WO2022192529A1 - Activation de zymogènes par des enzymes protéases immobilisées - Google Patents

Activation de zymogènes par des enzymes protéases immobilisées Download PDF

Info

Publication number
WO2022192529A1
WO2022192529A1 PCT/US2022/019738 US2022019738W WO2022192529A1 WO 2022192529 A1 WO2022192529 A1 WO 2022192529A1 US 2022019738 W US2022019738 W US 2022019738W WO 2022192529 A1 WO2022192529 A1 WO 2022192529A1
Authority
WO
WIPO (PCT)
Prior art keywords
transglutaminase
protease
immobilized
activated
tgase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2022/019738
Other languages
English (en)
Inventor
Kamil S. GEDEON
William Shindel
Steven Joseph Walsh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Curie Co Inc
Original Assignee
Curie Co Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Curie Co Inc filed Critical Curie Co Inc
Publication of WO2022192529A1 publication Critical patent/WO2022192529A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • Immobilized enzymes are typically more robust and more resistant to environmental changes compared to free enzymes in solution. More importantly, the heterogeneity of immobilized enzyme systems allows an easy or more simplified recovery of both enzymes and products, multiple re-use of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs.
  • a method for activating a zymogen of a transglutaminase comprising contacting the zymogen with at least one immobilized protease to produce an active form of transglutaminase and, optionally, separating the activated transglutaminase from the immobilized protease.
  • an activated transglutaminase produced by contacting a zymogen with at least one immobilized protease to produce an active form of transglutaminase and, optionally, separating the activated transglutaminase from the immobilized protease.
  • the transglutaminase is a microbial transglutaminase.
  • the microbial transglutaminase is a Streptomyces mobaraensis transglutaminase or a variant thereof.
  • the at least one immobilized protease is selected from the group consisting of bacterial proteases and fungal proteases.
  • the bacterial protease is subtilisin.
  • FIG. 1 shows SDS-PAGE analysis of S. mobarensis Tgase, as described in Example 2. Lane 1 - mature Tgase; Lane 2 - zymogen (pro-Tgase); Lane 3 - clarified lysate containing zymogen (crude pro-Tgase); Lane 4 - clarified lysate treated with immobilized protease for 60 minutes; Lane L - protein ladder. Expected molecular weight of the zymogen (pro-Tgase) is 43.6 kDa and the expected molecular weight of the mature Tgase is 38.9 kDa.
  • composition or the specified steps of a methods, and those additional materials or steps that do not materially affect the basic characteristics of the material or method.
  • Optional or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.
  • zymogen refers to an inactive precursor of an enzyme, which may be converted into an active/activated or mature enzyme by catalytic action, such as via proteolytic cleavage of a pro-sequence.
  • a “pro-sequence” refers to a polypeptide sequence within an expressed protein, e.g., a zymogen, which is typically cleaved from the protein to produce an active protein, such as an enzyme.
  • a pro-sequence may be essential for correct folding of the protein.
  • cleavage of the pro-sequence results in transition of an inactive enzyme to active or activated enzyme.
  • the terms "mature”, “active”, and activated” are used interchangeably herein.
  • An activated form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or enzyme without a signal, silencing, or chaperoning propeptide sequence. Additionally, the mature enzyme may be truncated or extended relative to the mature sequence while maintaining the desired catalytic activity (e.g., cross-linking activity).
  • T ransglutaminases are a family of enzymes that catalyze the formation of an isopeptide bond between a primary amine, for example, the e-amine of a lysine molecule, and the acyl group of a protein- or peptide-bound glutamine.
  • Transglutaminases may catalyze a transamidation reaction between glutamyl and lysyl side chains of target proteins. Proteins possessing Tgase activity have been found in microorganisms, plants, and animals. Tgases are widely distributed in various organs, tissues, and bodily fluids.
  • Tgases also form extensively cross-linked, generally insoluble, protein biopolymers that are needed for an organism to create barriers and stable structures.
  • an “activated Tgase” is a Tgase having or capable of reacting with amino acids, peptides and/or proteins.
  • Tgases of microbial origin are calcium- independent, which represents a major advantage for their practical use.
  • Microbial transglutaminase (Tgase, EC 2.3.2.13) is one of the most extensively studied industrial enzymes for protein functionalization and protein cross-linking because of its ability to polymerize or functionalize proteins through the formation of a stable £-(y-glutamyl)lysine isopeptide bond without the constraint of a consensus sequence or additional cofactors.
  • An “immobilized protease” refers to immobilization of a protease to/on a matrix or support.
  • a “protease” (also called a peptidase or proteinase) refers to enzymes capable of cleaving peptide bonds.
  • Proteases are any of various enzymes, such as endopeptidases and exopeptidases, that catalyze the hydrolytic breakdown of proteins into peptides and amino acids.
  • Proteases can be classified into seven broad groups: serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, and asparagine peptide lyases.
  • Proteases can be found in animals, plants, bacteria, fungi, archaea, and viruses.
  • the terms “protease, “peptidase”, and “proteinase” are used interchangeably herein.
  • Subtilisin is a protease initially obtained from Bacillus subtilis.
  • peptides are used interchangeably herein and refer to a polymer of amino acids joined together by peptide bonds.
  • a “protein” or “polypeptide” comprises a polymeric sequence of amino acid residues.
  • the single and 3- letter code for amino acids as defined in conformity with the lUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), well known to those skilled in the art, is used throughout this disclosure.
  • the single letter X refers to any of the twenty amino acids. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
  • Mutations can be named by the one letter code for the parent amino acid, followed by a position number and then the one letter code for the variant amino acid.
  • mutating glycine (G) at position 87 to serine (S) is represented as "G087S” or "G87S”.
  • a position followed by amino acids listed in parentheses indicates a list of substitutions at that position by any of the listed amino acids.
  • 6(L, I) means position 6 can be substituted with a leucine or isoleucine.
  • a slash (/) is used to define substitutions, e.g., F/V, indicates that the position may have a phenylalanine or valine at that position.
  • amino acid refers to the basic chemical structural unit of a protein, peptide, or polypeptide.
  • corresponding to or “corresponds to” or “correspond to” or “corresponds” refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide.
  • corresponding region generally refers to an analogous position in a related protein or a reference protein.
  • variant proteins encompass “variant” or “mutant” proteins, which terms are used interchangeably herein.
  • Variant proteins differ from another (/.e., parental) protein and/or from one another by a small number of amino acid residues.
  • a variant may include one or more amino acid mutations (e.g., amino acid deletion, insertion or substitution) as compared to the parental protein from which it is derived.
  • variants may have a specified degree of sequence identity with a reference protein or nucleic acid, e.g., as determined using a sequence alignment tool, such as BLAST, ALIGN, and CLUSTAL.
  • variant proteins or nucleic acid may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% amino acid sequence identity with a reference sequence and integer percentage therebetween [0036]
  • wild-type in reference to an amino acid sequence or nucleic acid sequence indicates that the amino acid sequence or nucleic acid sequence is a native or naturally-occurring sequence.
  • naturally-occurring refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature.
  • non-naturally occurring refers to anything that is not found in nature (e.g., recombinant/engineered nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).
  • derived from encompasses the terms “originated from,” “obtained from,” “obtainable from,” “isolated from,” “purified from,” and “created from,” and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to another specified material.
  • isolated refers to a material (e.g., a protein, nucleic acid, or cell) that is removed from at least one component with which it is naturally associated.
  • these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.
  • An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • a method for activating a zymogen of a transglutaminase comprising contacting the zymogen with at least one immobilized protease to produce an active form of transglutaminase and, optionally, separating the activated transglutaminase from the immobilized protease.
  • the transglutaminase is a microbial Tgase
  • the microbial Tgase is a bacterial transglutaminase, such as a Streptomyces Tgase
  • the bacterial Tgase is from Streptomyces mobaraensis, or a variant thereof.
  • Streptomyces mobaraensis belongs to a large group of Gram-positive, filamentous soil bacteria with a complex life cycle.
  • Transglutaminase variants and methods of producing such variants are disclosed, for example, in PCT Publication Numbers WO 2016/170447, published on October 27, 2016, and WO 2019/094301 , published on May 16, 2019.
  • zymogen of a transglutaminase requires cleavage of a pro-sequence from a zymogen form of the enzyme, resulting in mature, catalytically active enzyme.
  • a zymogen of a Tgase is contacted with at least one immobilized protease to produce an activated form of a Tgase, and optionally, separating the activated Tgase from the immobilized protease.
  • Any protease that produces an activated form of a Tgase from the zymogen of a Tgase may be used as described herein.
  • Nonlimiting examples of proteases that may be immobilized for use in cleaving a pro-sequence from a Tgase as described herein include, but are not limited to, bacterial and fungal proteases.
  • the bacterial protease is a subtilisin.
  • trypsin proteinase K; proteinase T; Dispase® I; Dispase® II; Papain®; Bromelain®; Ficin®; Actinidine®; Protex 6L, Multifect® PR 6L; Protex® 7L, Multifect® PR 7L; Protex® 14L, Multifect® PR 14L; Protex® 15L, Multifect® PR 15L; Protex® 30L, Multifect® PR 30L; Protex® 40L, Optimase® PR 40L; Protex® 50FP, Multifect® PR 50G; Protex® 51 FP, Multifect® PR 51 G; Protex® 89L, Optimase® PR 89L, Alcalase®, Savinase®, Everlase®, Esperase®, Flavourzyme®, Neutrase®.
  • immobilized proteases such as Alcalase®, Savinase®, Everlase®, or Esperase®, are commercially available as a protease pre-immobilized and conjugated to a support matrix.
  • Conditions for contacting the zymogen of a Tgase with an immobilized protease include any conditions that are suitable for activity of a selected protease(s). Such conditions are readily ascertainable by one of skill in the art.
  • Activated zymogens of any of the Tgases described herein may be used in a variety of applications such as, but not limited to, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, energy (e.g., fracking fluid), plastic, packaging, and agricultural products.
  • the activated Tgase may be incorporated into HVAC systems, cooling ponds, water purification systems, or may be used in an industrial application, such as, but not limited to, pulp and paper processing.
  • the activated zymogens of Tgases may be used in healthcare products, personal care or cosmetic formulations, packaging (food, cosmetic, and pharmaceuticals), textile and leather production, paints and coatings, and marine applications including water treatment and purification.
  • the activated zymogens of Tgases may be employed for permanently modifying proteins of interest, by way of example keratin and collagen, with dyes or proteins.
  • the activated zymogen of any of the Tgases described herein is included in a personal care product, such as, but not limited to, bar soap, liquid soap (e.g., hand soap), hand sanitizer (including rinse off and leave-on alcohol based and aqueous- based hand disinfectants), preoperative skin disinfectant, cleansing wipes, disinfecting wipes, body wash, acne treatment products, antifungal diaper rash cream, antifungal skin cream, shampoo, conditioner, cosmetics (including but not limited to liquid or powder foundation, liquid or solid eyeliner, mascara, cream eye shadow, tinted powder, "pancake” type powder to be used dry or moistened, make up removal products, etc.), deodorant, antimicrobial creams, body lotion, hand cream, topical cream, aftershave lotion, skin toner, mouth wash, toothpaste, sunscreen, and baby products such as, but not limited to, cleansing wipes, baby shampoo, baby soap, and diaper cream.
  • a personal care product such as, but not limited to, bar soap, liquid soap (
  • an activated zymogen of a Tgase may be included in a wound care item, such as, but not limited to, wound healing ointments, creams, and lotions, wound coverings, burn wound cream, bandages, tape, and steri-strips, and medical articles such as medical gowns, caps, face masks, and shoe-covers, surgical drops, etc.
  • the activated zymogen, of any of the Tgases described herein may be included in an oral care product, such as mouth rinse, toothpaste, or dental floss coating, a veterinary or pet care product, a preservative composition, or a surface disinfectant, such as a disinfectant solution, spray or wipe.
  • an oral care product such as mouth rinse, toothpaste, or dental floss coating
  • a veterinary or pet care product such as a veterinary or pet care product
  • a preservative composition such as a veterinary or pet care product
  • a surface disinfectant such as a disinfectant solution, spray or wipe.
  • a product or composition which includes an activated zymogen of any of the Tgases as described herein may further include one or more additional enzymes selected from acyl transferases, alpha-amylases, beta-amylases, alpha- galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1 ,4- glucanases, endo-beta-mannases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases,
  • an activated zymogen of any of the Tgases described herein may be included in a product to be used for long-lasting application of functional ingredients including UV-blocking sunscreens, and/or coloring agents, such as pigments or dyes.
  • such activated zymogens may be incorporated in a product to be applied topically and which bonds to the skin of an individual, such as a UV-blocking (sunscreen) product, or a cosmetic product.
  • the activated zymogen may be used to provide permanent application of color to the skin of an animal such as in leather processing.
  • the activated zymogen may be used to provide a permanent application of color in food processing.
  • Non-limiting embodiments of the foregoing disclosed herein include: [0055] 1. A method for activating a zymogen of a transglutaminase, said method comprising contacting the zymogen with at least one immobilized protease to produce an active form of transglutaminase and, optionally, separating the activated transglutaminase from the immobilized protease.
  • transglutaminase is a microbial transglutaminase.
  • Streptomyces mobaraensis transglutaminase or a variant thereof.
  • Three different assays can be used to assess activity of an activated Tgase derived from a zymogen of Tgase and activated using at least one immobilized protease as described herein.
  • Transglutaminase activity was measured herein using a colorimetric hydroxamate activity assay (Folk and Cole (1965) J Biol Chemistry 240(7) :2951-2960). Briefly, the hydroxamate assay uses N-benzyloxycarbonyl-L-glutaminyl-glycine (ZQG) as a low molecular weight amine acceptor substrate and hydroxylamine as an amine donor. In the presence of transglutaminase, the hydroxylamine is incorporated to form Z- glutamylhydroxamate-glycine, which develops a colored complex with iron (III), detectable at 525 nm after incubation at 37 °C for 5-60 minutes.
  • ZQG N-benzyloxycarbonyl-L-glutaminyl-glycine
  • ZQG N-benzyloxycarbonyl-L-glutaminyl-glycine
  • the hydroxylamine is incorporated to form Z- glutamylhydro
  • Tgase L- glutamic acid g -monohydroxamate (Millipore Sigma) as standard.
  • One unit of Tgase is defined as the amount of enzyme that catalyzes formation of 1 pmol of the peptide derivative of g-glutamylhydroxylamine per minute.
  • Transglutaminase activity is monitored by measuring the fluorescence (excitation wavelength 332 nm; emission wavelength 500 nm) using a BioTek Synergy H1 microplate reader.
  • Transglutaminase-catalyzed covalent coupling (crosslinking) of monodansylcadaverine with N,N-dimethylcasein (high molecular weight substrate) produces a product that causes a shift in intensity and wavelength of fluorescence of the dansyl group now linked to the casein.
  • the relative transglutaminase activity is shown by increase of fluorescence intensity overtime.
  • the samples are then run on an Agilent 1100 series HPLC through a Zorbax Eclipse Plus C18 column at 20-65% 0.08% TFA in Acetonitrile. Percent conversion of substrates to cross-linked product was calculated from the HPLC trace data to assess activity of the Tgase activated from a zymogen using at least one immobilized protease.
  • the gene coding for the pro-Tgase was codon optimized for expression in E. coli based on the published amino acid sequence (Kanaji, et al. (1993) J. Biol. Chem.
  • the expression vector also contains the pMB1 origin of replication and a kanamycin resistance gene.
  • the resulting plasmid was transformed first into E. coli DH-10B, using standard methods known in the art. The transformants were isolated by subjecting the cells to kanamycin selection, as known in the art (See, e.g., US Pat. No. 8,383,346 and W02010/144103, both of which are incorporated by reference herein, in their entirety), and the sequence of the pro-Tgase gene was verified by Sanger sequencing.
  • the plasmid was recovered from a positive clone, using methods known in the art, and transformed into E. coli BL21 (DE3) for expression.
  • Cells were harvested by centrifugation at 8000 x g for up to 60 minutes. The supernatant was discarded, and the pellet was resuspended to 20% w/v in 50 mM tris(hydroxymethyl)aminomethane (Tris) HCI, pH 8. The cells were lysed using a high- pressure homogenizer at pressures from 15000-20000 psi. The crude lysates were clarified through centrifugation at 15000 x g for up to 60 minutes.
  • Tris tris(hydroxymethyl)aminomethane
  • the clarified lysate containing pro- Tgase was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE), by spectroscopy, and Tgase activity (as described in Example 1).
  • Results depicted in Figure 1 show that the zymogen of a Tgase, i.e., pro-Tgase, was expressed in E.coli.
  • the pro-Tgase may be secreted, for example, from a microbial strain that is known to those skilled in the art to secrete Tgase such as Streptomyces mobaraensis or Bacillus subtilis. The pellet is discarded, and the supernatant is recovered and assessed by SDS-PAGE, by spectroscopy, and activity (as described in Example 1 above).
  • Clarified lysate containing pro-Tgase obtained as described in Example 2 above, was treated with a panel of commercially available immobilized proteases (Chiralvision Immozyme Protease Kit containing 20 immobilized proteases, Product Number: IMMPROT- 2000).
  • the protease treated clarified lysates were incubated with shaking at 30-45 °C, pH 5- 8, for 0.1-24 hours. Protease loading was varied and optimized to deliver maximum activity within the fastest time.
  • the protease-treated enzymes were compared on an SDS-PAGE gel, by spectroscopy, and by activity (as described in Example 1 above) for active enzyme.
  • the products of activations described in Examples 3, 4, and 5 were analyzed using SDS- PAGE gels to visualize the ratio of Pro-Tgase to the active form of Tgase.
  • Results are set forth in Table 1 and show relative activity of activated pro-Tgase (fold increase over activity of expressed zymogen, which may have some native activation during cell culture).
  • Clarified lysate containing pro-Tgase was treated with either immobilized Protex® 30L (Multifect ®PR 30L); or immobilized Protex® 40L (Optimase® PR 40L) (Chiralvision; Product Numbers: IMMP30-COV-2, IMMP40-COV-2, respectively).
  • the protease-treated clarified lysates were incubated with shaking at 30 or 45 °C, pH 5-9 for 0.1-24 hours. Protease loading was varied and optimized to deliver maximum activity within the fastest time.
  • the protease treated enzymes were compared on an SDS- PAGE gel, by spectroscopy, and by activity (as described in Example 1) for active enzyme. Results are shown in Table 2.
  • Example 5 Substrate Specificity of Activated Pro-Tgase Using Immobilized Protease [0081] Clarified lysate containing Pro-Tgase was prepared as described in Example 2.
  • the lysate was diluted to a final concentration of 0.5-3 g/L Pro-Tgase at pH 5-8 using bis-tris propane, phosphate, tris-acetate, or similar buffer.
  • a sample of 5-30 mg of immobilized protease was added to a 0.75 mL glass vial. Then, 0.4-0.6 mL of the buffered the lysate was added.
  • a total of 22 immobilized proteases (Chiralvision Immozyme Protease Kit containing 22 immobilized proteases, Product Number: IMMPROT-2200) were tested. The mixture was incubated at 30-45°C, while mixing at 200-400 rpm. Product was sampled at time points from 0.1-24 hours for further testing.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne des procédés d'activation de zymogènes de transglutaminases impliquant l'utilisation d'au moins une protéase immobilisée ainsi que des transglutaminases activées fabriquées selon de tels procédés.
PCT/US2022/019738 2021-03-10 2022-03-10 Activation de zymogènes par des enzymes protéases immobilisées Ceased WO2022192529A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163159313P 2021-03-10 2021-03-10
US63/159,313 2021-03-10

Publications (1)

Publication Number Publication Date
WO2022192529A1 true WO2022192529A1 (fr) 2022-09-15

Family

ID=81308435

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/019738 Ceased WO2022192529A1 (fr) 2021-03-10 2022-03-10 Activation de zymogènes par des enzymes protéases immobilisées

Country Status (1)

Country Link
WO (1) WO2022192529A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993015234A1 (fr) * 1992-01-22 1993-08-05 Novo Nordisk A/S Facteur xiii active
WO2010144103A1 (fr) 2009-06-11 2010-12-16 Codexis, Inc. Synthèse en parallèle automatisée combinée de variants polynucléotidique
WO2011133704A2 (fr) * 2010-04-20 2011-10-27 Whitehead Institute For Biomedical Researh Protéines et polypeptides modifiés et leurs utilisations
US8383346B2 (en) 2008-06-13 2013-02-26 Codexis, Inc. Combined automated parallel synthesis of polynucleotide variants
WO2016170447A1 (fr) 2015-04-24 2016-10-27 Rinat Neuroscience Corp. Transglutaminases microbiennes recombinantes
WO2019094301A1 (fr) 2017-11-07 2019-05-16 Codexis, Inc. Variants de transglutaminase

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993015234A1 (fr) * 1992-01-22 1993-08-05 Novo Nordisk A/S Facteur xiii active
US8383346B2 (en) 2008-06-13 2013-02-26 Codexis, Inc. Combined automated parallel synthesis of polynucleotide variants
WO2010144103A1 (fr) 2009-06-11 2010-12-16 Codexis, Inc. Synthèse en parallèle automatisée combinée de variants polynucléotidique
WO2011133704A2 (fr) * 2010-04-20 2011-10-27 Whitehead Institute For Biomedical Researh Protéines et polypeptides modifiés et leurs utilisations
WO2016170447A1 (fr) 2015-04-24 2016-10-27 Rinat Neuroscience Corp. Transglutaminases microbiennes recombinantes
WO2019094301A1 (fr) 2017-11-07 2019-05-16 Codexis, Inc. Variants de transglutaminase

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
FOLKCOLE, J BIOL CHEMISTRY, vol. 240, no. 7, 1965, pages 2951 - 2960
HALEMARHAM: "THE HARPER COLLINS DICTIONARY OF BIOLOGY", 1991, HARPER PERENNIAL
KANAJI ET AL., J. BIOL. CHEM., vol. 268, no. 16, 1993, pages 11565 - 11572
MARX C K ET AL: "Purification and activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli and partial characterization of the active enzyme", ENZYME AND MICROBIAL TECHNOLOGY, STONEHAM, MA, US, vol. 42, no. 7, 5 June 2008 (2008-06-05), pages 568 - 575, XP022635369, ISSN: 0141-0229, [retrieved on 20080314], DOI: 10.1016/J.ENZMICTEC.2008.03.003 *
MING-TE YANG ET AL: "Crystal Structure and Inhibition Studies of Transglutaminase from Streptomyces mobaraense*", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 286, no. 9, 4 March 2011 (2011-03-04), pages 7301 - 7307, XP055857780, DOI: 10.1074/jbc.M110.203315 *
NAVEED MUHAMMAD ET AL: "Protease-A Versatile and Ecofriendly Biocatalyst with Multi-Industrial Applications: An Updated Review", CATALYSIS LETTERS, vol. 151, no. 2, 11 July 2020 (2020-07-11), pages 307 - 323, XP037366385, ISSN: 1011-372X, DOI: 10.1007/S10562-020-03316-7 *
PASTERNACK ET AL: "Bacterial pro-transglutaminase from Streptoverticillium mobarense", EUROPEAN JOURNAL OF BIOCHEMISTRY, PUBLISHED BY SPRINGER-VERLAG ON BEHALF OF THE FEDERATION OF EUROPEAN BIOCHEMICAL SOCIETIES, vol. 257, 1 January 1998 (1998-01-01), pages 570 - 576, XP002127089, ISSN: 0014-2956, DOI: 10.1046/J.1432-1327.1998.2570570.X *
SINGLETON ET AL.: "DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY", 1994, JOHN WILEY AND SONS
ZHANG DONGXU ET AL: "Microbial transglutaminase production: understanding the mechanism", BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS, INTERCEPT LTD., ANDOVER, GB, vol. 26, 1 January 2010 (2010-01-01), pages 205 - 221, XP009190210, ISSN: 0264-8725 *

Similar Documents

Publication Publication Date Title
US7795382B2 (en) Peptide tags for the expression and purification of bioactive peptides
US8748399B2 (en) Acid-cleavable linkers exhibiting altered rates of acid hydrolysis
US7794979B2 (en) Solubility tags for the expression and purification of bioactive peptides
CN103282016A (zh) 用于递送使用过乙酸的脱毛剂产品底物的含水稳定组合物
US7829311B2 (en) Ketosteroid isomerase inclusion body tag engineered to be acid-resistant by replacing aspartates with glutamate
CA3194036A1 (fr) Compositions et leurs procedes d'utilisation
KRAMER et al. Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding
US7998702B2 (en) Mutant arabinose promoter for inducible gene expression
Goshev et al. Characterization of the enzyme complexes produced by two newly isolated thermophylic actinomycete strains during growth on collagen-rich materials
Clark et al. Purification and characterization of a unique alkaline elastase from Micrococcus luteus
WO2022192529A1 (fr) Activation de zymogènes par des enzymes protéases immobilisées
US20250304927A1 (en) Pro-peptide variants for modulating activity of transglutaminases
Santra et al. Studies on Vibrio mimicus derived collagenase variants providing insights into critical role (s) played by the FAXWXXT motifs in its collagen-binding domain
Avdiyuk et al. Keratinolytic enzymes: producers, physical and chemical properties. Application for biotechnology
Goodfellow et al. Characterization of a maize root proteinase
CN115975995A (zh) 一种无胶原蛋白水解活性的角蛋白酶突变体
Maeda et al. Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate
US20240093162A1 (en) Immobilized proteases for activation of the zymogen form of transglutaminase
Chauhan et al. Production and partial purification of collagenase from Bacillus sp. isolated from soil sample
WO2024054236A1 (fr) Protéases immobilisées pour l'activation de la forme zymogène de la transglutaminase
Luo et al. Couple action of some proteases from feather keratin-degrading bacterium B. licheniformis CP-16
Thu et al. Studies on a novel recombinant lipase A from Bacillus subtilis FS2
Jankiewicz et al. An activated by cobalt alkaline aminopeptidase from Bacillus mycoides
Mattey et al. Enzymes with a low molecular weight
Kimura 4.2 Chemical and Genetic Mutation of Enzymes for Creating Novel Catalysts

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22716609

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 23.01.2024)

122 Ep: pct application non-entry in european phase

Ref document number: 22716609

Country of ref document: EP

Kind code of ref document: A1