Recombinant Enzyme

Modality

Recombinant Enzymes

Recombinant Enzyme for Therapeutic Use

Therapeutic enzymes obtained through biotechnological processes are widely used for several clinical applications, including enzyme replacement therapy, degradation of accumulated metabolites and toxin, cancer treatment, and genome editing. Several therapeutic enzymes can be produced in microbial expression system.

Application	Enzymes	Substance	Typical Products/Pipelines
Enzyme Replacement Therapy	Adenosine deaminase, ADA	Adenosine or deoxyadenosine	Elapegademase-lvlr (Revcovi)
Enzyme Replacement Therapy	Phenylalanine ammonia lyase, PAL	Phenylalanine	Pegvaliase-pqpz (Palynziq)
Degradation of Accumulated Metabolites	Uricase/Urate Oxidase	Uric acid	Peglticase (Krystexxa)Rasburicase (Fasturtec)
	Collagenase	Collagen “scar”	Collagenase clostridium histolyticum (Xiaflex, Qwo, Santyl)
	Truncated plasmin	Collagen, fibronectin, and laminin	Ocriplasmin (Jetrea)
	Truncated tissue plasminogen activator (tPA)	Plasminogen	Reteplase (Retavase)
	IgG protease	Immunoglobulin G (IgG)	Imlifidase (Idefrix)
	IgA protease	Immunoglobulin A (IgA)	PKU308/AP308IGAN IgA protease
Toxin Degradation	Carboxypeptidase G2	Methotrexate	Glucarpidase (Voraxaze)
Cancer treatment	Asparagine-specific enzyme	Asparagine	Asparaginase (Elspar)Calaspargase pegol-mknl (Asparlas)
Genome Editing	Cas9 nuclease	Target gene	Exagamglogene autotemcel (Exa-cel, CTX001)

Recombinant Enzyme as Material

There are several enzymes used in long coding RNA production (e.g., mRNA, saRNA, circRNA), antibody–drug conjugates (ADCs) production, and others. Microbial produced enzymes are more cost-effective than mammalian cell, especially E. coli expression system.

Application	Enzymes	Function
Enzymes for Linear RNA Production, RNase-free	Restriction endonucleases	Linearization of plasmid DNA (pDNA) to avoid generating longer transcript.
	T7 RNA polymerase (T7 RNAP)	Bind to the T7 promoter and generate specific RNA transcript; Play a key role during in vitro transcript (IVT) reaction.
	Vaccinia capping enzyme	Add Cap structures to the 5′ ends of IVT mRNA.
	Pyrophosphatase, inorganic (iPPase)	Prevent pyrophosphate during IVT reaction.
	RNase inhibitor, recombinant	Inhibit RNase activity during IVT reaction.
	DNase I	Remove the DNA template.
Enzyme for circRNA production, RNase-free	RNase R	Digest linear RNA and enrich circular RNA.
Enzyme-mediated glycan conjugation	Peptide-N-glycosidase (PNGase F)	Cleaves the amide bond between the first saccharide GlcNAc and the Asn297 side chainL; Release glycans from IgG antibodies.
	Bacterial transglutaminase (BTG)	Conjugate payloads site-speciﬁcally to generate ADCs.
	Sortase A	Catalyze the ligation of proteins.
	β1,4-galactosidase	Release all terminal galactoses and form a homogenous G0 isoform of the antibody.
	β1,4-galactosyltransferase (Gal-T)	Transfer a sugar residue with a chemically reactive functional group.
	α2,6-sialyltransferase (Sial T)	Incorporate terminal sialic acid residues into the native glycan structure of an antibody.
Enzyme-mediated glycan remodeling and glycoengineering	Endo-N-acetylglucosaminidase (ENGase)	Hydrolyze the β1,4-glycosidic bond between GlcNAcβ1–4GlcNAc of N-glycans; Remove N-linked glycans on the Fc region of IgG antibodies.
	Endoglycosidase S (EndoS)
	Glycosyltransferases (GTs)	Transfer N-glycan oxazoline to defucosylated IgGs.
Others	Enzymes produced by microbial strain	Customized need

Recombinant Enzyme as Reagent

Tag Removal Proteases

Fusion tags are often used to improve the solubility and stability of recombinant proteins of interest and make them easier to be purifypurified. The commonly used tags include His-tag, maltose-binding protein (MBP), glutathione-s-transferase (GST), etc.

Typically, a linker sequence is added between the fusion tag and target protein sequence to remove the tag. The removal of fusion tags requires site-specific proteases, such as enterokinase (EK), thrombin, tobacco etch virus protease (TEVp), human rhinovirus protease 3C (HRV3C), small ubiquitin modifying protein (SUMO) protease, tobacco vein mottling virus (TVMV) protease, and carboxypeptidase A/B (CPA/CPB).

Type	Enzymes	Recognition Site
Tag removal endoproteases	Enterokinase (EK), enteropeptidase	DDDDK↓
	thrombin	LVPR↓GS
	TEV protease	ENLYFQ↓G
	HRV3C protease	LEVLFQ↓GP
	SUMO protease	SUMO tertiary structure
	TVMV protease	ETVRFQG↓S
Tag removal exoproteases	Carboxypeptidase A (CPA)	C-terminal amino acids, except Pro, Lys and Arg
Tag removal exoproteases	Carboxypeptidase B (CPB)	C-terminal Lys and Arg

Other Proteases, Nucleases and Amidases

Application	Enzymes	Function
Other proteases	Protease K	A serine protease that digests proteins by hydrolyzing peptide bonds.
	IdeS, IgG protease	IgG degrading enzymes (Ides) that cleave at a specific site of immunoglobulin G (IgG), generating Fab and Fc fragments
	IgA1 protease	A proteolytic enzyme that cleaves a specific site in the human immunoglobulin A1 (IgA1) hinge region sequence.
Nuclease	Nuclease	Cleaves nucleic acids (DNA or RNA) by hydrolyzing the phosphodiester bonds.
Nuclease	Restrict enzyme	An endonuclease that cleaves DNA at or near specific sites.
Amidase	PNGase F	Cleaves between the innermost N-acetylglucosamine (GlcNAc) and asparagine residues of high mannose, hybrid, and complex oligosaccharides.

Yaohai Bio-Pharma Offers One-Stop CDMO Solution for Recombinant Enzymes

Reference:

[1] Hennigan JN, Lynch MD. The past, present, and future of enzyme-based therapies. Drug Discov Today. 2022 Jan;27(1):117-133. doi: 10.1016/j.drudis.2021.09.004.

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