Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1986-06-04
1988-05-03
Shapiro, Lionel M.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435931, C12N 924, C12R 1785
Patent
active
047420081
DESCRIPTION:
BRIEF SUMMARY
This invention concerns a procedure for the production of .beta.-glucanase through the cultivation of a microorganism in a nutrient medium under optimal conditions.
We refer to Norwegian patent application No. 82 4321 concerning the use of microorganisms of the species Rhizomucor pusillus (Lindt) Schipper as being suitable for the above process, as well as an isolated and registered strain of the stated microorganism CBS 551.82 (Centraalbureau voor Schimmelcultures, Nederland) being particularly suitable for the production of the enzyme .beta.-glucanase.
The purpose of this invention is to optimize fermentation such that maximum enzyme yield within the shortest possible time is achieved, yet under the same conditions as otherwise stated in Norwegian patent application No. 82 4321, that is, to conduct fermentation under thermophilic conditions and wherein the microorganism is thermostable under the said conditions. In addition to the significance that the microorganism (Rhizomucor pusillus (Lindt) Schipper CBS 551.87) has for a positive result, it should also be mentioned that the composition of the nutrient medium, the air delivery rate and apparatus design are all important factors for the achievement of optimal conditions for fermentation.
Vegetable meal and starchy substances, as well as sugars, are commonly used as carbon sources for fermentation purposes. However, not all carbon sources have proven to be suitable for this purpose. The cause of this has not been definitely ascertained, but different factors appear to have an influence on the transformation of sugar into energy. If the source of carbon is to be used as an energy source, the following factors at least seem to be of significance: the solubility of the source of carbon and its ability to be taken up through the system of cell wall and membrane.
It now appears, astonishingly enough, that if we apply a two-stage fermentation with lactose as the source of carbon in a fermentation process using the previously mentioned microorganisms, then the enzyme yield increases several times when all conditions are met.
Under batch conditions, the fermentation exhibits the traditional growth pattern with a lag phase, exponential growth and a stationary phase where enzyme production is greatest in the stationary phase. It is important that the fermentation is carried out under aerobic conditions. This places great demands on the stirring machinery and on the stirring speed. If the stirring is too vigorous, then the fungal hyphae (microorganisms) are disintegrated, and if it is too weak, oxygen is not distributed sufficiently in the fermentor. Both factors reduce enzyme formation. Furthermore, too vigorous stirring combined with a low oxygen content promotes a threadlike structure in the fungal hyphae, whereas a more pellet-like structure is desired.
For a better understanding of the invention we refer to Examples 1-8.
EXAMPLE 1
(a) 300 ml of an inoculum of Rhizomucor pusillus (Lindt) Schipper CBS 551.82 was mixed in a 14 l fermentor with 10 l of a salt medium of the following composition:
Conc. H.sub.2 SO.sub.4 : 1.5 ml/l source and 13.4 g/l NH.sub.4 Cl as the nitrogen source. Berol 374 from Berol Kemi AB, Sweden, was used as an anti-foaming agent. The stirring rate was 600 rpm, the temperature 40.degree. C. and pH 4.5. The fermentor's air delivery was set to 3 vvm, that is, 30 l air/min. (1 vvm is 1 1 air per 1 nutrient medium per min.). Fermentation time was 45 hours. (b) An inoculum of the above mentioned 10 l nutrient medium was mixed in a 300 l fermentor with 200 l of the salt medium described above. of 40 g/l barley meal and 13.4 g/l NH.sub.4 Cl at pH 4.5 and a temperature of 40.degree. C., and a stirring rate of 410 rpm. The fermentor's air delivery rate was 0.2 vvm, or 40 l air/min. The enzyme activity was 2.5 EU/ml after 79 hours and 2.7 EU/ml after 92 hours according to Dygert's method (Analytical Biochemistry, vol. 13 (1965) pp. 367-374).
EXAMPLE 2
An inoculum from a 14 l fermentor holding the conditions described in Example 1 was put into
REFERENCES:
patent: 3880742 (1975-04-01), James et al.
patent: 4275163 (1981-06-01), Gallo
patent: 4399221 (1983-08-01), Schneider et al.
patent: 4588690 (1986-05-01), Nissen et al.
Hovland Jon
Nissen Bernt
Norsk Hydro a.s.
Shapiro Lionel M.
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