CS274362B1 - Method of fungal laccase preparation - Google Patents

Abstract

The principle of the solution consists in the fact that the source of laccase enzyme /EC 1.10.2/ used is the substrate in the industrial cultivation of oyster /Pleurotus species/ for example Pleurotus ostreatus, Pleurotus pulmonarius, Pleurotus cornucoipae, Pleurotus eryngii, or their hybrids, which is pressed before planting of the sporocarps, and is followed by the laccase enzyme isolation from the pressed juice by one of the known methods, or it is industrially applied in liquid state without being isolated. The solution can be used in biotechnology, mainly in industrial applications for removing phenol compounds from wastewaters, then in enzyme analytics, or eventually in organic chemistry in enzymatic reactions of some phenol compounds.

Description

The invention relates to fungal laccase and to a process for its preparation in the industrial production of Pleurotus species.

The enzyme lactase (EC 1.10.3.2) belongs to the group of polyphenol oxidases, which oxidizes a very wide range of substrates including monophenols, triphenols, ascorbic acid as well as ortho-para-diphenols (AM Mayer and E. Harel: Phytochemistry 18, 193 (1979)) . It is found in plants but also in many organisms from which it can be isolated in an appropriate way. It is an extracellular enzyme that organisms secrete into the culture medium. While in live plant cells, the lactase is found at very low concentrations in fungi, its concentration is substantially higher. Lactarius, Polyporus, Psalliota, Glomerela, Podospora, Botritis, Pleurotus, Neurospora, Polystictus, and Russula are the most preferred isolates from fungi. Most of the fungi mainly causing white rot of wood produce laccase (T.Kent Kirk: Ann. REV. Microbiol. 41, 465 (1987)), which has a significant effect on altering the chemical structure of lignin, resulting in its lighter solubility in white delignification rotten wood. Since laccase catalyzes the same type of reaction as peroxidase and tyrosinase, there is the possibility of using laccase to purify waste waters contaminated with phenols and substituted phenols (K. L. Shuttleworth and J. M. Bollag: Enzyme Microb. Technol. 8, 171 (1986)). The industrial use of laccases in this respect has hitherto been mainly hampered by economic reasons, as the preparation of laccases for industrial applications has not been solved.

The above-mentioned drawbacks are solved according to the invention, which allows the large-scale production of laccases also for industrial use.

The principle of the invention is that the enzyme lactase (EC 1.10.3.2) is isolated from a substrate in the industrial cultivation of Pleurotus species, preferably Pleurotus ostreatus, Pleurotus pulmonarius, Pleurotus cornucopise, Pleurotus eryngii, or hybrids thereof, such that the substrate used is advantageously, prior to the deposition of the fruiting bodies, the crude enzyme laccase is isolated from the squeezed juice by one of the known methods, or it is applied industrially in a liquid state without isolation thereof.

It has been found that the activity of laccase in the squeezed juice of the substrate used in the cultivation of the glass (straw, cotton waste, rapeseed straw, rope and hemp waste, sawdust) is very high. The activity of laccase measured on syringaldazine as a substrate (J.M. Harkin and J.R. Obst: Sciece 180, 296 (1973)) is in the range of 16,000 to 32,000 U / ml, which is substantially higher than in previously published papers. Depending on the substrate used, it can be directly pressed to obtain 40 to 50 liters of juice per 100 kg of spawned substrate. The compacted solid residue can be used as before for feeding as an additive to multi-component feed mixtures. Furthermore, it has been found that by concentrating the pressed juice on a vacuum evaporator at a temperature of 60 to 70 ° C 8 times, the laccase activity is lost by only 10 to 20% of the original activity.

Lyophilization of the untreated squeezed juice from the spawned substrate yields a dry enzyme preparation having a protein nitrogen content of 2 to 3.5% and an activity of 200 to 600 U / mg. The fungal laccase prepared according to the novel process has a pH optimum in the range of 5.0 to 6.0.

The main advantage of the novel laccase enzyme product is that it can be prepared from the waste produced by the industrial cultivation of livestock by simply molding the spawned substrate, and the molded residue can be used to prepare feed mixtures. The proposed process makes it possible to prepare laccase at a much lower cost, which is an essential prerequisite for its various industrial applications.

Example 1

100 kg of wheat straw substrate prior to the deposition of oyster mushroom fruiting bodies with a dry matter content of 20 to 30% is pressed on a continuous screw press to obtain 40 to 50 liters of pressed juice having an activity of 16 to 32,000 U / ml laccase. The squeezed juice is a deep tea color, is not cloudy, and can be used directly in suitable industrial applications.

CS 274 362 Bl

Example 2

The procedure as in Example 1 is followed, except that the pressed juice is concentrated to a eighth of the original volume on a vacuum evaporator, cooled to 4 ° C and the laccase is precipitated by pouring the concentrated solution into an equal volume of acetone cooled to -10 ° C. and the filter is drained with another portion of cooled acetone. The acetone is distilled off from the product in vacuo to give a dry powder of the crude laccase enzyme with a nitrogen content of 3.4% and an activity of 420 U / mg.

Example 3

The procedure as in Example 1, except that the squeezed juice is lyophilized to give a preparation of 600 U / Mg activity.

Example 4 juices.

The procedure as in Example 2 is followed, except that the lacquase is precipitated from the concentrated compressed by dissolving 350 g of solid ammonium sulfate per liter of thickened juice. The precipitated laccase is filtered or centrifuged and finally dehydrated with cold acetone. The product obtained is lighter and has an activity of 250 U / mg.

Example 5 straw

Procedure as in Example 1. Pressing yields 45 except that sputtered rapeseed liters of pressed juice having a laccase activity of 25,000 U / ml are used as the substrate.

Example 6

The process as in Example 1 except that wood sawdust is used as the spawned substrate. Pressing gives 40 liters of juice with a laccase activity of 22,000 U / ml.

Example 7

Procedure as in Example 1 except that a Pleurotus pulmonarius fungus is inoculated onto the substrate. By pressing the substrate, 42 liters of juice having a laccase activity of 28,000 U / ml are obtained.

Example 8

Procedure as in Example 1 except that a Pleurotus cornucopise fungus is inoculated onto the substrate. Pressing yields 50 liters of juice of laccase activity of 29,500 U / ml.

Example 9

Procedure as in Example 1 except that the fungus Pleurotus eryngii is inoculated onto the substrate. Pressing yields 42 liters of juice with a laccase activity of 25,000 U / l.

Example 10

The procedure as in Example 1, except that the fungus Pleurotus eryngii is inoculated onto the substrate and the substrate is compressed after the first crop of the fetuses. 50 liters of juice of laccase activity of 2700 U / l are obtained.

The invention has application in biotechnology, especially in industrial applications in the removal of phenolic compounds from wastewater, further in enzyme analysis, or in organic chemistry in the enzymatic reactions of some phenolic compounds.

Claims (1)

OBJECT OF THE INVENTION Process for the preparation of fungal laccase, characterized in that a substrate for the industrial cultivation of livestock, Pleurotus species, e.g. it isolates the pure enzyme in a known manner.