Enzyme Development

Due to their defined substrate and product specificity as well as high chemo selectivity, enzymes are important biocatalysts for synthesis of fine chemicals, aromatics and pharmaceuticals [1]. Enzyme development is necessary when a certain industrial application demands a specific chemical conversion under mild reaction conditions.

There are two essential steps in enzyme development, which are handled at Fraunhofer IGB. First step involves identification of suitable enzymes or enzyme variants from nature. Second step aims for improvement of known enzymes through implementation of structural changes with directed evolution or rational design. Improving enzyme stability, activity or substrate specificity is commonly of interest for most technical applications. For this the enantioselectivity of an enzyme plays an important role to ensure the chiral purity of the desired reaction product. Also enzyme solubility after expression is an important target for optimization to enhance enzyme yields after purification and downstream processing.

Selection

A well-established method for identification of new enzymes is based on screening of microbiotopes. For this microorganisms from biotopes are cultivated on defined media with substrate limitation, e.g. containing only one carbon source. This carbon source is an imperative nutrient supply for the microorganisms on the one hand and simultaneously represents the substrate for the desired enzyme reaction on the other hand. Thus, only microorganisms are allowed to grow which produce enzymes for substrate uptake and conversion for implementation into their growth metabolism and are selected thereby.

After isolation of respective microorganisms, enzymes are identified which are responsible for substrate conversion. One possibility for enzyme identification in microorganisms is by establishing genome libraries, a large quantity of genes on DNA fragments which are cloned in enzyme expression vectors. With help of enzyme activity assays, clones are identified bearing respective enzyme gene from genome library. A minor extensive possibilities for enzyme screening is the search for sequence homologues from databases of already sequenced genome fragments [2]. Such DNA-sequences can be amplified from the genome of microorganisms or direct in vitro synthesis of genes.

Fraunhofer IGB daily performs various transformation and recultivation of microorganisms as well as activity assays of enzymes for finding enzymes having desired specific catalytic activities for required chemical reactions.

Enzyme engineering

Enzyme engineering implies application of different molecular biological methods for enzyme optimization. Experiments for enzyme engineering are typically divided into three stages:

  • Diversification: Establishing gene libraries with numerous variants of one enzyme by application of error-prone-PCR, DNA-shuffling or rational design
  • Screening: Investigation of variants for improved catalytic properties
  • Verification: Amplification and evaluation of improved biocatalyst’s gene sequences (DNA-sequencing, enzyme modelling)

One tool for enzyme engineering is directed evolution. For this, a DNA-sequence encoding for the enzyme is mutated randomly (through error-prone-PCR or DNA-shuffling) for generation of a large number of new enzyme variants. With help from appropriate screening assays it is possible to detect enzyme variants with improved properties over wild type enzyme. Exchange of few amino acids in enzyme sequence might have large impact on internal protein interactions and thus on enzyme structure and thus on enzymes flexibility and stability.

At Fraunhofer IGB, a thermostable oxidoreductase was optimized by directed evolution. After diversification of its gene sequence by error prone PCR, further screening and sequence analysis was performed several sequent rounds to find different mutations and combinations thereof which enhanced the enzymes activity by factor ca. 50. Thus developed enzyme variants have improved affinity for desired substrates as well as higher specific activity for enhanced product formation [3].

Besides directed evolution there is one additional approach for diversification, the rational design of enzymes. For this amino acids in the enzyme are selectively exchanged by targeted mutations within the respective gene sequence to enhance biocatalytic properties. In contrast to directed evolution approach, rational design demands exact knowledge of three-dimensional enzyme crystal structure or at least availability of at least one meaningful enzyme model.

Fraunhofer IGB possesses competences to perform computer-based enzyme modelling on the one hand. Based on those enzyme models or enzyme crystal structures and suitable software, Fraunhofer IGB scientists can calculate targeted enzyme modifications which lead to improvement of enzymes biocatalytic properties on the other hand. For instance, impact of amino acid residues of a thermostable oxidoreductase on specific activity, cofactor affinity and stability of the enzyme could be analyzed with help of enzyme modelling [3].

To counteract the enormous personal and time effort by searching enzymes with appropriate biocatalytic properties, Fraunhofer IGB applies modern robotics for high throughput processing.

In preferably shortest amount of time large numbers of enzyme variants can be tested with respect to their catalytic functionality under desired process conditions.

For this, modern systems are available at the Straubing Branch, e.g. a pipetting robot and several picking stations. With those instruments it is possible to automatically analyze up to 10,000 variants per day.

Services

Fraunhofer IGB provides the following services in the field of enzyme development for biochemical reactions:

  • Method evaluation
  • Enzyme screening
  • Molecular biological and technical optimization of enzymes and enzyme reactions
  • Commissioned synthesis of fine chemicals
  1. Hofer, M.; Strittmatter, H.; and Sieber, V. Biocatalytic Synthesis of a Diketobornane as a Building Block for Bifunctional Camphor Derivatives. ChemCatChem, 2013. 5(11): p. 3351-3357.
  2. Reiter, J., et al., Enzymatic cleavage of lignin β-O-4 aryl ether bonds via net internal hydrogen transfer. Green Chemistry, 2013. 15(5): p. 1373-1381.
  3. Steffler, F.; Guterl, J.-K.; and Sieber, V. Improvement of thermostable aldehyde dehydrogenase by directed evolution for application in Synthetic Cascade Biomanufacturing. Enzyme and Microbial Technology, 2013. 53(5): p. 307-314.