Molecular machines

A technology of cofactors, complexes, applied in chemical instruments and methods, immobilized on/in organic carriers, enzymes, etc.

Inactive Publication Date: 2018-07-17
COMMONWEALTH SCI & IND RES ORG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, one of the major limitations of current enzyme systems applied to energy-intensive synthetic reactions is the cost of providing a continuous supply of diffusible cofactors or cosubstrates (Zhao et al., 2003)

Method used

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Embodiment 1

[0297] Example 1 - Construction and demonstration of a dual-enzyme fusion protein

[0298] 22 enzymes were evaluated for the synthesis steps of DHAP from glycerol (region-specific phosphorylation and oxidation) and proper cofactor recycling. The four best enzyme combinations were then used to synthesize dual-enzyme fusion proteins. Each fusion protein produced is a single molecule encoding two functionalities (DHAP synthesis step and cognate cofactor recycling).

[0299] By combining the gene encoding the related enzyme with the encoding containing GlySerSer repeat sequence (GSS) n (GSS) 3 C(GSS) 3 ) to generate a dual-enzyme fusion protein.

[0300] Bienzymatic Fusion 1 (BiF1) contains the optimal enzymes for glycerol-3-phosphate production and ATP regeneration (Thermococcus Kagoshima glycerol kinase [TkGlpK] and Mycobacterium smegmatis ATP kinase [MsAK]).

[0301] Bienzymatic Fusion 2 (BiF2) contains the optimal enzymes to generate DHAP from glycerol-3-phosphate and r...

Embodiment 3

[0412] Example 3 - Nanofactory Comprising Three Nanomachine Flow Reactors

[0413] Preparation of Sepharose Beads with Immobilized 1,1,1-Trifluoro-3-((6-Mercaptohexyl)thio)propan-2-one (TFK)

[0414] Add saturated NaHCO to a slurry of vinylsulfone-activated agarose (800 mL, 600-800 mmol vinylsulfone groups, 50% slurry in 1:1 ethanol / water) 3 Aqueous solution (80 mL), 1,1,1-trifluoro-3-((6-mercaptohexyl)thio)propan-2-one (104 mg, 0.4 mmol) dissolved in ethanol (4.8 mL). The mixture was stirred slowly overnight at room temperature. Excess reaction sites were blocked by addition of 2-mercaptoethanol (11.2 mL, 80 mmol), and stirring was continued for 6 hours. The resin was then thoroughly washed with 50% ethanol / water until no noticeable odor. Beads were stored as a 1:1 slurry in 50% ethanol / water.

[0415] Triple multienzyme reactor using fusion enzymes immobilized on TFK-derived agarose beads

[0416] TriF2 with tethered mNAD (EcG3PD-CaNOX-AaE2), galactose oxidase M3-5 -...

Embodiment 4

[0431] Example 4 - Biocatalytic Flow Reactor

[0432] D-buckwheat alkali nanofactory

[0433] The functionality of immobilized nanomachines in a reactor that retains and recycles cofactors for flow biocatalysis was demonstrated via the production of the important commercially relevant antidiabetic drug D-buckwine. D-buckwine can be enzymatically converted from glycerol via two regiospecific cofactor-dependent steps (ATP-dependent phosphorylation and NAD-dependent oxidation) and stereospecific aldol condensation) followed by chemical cyclization. produce( Figure 36 ).

[0434] phosphorylation transfer reactor

[0435] In order to prepare the TriF1 phosphotransfer reactor ( Figure 36 In step 1), 40 mg of TriF1 protein (296 nmol) was immobilized on 25 g of Sepharose-Hexyl-DVS-TFK beads. Immobilized TriF1 was treated with TCEP, washed with degassed spray PBS containing 0.5 mM EDTA, and then incubated at 4 °C with 6 equivalents of ADP-2AE-PEG 24 -NAD was reacted for 6 hou...

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Abstract

The present disclosure relates to isolated enzyme complexes comprising a tethered cofactor and at least two enzymes paired to catalyse an enzymatic reaction and recycle the cofactor.

Description

field of invention [0001] The present disclosure relates to isolated enzyme complexes comprising a tethered cofactor and at least two paired enzymes to catalyze an enzymatic reaction and recycle the cofactor. Background of the invention [0002] Biocatalysts have the potential to significantly reduce waste and energy costs generated in organic synthesis. In part, this is because the delicate selectivity of biocatalysts, many of which operate at low temperatures and pressures, reduces unwanted by-product formation, which has the added benefit of simplifying downstream separations. In fact, the number of organic syntheses in which enzymes are used as catalysts is rapidly increasing due to their excellent stereo and regiospecificity under mild pH and temperature conditions (Leonidat et al., 2001). [0003] Various industrial processes are now performed by immobilizing enzyme catalysts in flow reactors. Immobilization of enzyme catalysts in flow reactors has many advantages, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/18
CPCC07K2319/00C12N9/00C12N9/0006C12N9/0036C12N9/1205C12N9/18C12N11/06C12N11/18C12Y101/01094C12Y106/03001C12Y207/0103C12Y301/01Y02P20/50C12P9/00
Inventor C·斯科特C·哈特利C·威廉斯Q·邱凯萨J·朔布莱N·蒂内尔N·弗伦克
Owner COMMONWEALTH SCI & IND RES ORG
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