New technical enzymes for a green chemistry

Enzymes as biocatalysts offer a number of advantages for a sustainable chemical industry, such as mild reaction conditions, biodegradability, high selectivity and therefore low by-product formation. Fraunhofer IGB has been working for more than ten years on the screening for new, industrially usable enzymes as well as their optimization and production. Numerous projects with companies in the chemical and pharmaceutical industries have already been successfully completed.

Identification of suitable biocatalysts for a sustainable industry

Enzymes are already widely used in the food industry, the textile industry, the cleaning and detergent industry, the chemical industry and the pharmaceutical industry. New enzymes with tailor-made properties are constantly being sought for increasing sustainability in these branches of industry. The main focus is on the identification of new enzymes with a broad substrate and product spectrum, high stability against extreme temperatures and pH values and increased longevity.

• Identification of new enzymes with desired properties, e.g. for epoxidation, lignin modification and chitin monomerization. Methods such as enrichment cultures of organisms, metagenome screening, in-silico screening and functional assays are used.
• Establishment of stable, scalable expression systems with high yields, for example in bacterial systems such as E. coli and eukaryotic systems such as the yeasts Komagataella pastoris, Kluyveromyces lactis
• Production of enzymes on a 1 to 30-liter scale at Fraunhofer IGB, design and optimization of fermentation strategies (batch, fed-batch, continuous processes)
• Design and planning of scale-up experiments for fermentations up to 10 m³, scale-up at and in cooperation with Fraunhofer CBP

Fraunhofer IGB deals with the production and optimization of enzymes from various classes of enzymes, in particular hydrolytic enzymes such as lipases, phospholipases and proteases, and oxidoreductases such as dismutases, dehydrogenases and peroxidases.

Phospholipase C

Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to form diacylglycerols and water-soluble phosphorylethanolamine. PLC is used on a large scale for refining vegetable oils in order to achieve a faster and almost complete phase separation.

Lipases

Lipases take up about 5 percent of the world enzyme market due to their versatile application possibilities. Lipases catalyze the hydrolysis of fats (triglycerides) in glycerol and fatty acids. Depending on the reaction conditions, lipases can catalyze further reactions, such as esterification, transesterification, alcohol lysis, acid hydrolysis, aminolysis, acylation, perhydrolysis and epoxidation.

Proteases

Proteases (EC 3.4) are among the most important enzymes in industry. Approximately 60 percent of all commercial enzymes used worldwide are proteases. In industry, proteases are mainly used in pharmaceuticals, medicine and the food industry. Plants offer a broad spectrum of different proteases with different properties. However, in contrast to the production of microbial proteases, the use and production of plant proteases for industry is limited by the often low yields and difficult extraction conditions. Biotechnological processes and the recombinant production of plant enzymes in microbial expression systems, such as yeasts, make large quantities of plant enzymes accessible to industry.

Formaldehyde dismutase

A formaldehyde dismutase from Pseudomonas sp., which has already been successfully investigated for several years from the wild-type strain at Fraunhofer IGB, has a cofactor but also an integrated mechanism for regenerating it, so that this oxidoreductase is well suited for large-scale processes. The substrate formaldehyde and the products methanol and formic acid are important platform chemicals that are processed in large quantities in the chemical industry. Fraunhofer IGB is currently working on the heterologous expression of this enzyme in Escherichia coli. Another oxidoreductase, methanol dehydrogenase, is of industrial relevance, as only a few alcohol dehydrogenases have significant activity against the platform chemical methanol. The work at Fraunhofer IGB includes the heterologous expression of the enzyme and its immobilization and stabilization for use in industrial processes. Together with formaldehyde dismutase, this enzyme will be used to produce methanol from biogas.

Peroxidases

Peroxidases, also belonging to oxidoreductases, are investigated at Fraunhofer IGB with regard to their ligninolytic activities. In addition to peroxidases secreted by white rot fungi to degrade lignin, Fraunhofer IGB is working on new bacterial peroxidases and their role in the lignin degradation process. Bacterial dyp-type peroxidases are a separate class of haem peroxidases and are found in fungi and bacteria. Some of these dyp-type peroxidases are thought to be involved in the degradation of lignin. At Fraunhofer IGB, bacterial dyp-type peroxidases are first expressed heterologously on a laboratory scale in Escherichia coli and the enzyme yield is optimized. Furthermore, the enzymes are purified and their catalytic properties are investigated. A later application can be in the area of lignin modification or the oxidation of aromatic substances in waste water treatment.

Chitinases

Chitinases are another class of industrially relevant enzymes produced at Fraunhofer IGB. These are to be used for the monomerization of chitin, whereby a stream of recyclable materials is to be generated from the waste material chitin (see "Production of recyclable materials from waste streams").

At Fraunhofer IGB, we identify tailor-made recombinant enzymes with specific substrate spectra or special catalytic properties for a wide variety of applications.

For enzyme screening, we use conventional methods by isolating and enriching microorganisms. The aim is to find new enzymes in cultivatable organisms. On the other hand, molecular methods such as screening via metagenomic gene banks also play a role, enabling the detection of non-cultivable microorganisms. The metagenomic banks serve as a platform for the rapid identification and optimization of new enzymes. Another strategy for identifying new enzymes is the systematic use of genomic information from sequencing projects (in-silico screening). The basis for this is the often existing similarity of the gene sequences of enzymes with similar properties.

Enzymes that can already be produced in our laboratories are optimized for a wide range of applications using molecular-evolutionary techniques. We can produce and evaluate these in heterologous systems of up to 30 liters. Enzyme production on a large scale up to 10 m³ including upscaling of the process is carried out in cooperation with Fraunhofer CBP.

Fraunhofer IGB offers a broad spectrum of technical enzymes. Examples of technical enzymes already produced at the institute and their possible fields of application are shown in the adjacent table.

For phospholipase C, codon optimization was performed at Fraunhofer IGB in order to achieve high yields in its heterologous expression in the yeast Kluyveromyces lactis. At present, additional master and process optimizations are being carried out in order to further increase yields.

A fungal lipase identified at Fraunhofer IGB is currently recombinantly expressed in the methylotrophic yeast Komagataella pastoris (formerly Pichia pastoris). The recombinant lipase has already been produced to the 1 m³ scale in the multifunctional plant at Fraunhofer CBP in Leuna.

Plant proteases are recombinantly expressed in yeasts at Fraunhofer IGB. The activity of a recombinant protease has already been measured to be 1.4 times higher than that of a commercial enzyme.

The genomes of the ligninolytic bacterial strains Streptomyces sp. and Pseudonocardia sp. were sequenced and sequences from public databases were compared. In both strains, a large number of monooxygenases and dioxygenases could be identified that provide information on the aromatic metabolic pathways of these bacteria. In addition, three potential ligninolytic peroxidases were identified. A dyp-type peroxidase from Streptomyces sp. was expressed in E. coli and the enzyme yield was optimized. The peroxidase was purified and an activity towards different model substances was shown in enzyme assays.

In order to recombinantly produce formaldehyde dismutase, the associated gene was isolated from Pseudomonas sp. in a first step and the gene sequence analyzed. Comparisons of the IGB-own dismutase with a sequence known in the literature showed a high correspondence between these genes. The recombinant production of formaldehyde dismutase in different E. coli strains already showed higher yields with easier cultivation than in the wild-type strain. Recombinant production has significantly increased the long-term stability of the dismutase, among other things. Work is currently underway on process optimization for enzyme production and the targeted modification of the enzyme for subsequent immobilization.

Grumaz C, Rais D, Kirstahler P, Vainshtein Y, Rupp S, Zibek S, Sohn K. 2017. Draft genome sequence of Pseudonocardia autotrophica strain DSM 43083, an efficient producer of peroxidases for lignin modification. Genome announcements, No. 5.  https://doi.org/10.1128/genomeA.01562-16

Blaschke L, Wagner W, Werkmeister C, Wild, M, Gehring A, Rupp S, Zibek S. 2017. Development of a simplified purification method for a novel formaldehyde dismutase variant from Pseudomonas putida J3. in Journal of Biotechnology, Vol. 241, pp. 69-75. https://doi.org/10.1016/j.jbiotec.2016.11.007