Automated bionanocatalyst production
The present invention provides machines, compositions and methods for producing bionanocatalysts (BNCs) comprising one or more enzymes selected from a broad spectrum of industrially and medically important enzymes. The BNCs are self-assembled and magnetically immobilized enzymes. The machines, compositions, and methods are fully scalable from bench top to industrial manufacturing volumes.
1. An automated method of producing self-assembled bionanocatalysts (BNC), comprising combining an MNP preparation with an enzyme preparation in an automated machine, wherein said machine comprises an MNP disruptor and a BNC mixer, wherein said BNC mixer comprises a mixing tee or a mixing coil;
wherein said automated machine performs the steps of
(a) disrupting MNPs in said MNP preparation with said MNP disruptor; and
(b) mixing said enzyme preparation with said disrupted MNP preparation using said BNC mixer;
wherein said mixing step causes a plurality of enzymes from said enzyme preparation to become magnetically immobilized within said MNPs to form said BNCs; wherein said magnetically immobilized enzymes are operable to catalyze the conversion of a diffusible substrate to a diffusible product.
2. The method of claim 1 , wherein said BNCs are produced in a volume of less than about 1 ml.
3. The method of claim 1 , wherein said BNCs are produced in a volume of between about 1 ml and 10 ml.
4. The method of claim 1 , wherein said BNCs are produced in a volume of between about 10 ml and 100 ml.
5. The method of claim 1 , wherein said BNCs are produced in a volume of between about 100 ml and 1 liter.
6. The method of claim 1 , wherein said BNCs are produced in a volume of between about 1 liter and 10 liters.
7. The method of claim 1 , wherein said BNCs are produced in a volume of between about 10 liters and 100 liters.
8. The method of claim 1 wherein said BNCs are produced in a volume of between about 100 liters and 1000 liters.
9. The method of claim 1 , wherein said BNCs are produced in a volume of between about 1 kiloliter and 10 kiloliters.
10. The method of claim 1 , wherein said BNCs are produced in a volume of between about 10 kiloliters and 20 kiloliters.
11. The method of claim 1 , wherein said BNCs are produced in a volume of between about 20 kiloliters and 50 kiloliters.
12. The method of claim 1 , wherein said BNCs are produced in a volume of greater than about 50 kiloliters.
13. The method of claim 1 , further comprising the step of templating said BNC into a stabilizing level 2 assembly.
14. The method of claim 1 , wherein said machine further comprises:
a) an enzyme container;
b) a magnetic nanoparticle (MNP) container;
c) an enzyme pump; and
d) an MNP pump;
wherein said enzyme container is operable to hold an enzyme preparation; wherein said MNP container is operable to hold an MNP preparation; wherein said MNP pump performs the step of sending said MNP preparation to said MNP disruptor; wherein said MNP pump performs the step of sending a plurality of disrupted MNPs from said MNP disruptor to said BNC mixer; wherein said enzyme pump performs the step of sending said enzyme preparation to said BNC mixer.
15. The method of claim 1 , wherein said MNP disruptor is a sonicator.
16. The method of claim 15 , wherein said sonicator further comprises a sonicator coil and a sonication container, wherein said sonicator coil is operable to sonicate said MNPs within said sonication container.
17. The method of claim 15 , wherein the sonicator is an in-line sonicator.
18. The method of claim 15 , further comprising a cooling system operable for cooling said sonicator.
19. The method of claim 18 , wherein said cooling system is a water cooling system.
20. The method of claim 1 , wherein said MNP disruptor is operable to mechanically disrupt said MNPs.
21. The method of claim 1 , wherein said MNP disruptor is operable to magnetically disrupt said MNPs.
22. The method of claim 1 , wherein said MNP disruptor is operable to thermally disrupt said MNPs.
23. The method of claim 15 , wherein said enzyme pump sends said enzyme preparation to said BNC mixer via mechanical or gravitational force.
24. The method of claim 15 , wherein said MNP pump sends said MNP preparation to said MNP disruptor via mechanical or gravitational force.
25. The method of claim 15 , further comprising sending said BNCs to a container containing a magnetic scaffold preparation and mixing said magnetic scaffold preparation with said BNCs in said magnetic scaffold container to produce BNCs in a level 2 assembly.
26. The method of claim 25 , wherein said magnetic scaffold container mixes said BNCS and said magnetic scaffold preparation mechanically.
27. The method of claim 25 , wherein said magnetic scaffold container mixes said BNCS and said magnetic scaffold preparation magnetically.
28. The method of claim 25 , wherein said magnetic level 2 assembly is a Magnetic Microparticle (MMP).
29. The method of claim 25 , further comprising a templator that stabilizes said BNCs within said level 2 assembly.
30. The method of claim 25 , wherein said level 2 assembly is magnetic.
31. The method of claim 1 , further comprising the step of optimizing the chemical conditions, salts, or pH for BNC formation within said machine.
32. The method of claim 1 , wherein said enzyme is an isomerase.
33. The method of claim 1 , wherein said enzyme is selected from the group consisting of hydrolases, hydroxylases, nitrilases, hydratases, transaminases, ene reductases (EREDS), imine reductases (IREDS), and oxynitrilases.
34. The method of claim 1 , wherein said MNPs comprise a ferrite.
35. The method of claim 34 , wherein said ferrite comprises an iron oxide species.
36. The method of claim 1 , wherein said MNPs comprise maghemite.
37. The method of claim 1 , wherein said MNPs comprise magnetite.