Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes

Definitions

• the invention concerns the improvement of fermentation processes in order to increase the yield of the biological products or valuable substances produced by means of microorganisms in these fermentation processes.
• the fermentation of microorganisms is used, for example, to produce biological products or valuable substances, such as proteins, enzymes, or other valuable chemical substances, including those having pharmaceutical action.
• the microorganisms require nutrient media for both the intracellular and the extracellular formation of the products or valuable substances, which generally contain a so-called carbon source (“C-source”), a nitrogen source (“N-source”), and so-called supplements, such as salts and vitamins.
• C-source carbon source
• N-source nitrogen source
• supplements such as salts and vitamins.
• the N-source can be low- molecular- weight chemical substances such as ammonium salts or urea, but also high-molecular- weight nitrogen-containing substrates, such as proteins of natural origin, e.g., soybean meal, cottonseed proteins, cereal proteins, proteins of animal origin such as fish meal, etc.
• N-sources of vegetable origin can have more or less high starch content and thus serve as the C-source at the same time.
• carbohydrates are used as C- sources, primarily mono-, di-, tri- and oligosaccharides, but also polymeric carbohydrates like cereals, potato starches, tapioca starches, or yam root starches.
• Certain C-sources, such as glucose, fructose, dextrin are very easily metabolized by the microorganisms and therefore result in an explosive multiplication of the microorganisms, accompanied by rapid oxygen depletion in the fermentation nutrient solution.
• the oxygen depletion entails a rearrangement of the microorganism metabolism, so that the desired fermentation products are no longer produced, or are produced only in insufficient amount. In the worst case, the microorganisms die because of lack of oxygen. Furthermore, glucose and other mono- or disaccharides in the microorganism cell very often hinder the intracellular induction of enzymes for the secretion of a desirable valuable substance, enzymes for starch breakdown (e.g. , amylase), or other enzymes. Thus, as a result, the desired production of the biological product or valuable substance is often reduced to low quantities, or is even partly or totally stopped by the microorganisms.
• C- sources of higher molecular weight such as the already-mentioned carbohydrates or starches.
• all vegetable starch sources can be used as potential C- sources.
• the starch products used for this purpose are not isolated from the plants, but instead the vegetable raw materials used for production of starch, such as ground-up fruit or the storage organs of plants, like root nodes or rhizomes, are used as is.
• Such polymeric C-sources usually involve the meal of cereals, such as wheat meal or corn meal, potato meal or tapioca meal of various grain sizes.
• the meals and starches are very often subjected to further enzymatic treatment before the fermentation, in order to lower the viscosity of the fermentation medium.
• this should reduce the tendency of the starches to gel in water, thus ensuring that the fermentation medium can be easily agitated, and on the other hand it intensifies the transport of oxygen and carbon dioxide essential for the fermentation of the microorganisms.
• the polymeric C-sources like starch or starch-containing meals cannot be used without pretreatment, since they would otherwise result in highly viscous suspensions or solutions and possibly even form gels.
• the polymeric carbohydrate sources are therefore generally broken down with enzymes (such as amylases or glucoamylases) to such an extent that the proper degree of viscosity is obtained in the fermentation medium, while avoiding a disadvantageous repression of the substrate by low-molecular carbohydrate fragments.
• enzymes such as amylases or glucoamylases
• Costly measurement and control equipment is required, and the analysis expense for the fermentation process, e.g., the monitoring of the uptake of substrate by the microorganisms, also increases greatly.
• the apparatus expense also increases, since additional receptacles are needed.
• the process becomes more expensive through the frequent addition of raw material, since the raw materials must be sterilized in batches or continuously, and despite these steps the risk of contamination with foreign microbes is still substantially higher.
• the objective was to provide a simple and economical fermentation method in which the yield of substrate is improved with respect to the C-source, without having to modify an already existing fermentation technology with respect to apparatus, measurement and control equipment, or analysis expense.
• the fermentation method should result in an increased yield of the biological products or valuable substances produced in the fermentation process, such as the enzymes produced.
• the purpose can be achieved by the use of chemically modified carbohydrate sources.
• the invention concerns a method of fermentation of microorganisms for the production of biological 4 products or valuable substances, in which one employs a nutrient medium for use by the microorganisms in the fermentation, which contains a chemically modified carbohydrate source, in particular, a chemically modified oligomeric or polymeric carbohydrate source as the C-source substrate.
• a chemically modified carbohydrate source in particular, a chemically modified oligomeric or polymeric carbohydrate source as the C-source substrate.
• the chemically modified carbohydrate source can be based on any conventional carbohydrate-containing substrates which can be modified in familiar chemical ways.
• Such basic carbohydrates are, therefore, mono-, di-, tri- and oligosaccharides and, in particular, polymeric carbohydrates, whereby essentially all vegetable starch sources come into consideration; for example, meals of corn, rice, cereals, potatoes, tapioca or yam root, as well as the starches obtained from these plants after chemical modification, can be used within the context of the invention.
• the chemically modified carbohydrate source used in the method of the invention can be used in the fermentation process either as the sole carbohydrate source or to replace or supplement only a desired portion of a conventional nonmodified carbohydrate source.
• the chemical modification in this case occurs at the free hydroxy-groups of the glucose building blocks of the particular carbohydrate source.
• These hydroxy- groups allow, for example, etherifications, esterifications, oxidations and cross- linking with bifunctional or polyfunctional organic compounds. Examples are: for etherifications, hydroxyethylation with ethylene oxide; for esterifications, acetylation, e.g., with acetylchloride or acetanhydride; or for cross-linking, reaction with epichlorohydrin or phosphoric acid chlorides. Even small degrees of modification are enough to achieve the effects of the invention by substantially increasing the yield of valuable substances or enzymes.
• the degree of modification is understood to be the number of chemically modified hydroxy-groups per glucose unit of the carbohydrate source in mol/mol.
• the degree of modification is advisedly 0.005-4 mol/mol, and in particular 0.01-1 mol/mol. In this context, the practitioner can determine the advisable degree of modification for the particular fermentation process with a little experimentation. 5
• Suitable chemically modified carbohydrate sources which can be used in the fermentation method of the invention are therefore, in particular, those whose carbohydrate source is chemically modified and/or derivatized.
• this carbohydrate source serving as the C-source substrate is derivatized by esterification or etherification or chemically transformed by oxidation or cross- linking.
• Preferred chemically modified carbohydrate sources for this are characterized in that they are derivatized by esterification, in particular, by acetylation of free carbohydrate hydroxy-groups.
• esterified, especially acetylated starch with even relatively low degrees of derivatization
• the degree of esterification of the esterified or acetylated carbohydrate sources preferably the degree of esterification or acetylation of starch used according to the invention, can be determined by saponifying a certain quantity of a specified esterified or acetylated carbohydrate source with a likewise specific excess of alkaline base, such as sodium hydroxide, and then quantitatively determining the unused quantity of the base.
• alkaline base such as sodium hydroxide
• the degree of substitution (DS), i.e., the degree of esterification or acetylation, can then be calculated from the molar quantity of consumed base as the number of ester or acetyl groups per glucose unit of the particular esterified or acetylated carbohydrate source.
• the degree of esterification or degree of acetylation, determined by saponification of the esterified carbohydrate source as the number of substitution groups per glucose unit (DS) is 0.005-4 mol/mol, and in particular 0.01-1 mol/mol.
• esterification or acetylation degrees of 0.02-0.15 mol/mol are especially preferred.
• a chemically modified carbohydrate source is used, based on an (isolated) starch or a natural starch source, and in particular, on pure starch or starch-containing meal.
• an (isolated) starch or a natural starch source is used, and in particular, on pure starch or starch-containing meal.
• One possible chemically derivatized, especially esterified or acetylated starch is acetyl starch. Examples are the tapioca starch acetyl ester with a degree of substitution DS of 0.025-0.032 mol/mol, which is commercially available under the name "FARAZYM T R " (e.g., that distributed by the AVEBE Co. of Germany, Meerbusch; produced by Avebe B.A., Foxhol, NL), 6 or a corresponding acetylated potato starch ("FARAZYM 76 R "), available from Avebe.
• FARAZYM T R tapioca starch acety
• the method according to the invention enables a better utilization of substrate by simple means when using chemically modified carbohydrate sources (C-sources), in order to achieve an increased yield of valuable substances produced by fermentation, such as biological products like enzymes. Furthermore, the method according to the invention offers the advantage that it can be used in existing fermentation plants without changing the technology or the apparatus or analysis expense, or the monitoring and metering expense. Difficult dosing technologies are avoided. Another advantage of the invention is that it can be used for any kind of fermentation, and thus allows the use of advantageous chemically- modified carbohydrate sources for a number of microorganisms conventionally used in fermentation.
• the method according to the invention has diverse uses, especially for microorganisms of the fungus and bacteria families, especially microorganisms which are used in production of enzymes.
• microorganisms are the genus Aspergillus, Rhizopus, Trichoderma, Penicillium and Bacillus.
• Special examples for the genus Bacillus are, in particular, Bacillus licheniformis, Bacillus subtiiis, Bacillus alcalophilus, Bacillus lentus, Bacillus amyloliquefaciens, which have been sufficiently described in the technology of enzyme production.
• the advantages of the invention can also be achieved with optimized, e.g., genetically altered microorganisms, especially those which are used on an industrial scale for the production of enzymes.
• DU is meant the enzymatic activity in Delft Units, with 1,000 DU corresponding to the proteolytic activity producing an extinction difference (1 cm light path; 275 nm; compared to blind sample test) of 0.4000 in a volume of 1 ml of a 2-W/W-% enzyme solution after breakdown of casein.
• Ash (measured as dry substance) ⁇ . 2 mg/g
• potato starch and tapioca starch can be obtained, for example, from the firm AVEBE Germany (in Meerbusch).
• the acetylated tapioca starch used according to the invention revealed a striking improvement in activity formation, which lies clearly above the result obtained with the standard potato starch and also with the native tapioca starch.
• the method can also be advantageously used for other fermentations, e.g., for production of enzymes such as proteases, lipases, amylases, cellulases, glucosidases, amyloglucosidases, etc., and shall be further explained in the following example.
• enzymes such as proteases, lipases, amylases, cellulases, glucosidases, amyloglucosidases, etc.

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Citations (3)

• 1996
  • 1996-06-20 DE DE19624624A patent/DE19624624A1/de not_active Withdrawn
• 1997
  • 1997-06-20 WO PCT/US1997/010639 patent/WO1997048816A1/en active Application Filing
  • 1997-06-20 AU AU34035/97A patent/AU3403597A/en not_active Abandoned

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