes include threonine deaminase, isopropylmalate synthase, and optionally branched chain amino acid transaminase.

Microorganisms are genetically engineered to synthesize caffeic acid from simple carbon sources via a tyrosine intermediate by means of a dual pathway that utilizes both endogenous and engineered enzymatic activities.

Conversion in vitro of X-Gly to X-alpha-hydroxy-Gly or X—NH₂ (X being a peptide or any other compound having a carbonyl group capable of forming a covalent bond with glycine) is accomplished enzymatically in the presence of keto acids, or salts or esters thereof, to provide a good yield without the necessity of catalase or similar enzymatic reaction enhancers. Peptidylglycine α-amidating monooxygenase (PAM) is a preferred enzyme for catalyzing the conversion. Alternatively, peptidylglycine α-hydroxylating monooxygenase (PHM) is utilized to convert X-Gly to X-alpha-hydroxy-Gly which may be recovered, or optionally may be simultaneously or sequentially converted to an amide by either a Lewis base or action of the enzyme peptidyl α-hydroxyglycine α-amidating lyase (PAL). Both PHM and PAL are functional domains of PAM.

The invention discloses a high yield L-leucine engineering bacterium and an application of the bacterium.The invention provides a method for preparing a recombinant bacterium. The method comprises the following step of importing an alpha-isopropylmolic acid synthetase mutant encoding gene into a target bacterium to form the recombinant bacterium, wherein an alpha-isopropylmolic acid synthetase mutant is enzyme obtained by mutating 494th arginine of alpha-isopropylmolic acid synthetase into histidine, 497th glycine into aspartic acid and 499th leucine into valine.The high yield L- leucine engineering bacterium, particularly corynebacterium glutamicum MD0032, has higher L-leucine yield than bacterial strains produced at home at present, has a unique identification label and is a L-leucine production strain with a high production and application value.

The amino acid sequence of an aspartate ammonia lyase mutant, an amino acid sequence is SEQ ID NO: 3, and compared with an initial aspartate ammonia lyase AspB, the enzyme activity of catalyzing acrylic acid to generate beta-alanine is remarkably improved. When the whole cell expressing the aspartate ammonia lyase mutant is used for catalyzing a 120g/L acrylic acid substrate to react to generate beta-alanine, the conversion rate exceeds 95%, and the aspartate ammonia lyase mutant has an industrial application prospect.

The invention discloses a construction and application of a recombinant strain converting L-threonine to L-2-aminobutyric acid, belonging to the field of bioengineering technology. The production method of the invention utilizes a recombinant bacterium expressing two plasmids to simultaneously realize the high-efficient expression of three enzymes, and comprises the steps of: conversion of L-threonine to L-2-aminobutyric acid, coupled with a coenzyme regeneration system, converts NAD+ into NADH, so that the concentration of NADH in the system is relatively stable, and the conversion can be carried out efficiently. Furthermore, the CO<2> converted from ammonium formate can be dissolved in ammonia water, which has little environmental pollution and industrial application value. The method has the advantages of mild conversion condition, strong specificity, low cost and short conversion time. The L-2-aminobutyric acid is prepared adopting the method, 40g/L L-threonine is added, The concentration of the obtained product L-2-aminobutyric acid is 43.3 g/L, and the conversion ratio was over 99.9%.

The invention discloses an aspartase mutant, which comprises the following mutations M321V, K324T and N326S on an amino acid sequence represented by SEQ ID NO.2, and has high enzymatic activity compared with wild-type aspartase in catalysis of (E)-4-(2,4,5-trifluorophenyl)2-butenoic acid. The invention also discloses applications of the aspartase mutant in preparation of (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid. According to the invention, with the application of the aspartase mutant in an enzyme catalytic reaction to prepare a sitagliptin chiral intermediate (R)-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid, the conversion rate is high, the stereoselectivity is high, the yield is high, the production cost is low, the environment is friendly, and industrial large-scale production is facilitated.

Belonging to the field of biochemical engineering, the invention discloses a high efficiency biosynthesis method of caffeic acid with catechol as the substrate. According to the invention, tyrosine phenol-lyase takes catechol, pyruvic acid and ammonia as the substrate to synthesize levodopa, tyrosine ammonia-lyase converts levodopa into trans-caffeic acid and NH3. Catechol, sodium pyruvate and ammonium chloride are relatively cheap compounds, therefore the caffeic acid biosynthesis method provided by the invention has large potential. Compared with the previous conversion methods taking L-tyrosine as the substrate, the substrate used by the method are cheaper and easier for large-scale production. Compared with chemical synthesis methods, the product of the method provided by the invention is single trans-caffeic acid, and further separation of an isomer is unnecessary.

The present invention provides engineered phenylalanine ammonia-lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia-lyase (PAL) polypeptides.

The invention discloses a recombinant escherichia coli capable of producing cinnamic alcohol and rosin, and a construction method and applications thereof. The construction method comprises followingsteps: phenylalnine ammonialyase PAL, p-hydroxycinnamic acid coenzyme A ligase 4CL, and cinnamamide coenzyme A reductase CCR are introduced into Escherichia coli so as to obtain the recombinant escherichia coli capable of producing cinnamic alcohol. In the construction method, Escherichia coli is taken as a model organism, the background is clear, growth speed is high, large scale fermentation culture is convenient to realize, and cost is low. The recombinant escherichia coli capable of producing cinnamic alcohol and rosin is obtained via construction of a cinnamic alcohol synthesis pathway, the cinnamic alcohol yield is 300mg/L; UDP glucosyltransferase is introduced into Escherichia coli further, so that organic combination of biological synthesis pathways of rosin and cinnamic alcohol isrealized, the highest rosin yield is 280mg/L. The construction method is capable of providing large scale industrialized production of cinnamic alcohol and/or rosin with foundation.

The invention discloses a saccharomyces cerevisiae recombinant bacterium. According to the saccharomyces cerevisiae recombinant bacterium, an RgTAL gene, an HpaB gene and an HpaC gene are integrated in a genome. The invention further discloses a construction method and application of the saccharomyces cerevisiae recombinant bacteria. The invention finally discloses a production method of caffeic acid. According to the invention, three exogenous genes required by caffeic acid synthesis are integrated into saccharomyces cerevisiae chromosomes; total synthesis from glucose to caffeic acid is achieved through knockout of competitive pathway genes, elimination of feedback inhibition steps and overexpression of rate-limiting enzymes, the shake flask yield reaches about 760 mg/L, the shake flaskyield is the highest level of the yeast yield reported at present, and a new method is provided for industrial production of caffeic acid.

The invention discloses escherichia coli capable of producing L-alanine through fermentation and application of the escherichia coli and belongs to the technical field of biological engineering. According to the escherichia coli, aspartase AspA and L-aspartic acid-beta-decarboxylase AsD are introduced into the escherichia coli capable of producing fumaric acid through the fermentation; furthermore, a YgaW gene is over-expressed to strengthen the transport ability of the L-alanine in recombinant bacteria, so that a novel strain capable of fermenting glucose to produce the L-alanine is constructed; when the recombinant strain is fermented in a fermentation tank for 45h, the yield of the L-alanine reaches 147g/L and the saccharic acid conversion rate is 79.0 percent.

The invention provides an immobilized enzyme as well as a preparation method and an application thereof. The immobilized enzyme comprises an enzyme and an amino resin carrier for immobilizing the enzyme, the enzyme is selected from any one of transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase and nitrilase, the amino resin carrieris the cross-linking agent modified amino resin carrier, and a cross-linking agent is a cross-linking agent treated by a high polymer. The amino resin carrier is modified by adopting the cross-linking agent treated by the high polymer, so that enzymes immobilized on the amino resin carrier can form netted cross-linking, the immobilization effect of the enzymes is more stable, and the cyclic utilization efficiency of the enzymes is improved.

The invention relates to a genetic engineering bacterium for producing L-isoleucine and an application thereof, which belong to the field of metabolic engineering. The genetic engineering bacterium isobtained by overexpressing an acetohydroxyacidsynthase encoding gene ilvBN<M>, a threonine dehydratase encoding gene ilvA, and a threonine operon thrABC releasing feedback inhibition of L-isoleucine,the acetohydroxyacidsynthase encoded by ilvBN<M> releases the feedback inhibition of L-isoleucine, and its activity is not significantly lower than that of the wild-type ilvBN-encoded acetohydroxyacidsynthase; The L-isoleucine genetic engineering bacterium of the invention has no nutritional deficiency, rapid growth, short fermentation cycle, high yield and high conversion rate, and after 26-36hfermentation, the concentration of L-isoleucine in a fermentation broth reaches 42.5-51.6g / L.

The invention discloses a genetically engineered bacterium for directly producing L-aspartic acid through fermentation. The classification naming of the genetically engineered bacterium is Escherichia coli CM-AS-115, and the preservation number of the genetically engineered bacterium is CCTCC NO: M 2016457. The bacterial strain relates to inactivation of multiple genes, evolution, metabolism and domestication are simultaneously carried out on the bacterial strain which knockouts the multiple genes, and a mutant strain, namely, CM-AS-105, which has a lower respiratory quotient under the aerobic condition and of which the highest dry cell weight is 60-70% of dry weight of an original strain W1485 is obtained; meanwhile, the bacterial strain further relates to over expressions of two genes, wherein the two genes comprise an enol phosphate type pyruvate carboxylase encoding gene (ppc) and an aspartase encoding gene (aspA), and the obtained bacterial strain is CM-AS-115. The genetically engineered bacterium for directly producing L-aspartic acid through fermentation achieves a way of completely adopting renewable biomass resources such as starch and cellulose as raw materials to ferment and prepare the L-aspartic acid, and the way is green and environmentally friendly.

The invention discloses a beta-alanine producing strain and a preparation method and application thereof. The beta-alanine producing strain (Escherichia coli) has a preservation number of CGMCC NO: 17830. The beta-alanine producing strain constructed by the invention can realize the effective accumulation of the beta-alanine in the fermentation liquor in the fermentation process without adding aspartic acid in the fermentation process, and improves the yield of the beta-alanine and the sugar conversion rate. The yield of the beta-alanine is up to 50g/L, the sugar-acid conversion rate is up to 40%, and the producing strain has the application potential of large-scale production.