HU227235B1 - Process for producing 1,3-propanediol by biotransformation - Google Patents

Abstract

1,3-Propane-diol is produced by using enzyme gained or partially gained by fermentation. The enzyme is retained by a membrane in membrane bioreactor, or is bound to solid carriers. Coenzymes are also retained by binding to carriers. The bioreactor can be operated in batch, fed-batch or continuous modes at a wide range of temperature and pH. The glycerine starting material can be converted partially into useful or different compound during regeneration of the coenzyme. The method utilizes inhibitors suppressing the operation of enzymes causing the occasional by-reactions.

Description

The process involves the biotechnological production of 1,3-propanediol using enzyme preparations from microorganism (s) in a membrane bioreactor by coenzyme regeneration, while other useful products can be formed in addition to the main product.

History background

There has been tremendous interest in the cost-effective production of 1,3-propanediol (1,3-PD) since the 1990s. This is because its copolymer with terephthalic acid (PTT) has much better properties than certain conventional plastics (polyethylene terephthalate (PET), polybutylene terephthalate (PBT)). It is a high-elastic, well-dyed, stain-resistant, low-electrostatic, shape-retaining fiber that can be used to make carpets, clothes, films, thermoplastics. 1,3-PD can be used as a solvent for inks and as a monomer for polyethers and polyurethanes. Thanks to its extensive use, it is now available

1,3-Propanediol has become one of the most economically efficient polymeric starting materials. Its derivatives are also utilized by medicine to prevent rejection after organ transplantation (WO 9822100).

1,3-PD can be produced both biologically and chemically. When it was discovered (1881), it was obtained by culturing microorganisms on glycerol, but synthetic methods became more economical using cheap petroleum-based raw materials. In a cost-effective process developed by Shell Chemicals, ethylene oxide is reacted with carbon monoxide and hydrogen under pressure. In addition to the previous 4,000 t / y plant, this process also put in place a 73,000 t / y capacity track in 2000. With a capacity of 10,000 t / year, Degussa produces 1,3-PD using classic synthetic technology. In their process, the hydrolysis of acrolein is followed by catalytic hydrogenation. DuPont also produces 1,3-PD with this technology, but after decades of research, it will launch a new plant in Loudon, Tennessee, in 2006, where it will implement biological, fermentation-based technology. During the development, the genes responsible for the conversion of glycerol> 3-hydroxypropionaldehyde to 1,3-PD from the natural strains and the glucose to glycerol conversion from other wild strains have been transferred to a gene manipulated bacterium (Escherichia coli), which results in normal sugar-based fermentation. produces 1,3-PD on a cheap carbon source (WO9821339).

Although it has been possible to produce 1,3PD efficiently on a cheap coal source, some of the starting sugar is always converted to biomass, so the whole raw material can never be converted into a product.

They use a genetically manipulated microorganism (GMO) against which society is increasingly critical and thus more demanding. In contrast, the present inventors disclose an enzymatic process in which the product is produced with enzymes of natural (possibly GMO) origin. The raw material is a by-product of the production of a potentially accumulating renewable energy source: glycerol. Since the development of the 1,3-PD fermentation process (1998), the production of biofuels, such as biodiesel, has been increasing in volume, and thus the present invention has the potential to utilize glycerol as a by-product.

GDHt, 1,3-PDOR and GDH also play a role in the fermentation biological process. These in vitro koenzimeikkel (NAD ⁺ and B ₁₂₎ were measured and glycerol added to the reaction mixture simultaneously 1,3-PD and DHA was formed. It is an object and an essential point of the invention that the second step of 1,3-PD formation requires a reduced nicotinamide dinucleotide (NADH) coenzyme, from which it forms an oxidized coenzyme with 1,3-PD, but that GDH also utilizes the spent coenzyme in the presence of glycerol. it is reduced back as the glycerol is oxidized to DHA. DHA is also widely used, especially in the cosmetics industry, as a tanning agent.

There are many examples of such coenzyme regeneration, such as Obon et al. (1998), who studied the NAD (H) regenerative membrane enzyme reaction of glucose dehydrogenase and lactic acid dehydrogenase.

Description of the Invention

In our process, three target enzymes cooperating with microorganisms (e.g., Enterobacter aerogenes, Citrobacter freundii, Clostridium butyricum, Thermotoga maritima, Lactobacillus) are prepared: glycerol dehydrate (GDHt, EC4.2.1.30), 1,3-propanediol -doreductase (1,3PDOR, EC 1.1.1.202), and glycerol dehydrogenase (GDH, EC 1.1.1.6). The enzyme GDHt using B ₁₂ coenzyme (or without) 3-hydroxy-propionaldehyde (3-HPA) to form the dehydration of glycerol, which is the

1,3-PDOR is reduced to 1,3-PD by the co-enzyme reduced nicotine adenine dinucleotide (NADH ₂ ), while the coenzyme is oxidized (to NAD ⁺ ). The oxidized coenzyme can be reduced in any coupled reaction by any NAD ⁺ enzymatic reaction. An example of this is glycerol dehydrogenase, which reduces the coenzyme to NADH ₂ using glycerol to form 1,3-dihydroxyacetone (DHA, i.e. 1,3-dihydroxypropan-2-one), which is used in the cosmetics and food industry. are used.

Another example is the use of formate dehydrogenase as a coupled reaction partner. This requires addition of glycerol to formic acid, which is oxidized by formate dehydrogenase to CO _{2 with} the formation of NAD ⁺ , and the resulting reduced coenzyme can again be involved in the second step of the production of 1,3-PD. These coupled enzymatic transformations according to the examples presented can be accomplished in both batch and continuous modes. The former requires a simpler reactor but with lower productivity, while the latter requires a more complex reactor vessel with higher productivity. In order to achieve high production capacity, a continuous process is definitely preferable. For this purpose it is advisable to

The reaction is carried out in a system in which the enzymes and (immobilized) coenzymes are retained by a continuous membrane in the reactor. An example is a system in which the reaction is carried out in a conventional reactor by continuous feed / removal, but the reaction mixture is passed through an outer membrane module where only small molecule products can leave, and the remaining enzymes and coenzymes are recycled to the reactor. Another possibility for continuous operation technology is to carry out the reaction in a reactor with one wall, preferably the lower one, bounded by a membrane and through which the product is removed.

1.

A further advantage is that when the tank mixer is close to the membrane, intensive mixing reduces the concentration polarization phenomenon and facilitates filtration. Given that most of the microorganisms listed contain photosensitive and oxygen sensitive enzymes, it is advisable to solve the material flow and reaction in the dark and under a nitrogen atmosphere.

Enzymes are prepared intracellularly with the microorganisms listed (e.g., Enterobacter aerogenes, Citrobacter freundii, Clostridium butyricum, Thermotoga maritima, Lactobacillus). Cells should be recovered from the fermentation broth by filtration or precipitation, but centrifugation is most appropriate. Subsequently, the sedimented cells should be washed twice with distilled water, but optimal washing with buffer. For this, any buffer between pH 6 and 9 is suitable, for example phosphate buffer or imidazole buffer, but preferably N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid) (HEPES) buffer is adjusted to pH 7.4. . The cells should then be disrupted with either glass beads or organic solvents (eg acetone, ethanol), but ultrasound is best. After cell destruction, cell debris must be removed by centrifugation again. The resulting solution contains a significant amount of cellular enzymes, and by-products corresponding to normal in vivo metabolism of cells may be formed, since the DHA formed may be further modified by phosphorylation. This can be prevented by using an inhibitor molecule. However, ATP that promotes phosphorylation and thus byproduct formation is also required for the regeneration of GDHt, an ATP-dependent protein in cells. Since the enzyme solution used in the method contains a significant proportion of the total protein of the cell, it also contains this enhancer complex, which is thus capable of repairing inactivated GDHt enzyme in vitro in the presence of ATP.

Reactor operating parameters are determined by the source microorganism of the enzymes, from 30 ° C to 90 ° C and pH 6.5 to 9.5 in the dark, generally excluding air, since the source of GDHt is photosensitive and oxygen sensitive depending on the microorganism strain. may.

Figure Index

Figure 1: Coenzyme regeneration bioreactor assembly containing a membrane module.

Figure 2 Conversion of glycerol to 1,3-PD and acetic acid (P1).

Figure 3: Nearly 1: 1 ratio of 1,3-PD and DHA formation in the presence of inhibitor (P2).

Figure 4 Bioconversion with 20-fold concentrated enzyme mixture solution (P3).

Figure 5: Production of 1,3-PD and DHA in a continuous coenzyme regeneration reactor (P5).

Examples

P1. Enzymatic conversion of glycerol to 1,3-PD and acetic acid

Fermentation of Enterobacter aerogenes according to Németh et al. (2003), after aerobic, intermittent cultivation of glucose and glycerol as a C / E source, yields the required 10-12 g / l cell mass after about 24 h after anaerobically fed glycerol. This must be separated from the medium by precipitation, filtration, but most appropriate centrifugation, and the cells harvested washed twice with distilled water or buffer, optimally with HEPES buffer. The cell suspension thus obtained must be subjected to cell destruction. This can be done with a solvent, a homogenizer, but ultrasonic cell lysis is most appropriate. After destruction, cell debris should be removed by optimal centrifugation. The resulting supernatant is the crude enzyme solution, which contains all the proteins of the cells as well as some nutrient components, coenzymes. Concentration of the enzyme solution may be advisable for faster product formation and capacity increase. There are several methods for doing this (reverse osmosis, precipitation / redissolution, etc.), but ultrafiltration is best because it is gentle on proteins. The enzymatic reaction buffer should be dissolved in glycerin necessary, B ₁₂ and coenzymes NAD ⁺ and then being combined in the reactor in the same volume of enzyme solution. EXEMPLARY initial szubsztrátkoncentrációval a 4 g / L, 0.28 g / L NAD ^+, and 0.235 g / l of an experiment B ₁₂ coenzyme concentration (Figure 2) A weighed quantity of the target enzymes enzyme solution contained the following proportions: GDHt 85 U / l; PDOR: 15 U / l; GDH: 1367 U / l for fermentation broth. The enzymatic reaction after 24 h was 88% conversion (46%

1,3-PD, 10% DHA, and 15% AcOH).

P2. Enzymatic conversion of glycerol to 1,3-PD and

DHA

Cells are prepared as described in P1, which are separated from the culture medium by precipitation, filtration but most suitably centrifugation, and the cells recovered are washed twice with distilled water or buffer, optimally with HEPES buffer. The cell suspension thus obtained must be subjected to cell destruction. This can be done with a solvent, a homogenizer, but ultrasonic cell lysis is most appropriate. After destruction, cell debris should be removed by optimal centrifugation. The resulting supernatant is a crude enzyme solution containing all the proteins of the cells as well as some nutrient components, coenzymes.

HU 227 235 Β1

For the enzymatic reaction, the required glycerol, B-, must be dissolved in buffer ₂ and NAD ⁺ coenzymes and then mixed with an equal volume of enzyme solution in the reactor. In order to allow DHA accumulation, the required DHA-> DHAP conversion leading to acetic acid formation must be inhibited by structural analogs of DHA, preferably irreversibly. ATP is required for the regeneration of GDH and therefore the reaction cannot be inhibited from this side. Based on the experiment presented in AP 1, mathematical modeling shows the effect of the concentration of an arbitrary (Clha) non-competitively acting inhibitor molecule on the final composition of the reaction mixture (Figure 3).

Based on the results, it can be stated that with a certain concentration of inhibitors a low 1: 1 ratio of glycerol and acetic acid can be achieved.

Product ratios of 1,3-PD and DHA.

P3. The enzymatic conversion of glycerol is high

1,3-PD and DHA

Cells are prepared as described in P1, which are separated from the culture medium by precipitation, filtration but most suitably centrifugation, and the cells recovered are washed twice with distilled water or buffer, optimally with HEPES buffer. The cell suspension thus obtained must be subjected to cell destruction. This can be done with a solvent, a homogenizer, but ultrasonic cell lysis is most appropriate. After destruction, cell debris should be removed by optimal centrifugation. The resulting supernatant is the crude enzyme solution containing all the proteins of the cells as well as some nutrient components, coenzymes. Concentration of the enzyme solution may be advisable for faster product formation and capacity increase. There are many methods for doing this (reverse osmosis, precipitation / redissolution, etc.), but ultrafiltration is best because it is gentle on proteins.

The enzymatic reaction buffer should be dissolved in glycerin necessary, B ₁₂ and coenzymes NAD ⁺ and then being combined in the reactor in the same volume of enzyme solution. Thus, significantly higher product concentrations can be achieved. Concentration of the enzyme solution described in P1-P2 at 20 x concentration can completely convert glycerol to 100 g / l within 28 h, resulting in more than 50% 1,3-PD with 7% AcOH and 27% DHA. ^).

P4. Enzymatic conversion of glycerol to pure 1,3-PD

Cells are prepared as described in P1, which are separated from the culture medium by precipitation, filtration but most suitably centrifugation, and the cells recovered are washed twice with distilled water or buffer, optimally with HEPES buffer. The cell suspension thus obtained must be subjected to cell destruction. This can be done with a solvent, a homogenizer, but ultrasonic cell lysis is most appropriate. After destruction, cell debris should be removed by optimal centrifugation. The resulting supernatant is a crude enzyme solution containing all the proteins of the cells as well as some nutrient components, coenzymes. If necessary, the enzyme solution can be concentrated here as well.

For the enzymatic reaction, the necessary glycerol formic acid, B-1, must be dissolved in buffer ₂ and NAD ⁺ coenzymes, as well as formate dehydrogenase, must be mixed in the reactor with the same volume of enzyme solution. Thus, only 1,3-PD will be present in the finished reaction mixture, but free CO ₂ must be assured during the reaction, otherwise pressure will increase which may suppress the coenzyme regeneration reaction. Thus, 62% PD is formed with 7% AcOH and 29% CO ₂ .

P5. Enzymatic conversion of glycerol to 1,3-PD and

To DHA in a continuously operated membrane reactor

Cells are prepared as described in P1, which must be separated from the medium by precipitation or filtration, but most suitably centrifuged, and the cells recovered are washed twice with distilled water or buffer, optimally with HEPES buffer. The cell suspension thus obtained must be subjected to cell destruction. This can be done with a solvent, a homogenizer, but ultrasonic cell lysis is most appropriate. After destruction, cell debris should be removed by optimal centrifugation. The resulting supernatant is a crude enzyme solution containing all the proteins of the cells as well as some nutrient components, coenzymes.

The enzymatic reaction buffer should be dissolved in glycerin necessary, B ₁₂ and coenzymes NAD ⁺ and then being combined in the reactor in the same volume of enzyme solution. For DHA accumulation, the conversion of DHA to DHAP required for acetic acid formation should be inhibited as irreversibly as possible with structural analogs of DHA. ATP is required for the regeneration of GDH and therefore the reaction cannot be inhibited from this side. Continuous operation can only be carried out efficiently if the coenzymes are kept in the reactor. The 1.5 kDa membrane coenzyme B ₁₂ has been retained in a good proportion, but the smaller coenzyme NAD ⁺ in this case should be immobilized, such as PEG ATP and polysaccharide such as agarose (Figure 5).

The results show that, with 8g / l glycerol feed, a steady-state steady state can be achieved without concentrating, where the effluent juice is 3.8g / l 1.3PD and 3.7g / l DHA contains total coenzyme retention and DHAK inhibitor. By concentrating the enzyme solution, a higher concentration of glycerol solution can be converted to the same product ratio after a quicker, steady state.

References

Róbert Paul, Wenger, Roland, and Cottens, Sylvain: Use of 1,3-Propanediol Derivatives to Prevent Rejection after Organ Transplantation. Patent (WO 9822100) 1999.

2. Gatenby A. et al. al .: Methods for the production of

1,3-propanediol by a recombinant organism. Patent (WO9821339) 1998.

3. Obon, J.M. Manjon, A. Iborra, J.L .: Retention and Regeneration of NAD (H) in Noncharged Ult4

HU 227 235 Β1 Rafiltration membrane reactors: application to L-lactate and gluconate production (1998) Biotechnology and Bioengineering 57 (5) 510.

4. Németh, Á. Kupcsulik, B. Sevella, B.: 1,3-Propanediol oxidoreductase production with Klebsiella pneumoniae DSM2026 World Journal of Microbiology and Biotechnology 19 (7) 659 (2003).

Claims (7)

PATENT CLAIMS A process for the enzymatic production of 1,3-propanediol (1,3-PD) glycerol dehydrate from wild "strains" of Enterobacter, Citrobacter, Clostridium, Thermotoga, Lactobacillus or genetically engineered variants of these or other microorganisms. , 1,3-propanediol oxidoreductase and glycerol dehydrogenase, characterized in that: (a) propagating the enzyme-producing micro-organism, b) digesting the cell mass of the microorganism, optionally isolating the enzymes and purifying them in whole or in part, c) contacting the mixture of enzymes prepared in step b) with glycerol while providing the coenzymes required for the enzyme function, thereby converting the glycerol to 1,3-propanediol. Process according to claim 1, characterized in that the process is carried out in a membrane reactor and / or the enzymes are fixed on a solid support and thus the enzymes are reused for several cycles. Process according to claim 2, characterized in that the coenzymes (co-substrates) required for the enzyme function are retained in the membrane-bounded reaction space of the membrane reactor bound to polyethylene glycol, polysaccharide or other support. 4. Process according to claims 1 to 3, characterized in that the coenzymes used in the conversion of glycerol to 1,3-PD are regenerated by a coupled regenerative reaction in which the target product In addition to 1,3-PD, other useful or inert products are formed. 5. A process according to any one of claims 1 to 3, wherein the formation of the unwanted by-product (s) is suppressed by the addition of a competitive, non-competitive or irreversible inhibitor of the enzyme responsible for the reaction responsible for its formation. 6. A process according to any one of the preceding claims, wherein the reactor is operated by a batch, substrate feed or continuous process. 7. Process according to claims 1 to 3, characterized in that the process according to the enzyme source (s) used has a temperature range of 25 ° C to 90 ° C and a pH range of 6.5 to 9.5 and preferably light and is generally in an airtight reactor.