How can enzymes be characterized
Objective and reason for the project
The introduction of modern enzyme technologies in the processing industry leads to the substitution of environmentally harmful, energy-intensive and high-emission chemical processes and chemically synthetically manufactured products with more ecologically compatible biotechnological alternatives. The key to a broad establishment of economically and ecologically advantageous processes lies in the availability of highly efficient and stable biocatalysts. Due to their ability to break down lignified biopolymers, fungi from the Basidiomycete class have a unique set of enzymes. The hundreds of potential extracellular Basidiomycete enzymes include: Glycosidases, peptidases and lipases.
The aim of the project was to make this previously unused biochemical potential available in the form of a platform technology for processes and products of white biotechnology. This includes the discovery and biochemical characterization of the following enzymes: proline-specific peptidases for use in the beverage industry, glycosidases with new catalytic properties also for use in the beverage industry, lipases and esterases for aroma biotechnology, the detergent, animal feed and food industries as well as polyvalent ones Peroxidases for the synthesis of fine chemicals. The suitability of the enzymes for the listed technical purposes should also be demonstrated.
The work on the discovery of secretory peptidases,? -1,3-glucanases and peroxidases from Basidiomycetes initially included a screening in emers and submerged cultures. Induction was carried out by adding cinerean (a? -1,3-glucan from Botrytis cinerea), proline-containing proteins or anthocyanins to the respective fermentation medium. A proline-specific peptidase from Wolfiporia cocos identified in this way was first purified by ion exchange chromatography and subsequent gel permeation chromatography. The purified enzyme was characterized biochemically in order to be able to assess whether the properties meet the requirements for use in the beverage industry. A glucanase from Lentinellus cochleatus was characterized with regard to its activity and compared with other commercially available glucans.
Results and discussion
The following results generated during the course of the project are particularly noteworthy: In the screening of edible Basidiomycetes, three of the targeted activities could be identified. A protease with proline-specific activity from Wolfiporia cocos, a? -1,3-glucanase from Lentinellus cochleatus and an anthocyanin-discoloring activity from Cyathus striatus were discovered. The proline-specific peptidase from W. cocos was purified, biochemically characterized and its technical applicability investigated. This enzyme has promising properties in terms of industrial use. For example, both the temperature optimum with 40-50 ° C and the pH optimum with pH 3.5-5.5 are in areas that predestine the enzyme for use in the beverage industry. The enzyme also has the stability required for industrial applications (approx. 80% residual activity after 6 months of storage). The substrate spectrum ranges from small proline-containing peptides to prolines of various origins (rice, wheat, barley, corn) and casein. The next steps will be to obtain the enzyme on a larger scale and to specify the application in the various areas. The? -1,3-glucanase from L. cochleatus shows its maximum activity at a pH value of 3.5 to 5.5 and a temperature of around 65 ° C. The chromatographic analysis, which can be used to track the degradation of glucan via oligomers to glucose, suggests that exo- and endoglucanase activities overlap in the fermentation supernatant. A more precise classification of these enzymes will result in the still pending characterization of the purified enzymes in follow-up projects. In the application tests, the expectation that a combination of endo- and exo-glucanase activity leads to a faster release of glucose than the isolated use of one of the activities was not confirmed. This is probably due to the fact that the exoglucan used does not accept the generated smaller glucan fragments as a substrate as well as high molecular glucan. Detailed investigations are still pending.
The chosen experimental procedure has proven itself, even if the fermentation times necessary for secretion of the enzymes were longer than expected and the biochemical characterization, for example through the complex analysis of the glucan degradation, turned out to be more labor-intensive than expected. Although the planned program could not be worked through in detail as a result, substantial progress was made. The most important project goals were achieved, whereby the character of the project, namely to show the biochemical potential of basidiomycete enzymes in the sense of a platform technology, was retained.
All project partners agree that the project was extremely successful. The cooperation between the project partners turned out to be very pleasant, constructive and cooperative. The cooperation partners were and are in regular contact in order to exchange information and results promptly.
Public relations and presentation
No results have been published so far. The patent situation in the area of proline-specific protease is currently being examined in detail. A final assessment is therefore not yet available. The current data suggests publications on proline-specific protease as well as on glucanase from Lentinellus cochleatus in peer-reviewed journals.
The project was able to more than confirm the expectation, based on the preliminary work of the University of Hanover, that basidiomycetes have a unique set of enzymes. The identification and characterization of the proline-specific protease from W. cocos has almost been completed, so that production on a larger scale and the development of methods for using the enzyme can begin. The application potential of the? -1,3-glucanase identified in L. cochleatus must be tested in further series of experiments. It remains to be seen whether the enzyme that was discovered in Cyathus striatus and capable of discoloring anthocyanins is a promising one
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