Compositions and methods for enzyme immobilization
Inventors
SHAKEEL, AMEER HAMZA • DAVIS, ZACHERY GEORGE • FRANK, Joseph Thomas • ZOMORODI, SEPEHR • POURTAHERI, PAYAM
Assignees
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Publication Number
US-11624061-B2
Publication Date
2023-04-11
Expiration Date
Abstract
The present disclosure relates to compositions of immobilized enzymes on the surface of achromosomal and/or anucleate cells and uses thereof. In particular, the present disclosure provides genetically engineered minicells with enzymes self-assembled on their surface. The immobilized enzymes on the surface of achromosomal and/or anucleate minicells, has agricultural, industrial, and environmental applications due to their improved stability durability and, reusability. Also, provided are methods for producing and purifying enzyme-immobilized minicells.
Core Innovation
The invention provides an industrially suitable anucleated cell-based enzyme immobilization and delivery platform using an intact anucleated cell derived from a protease deficient parental cell. The anucleated cell comprises at least one expressed self-assembled enzyme immobilized to the surface of the cell, and the platform further includes at least one fusion protein comprising at least one surface expressing moiety and at least one cell adhesion moiety.
The surface expressing moiety enables surface localization of the fusion protein, and the cell adhesion moiety supports cell adhesion to a desired surface. Surface expressing moieties include transmembrane or auto-transporter moieties, such as INP, BrkA, and AIDA, as well as exported bacterial proteins such as LamB, OprF, OmpA, Lpp, MalE, PhoA, Bla, and F1/M13 coat proteins.
The platform supports display of multiple enzymes, including multi-display and co-localization at a desired locus, where different expressed self-assembled enzymes maintain enzymatic activity at the locus. The multi-enzyme configuration is characterized by complementary or synergistic action, or by partitioning an overall enzymatic reaction into portions.
Claims Coverage
Across the independent claims, the inventive coverage centers on 4 inventive features: protease deficient parental cell-derived intact anucleated cells with surface-immobilized self-assembled enzymes, fusion proteins with surface expressing moieties and cell adhesion moieties, and multi-enzyme configurations with specified enzymatically active moieties.
Protease deficient parental cell-derived intact anucleated cell enzyme immobilization platform
An intact anucleated cell derived from a protease deficient parental cell, wherein the anucleated cell comprises at least one expressed self-assembled enzyme immobilized to the surface of said cell, and at least one fusion protein comprising at least one surface expressing moiety and at least one cell adhesion moiety.
Two surface-immobilized self-assembled enzyme fusion constructs with distinct enzymatically active moieties: lipase and non-lipase
An intact anucleated cell derived from a protease deficient parental cell, wherein the anucleated cell comprises at least two different expressed self-assembled enzymes immobilized to a surface of said cell, and each expressed self-assembled enzyme is a fusion protein comprising at least one surface expressing moiety and at least one enzymatically active moiety, with one enzymatically active moiety being lipase and the other not lipase.
Two surface-immobilized self-assembled enzyme fusion constructs with distinct enzymatically active moieties: glucose isomerase and non-glucose isomerase
An intact anucleated cell derived from a protease deficient parental cell, wherein the anucleated cell comprises at least two different expressed self-assembled enzymes immobilized to a surface of said cell, and each expressed self-assembled enzyme is a fusion protein comprising at least one surface expressing moiety and at least one enzymatically active moiety, with one enzymatically active moiety being glucose isomerase and the other not glucose isomerase.
Two surface-immobilized self-assembled enzyme fusion constructs with distinct enzymatically active moieties: protease and non-protease
An intact anucleated cell derived from a protease deficient parental cell, wherein the anucleated cell comprises at least two different expressed self-assembled enzymes immobilized to a surface of said cell, and each expressed self-assembled enzyme is a fusion protein comprising at least one surface expressing moiety and at least one enzymatically active moiety, with one enzymatically active moiety being protease and the other not protease.
The claims cover protease deficient parental cell-derived intact anucleated cells presenting expressed self-assembled enzymes immobilized on the cell surface, using fusion proteins that include surface expressing moieties and cell adhesion moieties, together with multi-enzyme arrangements defined by lipase, glucose isomerase, or protease versus corresponding non-matching activities.
Stated Advantages
Improved enzyme stability.
Improved enzyme durability.
Improved enzyme reusability.
Documented Applications
Agricultural uses.
Industrial uses.
Environmental uses.
Detergent.
Baking.
Pulp/paper.
Biofuels.
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