# US 12,180,465 B2 · Transglutaminase nanoflowers

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- Number: US 12,180,465 B2
- Document ID: US12180465B2
- Status: Granted
- Kind: B2
- Application number: 17/406,928
- Filed: 2021-08-19
- Published: 2022-02-24
- Granted: 2024-12-31
- Earliest claimed: 2020-08-20
- Assignees: Indian Institute of Technology Delhi
- Applicants: Indian Institute of Technology Delhi
- Inventors: Sunil Kumar Khare; Syeda Warisul Fatima
- Web page: https://patentgrep.com/patent/US12180465B2

## CPC

- C12N 11/14: Enzymes or microbial cells immobilised on or in an inorganic carrier
- C12N 9/1044: {Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII}
- C12Y 203/02013: Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The present invention relates to novel immobilized nanoflowers made of Transglutaminase (TGase), a method for their synthesis and applications thereof. The Transglutaminase nanoflowers of the present invention are catalytically more active without mass transfer limitation, stable and reusable, wherein the overall flower is in the nano-range, is homo-dispersed and small-sized, and wherein Transglutaminase functions both as a cross-linking enzyme and as a seeding template.

## Claims

### Claim 1

Immobilized Transglutaminase nanoflowers, comprising:
Transglutaminase and an inorganic metal, wherein the overall flower is in the nano-range, is homo-dispersed and small-sized, and wherein
the transglutaminase is a template for the formation of the flowers that are immobilize to the structure of the transglutaminase, and
wherein said nanoflowers can tolerate cold temperature and possess higher durability in terms of organic solvents, temperature, pH, storage and operational stability, having higher catalytic efficiency of 6.92 fold as compared to free Transglutaminase without any mass transfer limitation.

### Claim 2 (depends on 1)

The nanoflower as claimed in claim 1 wherein the size of the nanoflowers is <50 nm.

### Claim 3 (depends on 1)

The nanoflower as claimed in claim 1 wherein the source of Transglutaminase is extremophilic actinomycetes Streptomyces mobaraensis.

### Claim 4 (depends on 1)

The nanoflower as claimed in claim 1, wherein said inorganic metal is selected from the group comprising zinc, iron, manganese, magnesium, potassium, calcium and copper.

### Claim 5 (depends on 1)

The nanoflower as claimed in claim 1, wherein said inorganic metal is copper.

### Claim 6 (depends on 1)

The nanoflowers as claimed in claim 1, wherein said nanoflower is homo-dispersed and less than 50 nm in size.

### Claim 7 (depends on 1)

The nanoflowers as claimed in claim 1, wherein said nanoflowers have uniform cages/pockets in between the petals.

### Claim 8 (depends on 1)

The nanoflowers as claimed in claim 1, wherein said nanoflower is bio-catalytically active.

### Claim 9 (depends on 1)

The nanoflowers as claimed in claim 1, wherein nanoflowers display a 3-D structure.

### Claim 10 (depends on 1)

The nanoflowers as claimed in claim 1, wherein the activity of the nanoflower is increased 156.74% over free TGase.

### Claim 11 (depends on 1)

The nanoflower as claimed in claim 1, wherein the structure of the petals is porous in nature.

### Claim 12 (depends on 1)

The nanoflowers as claimed in claim 1, wherein the nanoflower retains the activity even after ten cycles.

### Claim 13 (depends on 1)

The nanoflowers as claimed in claim 1, wherein said nanoflowers display controlled and targeted release of bioactive drugs and heavy metals.

### Claim 14 (depends on 1)

A one-pot green synthesis process for designing the Transglutaminase nanoflower of claim 1, said process comprising the steps of:
a. solubilizing 200 ul CuSO4 in Milli-Q water;
b. adding to 30 ml of 2×PBS (pH-7.4) with 3 mg/ml enzyme Transglutaminase;
c. incubating the solution from step b for three days at 4° C.;
d. sonicating the suspension obtained from step c for 15 min;
e. drying and filtering to obtain the immobilized Transglutaminase nanoflowers.

### Claim 15 (depends on 14)

The process as claimed in claim 14, wherein the transglutaminase is a template for the formation of the flowers that are immobilize to the structure of the transglutaminase.