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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
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  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
  • C12N9/2411—Amylases
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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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  • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
  • C12P37/06—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by desacylation of the substituent in the 6 position
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel
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  • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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  • Y02W10/10—Biological treatment of water, waste water, or sewage

Definitions

• the present invention relates to a method of carrying out biological and chemical conversion processes which require the presence of at least one catalytically active substance, which may be, for example, enzymes, micro- organisms, whole cells, cell homogenates, organelles, or organic or inorganic catalysts.
• at least one catalytically active substance which may be, for example, enzymes, micro- organisms, whole cells, cell homogenates, organelles, or organic or inorganic catalysts.
• a further serious problem often occurring in biochem cal conversion processes of the aforementioned kind is that the end product of the conversion process, or intermediate products formed in different stages in a conversion process comprising a number of stages, often has a strong inhibiting effect on the process or the various process stages. In consequence hereof it is necessary to hold the concentration of the end product and such intermediate products as may be formed at a low level during the process, in order to obtain a high process yield and to enable high, concentrations of starting substrate and cataly ⁇ ic substance to be used.
• the object of the present invention lis to provide a novel and useful method of carrying out biochemical conversion processes of the aforedescribed kind in which the aforementioned problems can be resolved in a satisfactory manner.
• Fig. 2 illustrates schematically a conversion process carried out continuously in accordance with the method of the invention
• Figures 3A and 3B illustrate schematically a conversion process using a coenzyme dependent enzyme when applying the method according to the invention.
• Figures 4, 5 and 6 are diagrams illustrating the results obtained in tests carried out when applying the method according to the invention for converting cellulose to ethanol.
• the conversion process is carried out in a liquid system including at least two liquid phases which are so selected that the catalytic substance used is enriched in one of said phases.
• Figure 1A illustrates schematically one such biphase liquid system, including a bottom phase F1 and a top phase F2.
• the biphase system is so selected hat the catalytic substance K used, for example an enzyme present in solution, is enriched in the bottom phase F1.
• the starting substrate S for the conversion process may be uniformly distributed in the biphases F1 and F2, although there may be selected to special advantage a system in which also the substrate S is enriched in the same phase Fl as the catalytic substance K, as illustrated in Figure 1A.
• the top phase F2 contains neither the catalytic substance K nor the starting substrate S.
• the liquid system When carrying out the conversion process according to the invention, the liquid system is agitated so that the top phase F2 becomes finely dispersed in the bottom phase F1, as illustrated schematically in Figure 1B.
• the conversion process continues in the resultant dispersion or emulsion, within the fine droplets of the bottom phase F1, as illustrated schematically in Figure 1C which shows one such droplet.
• the starting substrate S and the catalytic substance K are both isolated in said droplets and are in extremely intimate contact with each other , thereby enabling the process to continue without diffus ion limitations or steric hindrance , even though the starring substrate should be macromolecular or particulate .
• the two phases F1 and F2 in the liquid system are so selected that the product P formed in the conversion proces s is also distributed to the top phase F2 , as illus trated schematically in Figures 1B and 1C.
• the product P pas ses to the top phase F2 as s aid product is formed in the convers ion proces s taking place in the finely divided droplets of the bottom phase F1.
• it is pssible to maintain a low concentration of the product P in the bottom phase F1 so as to enable any inhibiting effect of the product on the conversion: process to be eliminated or at leas t greatly restricted .
• the phases of the liquid system are s elected so that the product P formed is enriched in the top phase F2 , so that the concentration of product P is much greater in the top phase F2 than in the bottom phase F1 in which the convers ion process continues .
• the concentration of product P in the bottom phase F1 can be kept at such a low level as to have substantially no inhibiting effect on the process .
• the trans ition of product P from the bottom phase F1 to the top phase F2 becomes very effective when maintaining the bottom phase F1 in a finely dispersed state in the top phase F2, such as to provide a very large transition interface between the two phases.
• a biphase system which can be used when practicing the invention comprises mixtures of water and two mutually different polymers, which in this respect may both be non-ionic polymers or polyelectrolytes or a non-ionic polymer and a polyelectrolyte.
• biphase systems examples include dextran-polyethylene gly col-water, dextran-Ficoll-water, dextran-methylcellulose-water, dextran- polyvinylalcohol-water, Na dextran sulfate-polypropylene glycol-water, Na carboxymethyldextran-polyethylehe glycol-water with an addition of NaCl, Na-dextran sulfate- Na carboxymethyldextran-water.
• a useable biphase system may also comprise a mixture of water, a polymer and a component of low molecular weight, for example a salt.
• the method according the invention affords particularly important advantages over previously known methods when using macromolecular or particulate starting substrates. It will be understood, however, that the method according to the invention can also be applied in conjunction with the use of different starting substrates, for example gases or low molecular, soluble or insoluble substances.
• the product formed in the process may also be of a different kind, such as a gas, a low-molecular, macromolecular or particulate substance, and may be soluble or insoluble.
• mixtures of different starting substrates may also be used, and mixtures of different process products may be produced.
• the method according to the invention may be applied to particular advantage for carrying out continuous conversion processes.
• a reactor vessel 1 having a lower part la, which serves as a mixing chamber and in which the two phases of the liquid system are maintained intimately mixed by agitation, and an upper part 1b, in which no stirring or agitation takes place and in which the two phases thus separate from each other so that only the top phase E2 is present in at least the uppermost region of the part lb.
• This top phase F2 is removed, either intermittently or continuously, from the upper part of the reactor vessel through an outlet 2, and is passed to a treatment unit 3, in which the formed process product can be removed from the top phase F2 in a suitable manner, said top phase then being returned to the lower part of the reactor vessel through an inlet 4.
• the process starting substrate may be supplied to the lower part 1a of the reactor vessel in a suitable manner, not shown in detail.
• the method according to the invention can afford particularly important advantages over previo known methods when carrying out biochemical conversio processes with the use of an enzyme whose activity is dependent upon the presence of a coenzyme.
• the product formed in the desired conversion process is designated P 1 in Figure 3B, while the reference S 2 identifies a substrate requisite for regenerati the coenzyme, and the reference P 2 identifies a product formed when regenerating the coenzyme under the influence of the enzyme E 2 . It may be necessary also to add the substrate S 2 to the liquid system, and the phases of the liquid system may, if so is suitable, be selected so that the substrate S 2 is partitioned to the bottom phase F 1 and that both products P 1 and P 2 are partitioned to the top phase F 2 .
• the invention will hereinafter be further illustrated as applied to a process for the enzymatic degradation and conversion of particulate cellulose to ethanol. This process proceeds in accordance with the following reaction chain:
• the end product, ethanol has a highly inhibiting effect on the preceding reaction stages, while the intermediate product glucose, and still more the intermediate product cellobiose, have an inhibiting effect on respective preceding reaction stages.
• Tests have been carried out in accordance with the invention both on the first part of the reaction chain, namely the enzymatic degradation of cellulose to glucose, and on the latter part of the reaction chain, namely the fermentation of glucose to ethanol, and also on the complete reaction chain from cellulose to ethanol.
• aqueous biphase system prepared by mixing a volume of Dextran T-40 (16 % weight/weigh ) (from Pharmacia Fine Chemicals, Uppsala) dissolved in a 0.1 M sodium acetate buffer, pH 4.5, with 5 volumes of Dextran T-40 (16 % weight/weigh ) (from Pharmacia Fine Chemicals, Uppsala) dissolved in a 0.1 M sodium acetate buffer, pH 4.5, with 5 volumes of
• PEG-6000 (12 % weight/weight) (polyethylene glycol having a molecular weight of 6000 from Union Carbide, N.Y., USA) dissolved in the same buffer.
• Test 1 Fermentation of glucose to ethanol The fermentation of glucose to ethanol when using-baker's yeas was investigated, by adding 100 mg of yeast to 60 ml of the biphase system prepared in accordance with the above, and suspending said yeast in the system, after which 0.5 grams of glucose was added. The system was agitated by means of a agnetic agitator at a temperature of 30oC and samples (0.5 ml) were taken from the mixed phase system at regular intervals. These samples were centrifuged and the top phase analysed. The result can be seen from the diagram in Figure 4, where curve A shows the concentration of ethanol (mg/ml) in the top phase as a function of the reaction time (hours).
• Test 2 Enzymatic degradation of cellulose to glucose
• Test 3 Bioconversion of cellulose to ethanol
• the enzymatic saccharification of cellulose to glucose in the biphase system used in accordance with the invention was investigated in Test 2. Because of the high inhibiting effect of primarily cellobiose, the enzymatic saccharification of cellulose is generally characterized by a high initial rate, whereafter the process of saccharification continues very slowly.
• the curve C in Figure 5 shows, however, that in the test carried out according to the inyention the glucose content increased constantly during the four days over which the experiment was continued. At the same time the amount of reducing sugars decreased still more strongly, which shows that substantially equal quantities of glucose and cellubiose were produced in the system.
• the test was carried out over five days, during which no inhibiting effect of the ethanol could be observed.
• the ethanol production was substantially linear during the whole of the test period.
• the final value of about 5 mg/ml indicates that a practically complete conversion of cellulose to ethanol had been reached at the end of the test, assuming an equal distribution of ethanol in the system.
• the tests carried out show that a bioconversion of cellulose to ethanol in a biphase system in accordance with the present invention is very effective.
• a biphase system also enables the ethanol formed to be removed continuously from the top phase of the system, whereby a continuous transfer of ethanol from the bottom phase to the top phase is obtained, so that inhibiting concentrations of ethanol are prevented from occurring in the bottom phase, where the bioconversion process takes place.
• Conventional batchwise processes for manufacturing ethanol from cellulose by simultaneous saccharification and fermentation of the resultant glucose to ethanol have shown that the enzyme activity for cellulose degradation is inhibited to 50 % already at an ethanol concentration of 3 %.
• the method according to the invention will be exemplified with a number of additional examples.
• a biphase system was used consisting of 6 % (w/w) Dextran T-40 as bottom phase and 25 % (w/w) PEG 8000
• top phase polyethylene glycol with molecular weight 8000
• the volume ratio of the top phase to the bottom phase was 6:1.
• To this phase system were added 40 g/l glucose as substrate and 10 g/l peptone together with 10 g/l yeast extract as nutrition medium for the bacteria Clostridium acetobutylicum, ATCC 824. The temperature was 35°C.
• the bacteria were enriched in the bottom phase, whereas the substrate and the nutrition medium were distributed in both phases. After about 30 hours the bacteria had produced 8 g/l butanol and 3 g/l acetone to be compared with 6 g/l and 2 g/l, respectively, if no biphase system is used.
• Butanol and acetone can be distilled from the top phase and if more sugar is added to the system the production will start again. This will happen also if the top phase is replaced with a new top phase without any additional nutritive medium and more sugar is added to the system.
• Example 2 Production of polymer with the use of bacteria If the fermentation in Example 1 is left to continue on its own, the butanol is after 4 days converted to a polymer, which is not yet fully defined but which seems to be polyhydroxy butyric acid. The reason for this conversion is believed to be that the bacteria are present in an environment having a reduced water activity whereby their metabolism is changed.
• a biphase system was used consisting of 6 % (w/w) Dextran T-40, 7.5 % (w/w) PEG 8000 and 0,3 H TRIS buffer up to 100 % (w/w), pH 7.8.
• a continuously operating reactor of the general type illustrated in Fig. 2 was used. The reactor volume was 6 ml of which volume 3 ml was agitated whereas the remaining 3 ml was maintained unagitated. The volume ratio of the top phase to the bottom phase was 3:1.
• the bottom phase included 0,5 of a solution of penicillin acylase which was partitioned to the bottom phase.
• the top phase was pumped into the reactor at a rate of 1,8 ml/h.and included 70 g/l benzyl penicillin, which in the reactor was distributed in both phases.
• the reactor temperature was 37°C.
• the output flow from the reactor contained 14 g/l of 6-APA.
• a biphase system was used consisting of 6 % (w/w) Dextran T-40 and 7.5 % (w/w) PEG 8000.
• the volume ratio top phase to bottom phase was 3:1.
• Example 5 Biphase system in which one phase is also substrate
• a phase system was used consisting of 15 % (w/w) PEG 800 and 15 % (w/w) starch, which had been geled at 73oC in the presence of 0,6 % (v/w) of the amylase enzyme Termamyl ® (NOVO A/S, Copenhagen).
• the bottom phase contained also 10% (w/w) of yeast.
• the initial volume ratio top phase to bottom phase was 3:1.
• the starch was converted to 20 g/l ethanol.
• Example 6 Use of enzyme and coenzyme biphase system was used consisting of 6 % (w/w) PEG 20000 (molecular weight 20000), 10 % (w/w) Dextran T-10 (molecular weight 10000) and 0,03 M, pH 8,8, pyrophosphate buffer.
• the volume ratio top phase to bottom phase was 3:1.
• the enzyme was alcohol dehydrogenase, 50 units/ml in the bottom phase, and the coenzyme was NAD + , 8 mM.
• As substrate was used 200 mM ethanol, which was converted to 100 mM acetaldehyde.
• Example 7 Production of the enzyme amylase with the use of Bacillus subtilis The same phase system as in Example 4 was used.
• Bacillus subtilis produced the enzyme amylase after about 48 hours.
• the enzym was partitioned (enriched) to the top phase.
• Example 4 The same phase system as in Example 4 was used. As substrate one used 30 g/l glucose.
• the nutritive medium was chosen according to Jano et al, J. Ferment. Technol., Vol. 58, No. 3 (1380), p. 259.
• E. coli produced about 5 g/l acetic acid from 30 g/l glucose.
• the acetic acid was partitioned (enriched) to the top phase.
• Example 9 Production of ethanol with the use of Thermoanaerobacter ethanolicus
• Example 4 The same phase system as in Example 4 was used. As subsfrate one used not more than 10 g/l of a sugar chosen according to Wiegel J., Ljungdahl L.G.,
• the nutritive medium was selected according to Wiegel J., Ljungdahl L.G., Rawson J.R., (1979) J. Bacterial. 139:800-810.
• Example 10 Biological conversion in a polymer-salt system
• a biphase system was used consisting of 13,5 % (w/w) PEG 4000, 13,5 % (w/w) KgSO 4 7H 2 O and 100 mM TRIS HCl buffer, pH 7,0.
• the volume ratio top phase to bottom phase was 3:1.
• As substrate one used 14 mM phenol, 0,8 mM amino antipyrine and 1 mM H 2 O 2 .
• As enzyme one used peroxidase, which was partitioned (enriched) to the bottom phase.
• Example 11 Production of ethanol with the use of Zymomonas sp.
• Example 4 The same phase system as in Example 4 was used.
• substrate one used 10-25 % (w/w) glucose and the nutritive medium was selected according to Rogers P.L., Lee K.J., Tribe D.E. (1979) Biotechnol. Letters 4, p. 165-170 and p. 421-426.
• the use of the biphase system prevents the inhibiting effects of the substrate glucose as well as the product ethanol .
• the method according to the present invention can be applied to great advantage when carrying out a number of different biological and chemical conversion processes.
• one of the advantages with the method according to the invention is that it is pos e to use non-immobilised catalytic substances, such as enzymes and microorganisms for example, and despite this retain these catalytic substances in the process chamber, it will be seen that there is nothing to prevent the invention from being applied while using immobilised catalytic substances, if so desired.
• biphase or polyphase liquid system used has been exemplified in the aforegoing with reference to a system consisting of aqueous phases, although it will be understood that systems based on liquids other than water can also be used if said liquids are compatible with the starting substrate, catalytic substance and products present in the process in question.

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• 1981
  • 1981-10-16 EP EP81902941A patent/EP0063146A1/de not_active Withdrawn
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