New thermostable and catalytically-enhanced pectate lyase (L-11927)

Summary

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Microbial enzymes are routinely used across many economic industrial sectors for the development of environmentally-friendly processes. Enzymes with enhanced thermal stability and activity are highly sought after for use under industrial processing conditions (e.g., elevated temperature and density). Genetic engineering approaches were applied to create variants of a new Xanthomonas campestris pectate lyase. These variants with improved thermotolerance and increased activity hold high market potential for the industrial processing of pectinaceous compounds.

Applications

• Robust catalyst alternatives for the breakdown of pectinaceous materials under industrial process temperature.
• Extraction and clarification of fruit juices and wine.
• Treatment of industrial wastewater containing pectinaceous materials.
• Retting or bioscouring of natural bast fibers (e.g., hemp and flax), and cotton fabric.

Concept

There is increasing demand to replace some traditional chemical processes with biotechnological processes involving microorganisms and enzymes which not only provide an economically viable alternative but are also more environmentally friendly. Microbial pectinases are a family of enzymes that have shown great potential for a variety of industrial uses as they have the capacity to degrade pectin, a polysaccharide found in the tissues of most plants. Pectinolytic enzymes include pectate lyases. Given the growing understanding and interest in the mechanism of action of pectate lyases, new applications continue to be discovered for this enzyme. As the market develops, an increasing number of pectate lyases are being developed from a range of microorganisms and the available microbial genome database. A strategy that involved multiple sequence alignment and site-directed mutagenesis was used to create variants of a new X. campestris enzyme, pectate lyase II. Three different variants of the enzyme were produced in Escherichia coli: one with increased thermostability, one with increased catalytic activity, and one combining both features. The variants with enhanced catalytic efficiency exhibit a two- to five-fold increase in activity relative to the wild type enzyme; those with enhanced heat stability are active up to 50°C. Since many industrial processes take place at even higher temperatures, R&D is being conducted to further improve the thermostability of the enzyme.

Increasing amount of pectin released from hemp fibre preparation by the improved variants of pectate lyase (A31G, R236F and A31G/R236F) compared to the parent enzyme.

Benefits

Numerous fields of application
Pectate lyases have the potential of addressing significant unmet needs in fields such as wastewater treatment, biocomposites, animal feeds, pretreatment of textiles, wine and juice processing, detergents, paper de-inking and food ripening.

Enhanced thermostability and catalytic activity
Thermostability and increased catalytic activity are important features as they increase the rate of activity and the shelf-life of the enzyme while decreasing energy consumption and costs when processing substrates. Further, improved enzymatic activity allows the processing of a larger volume of substrate.

Increased production capacity and safety
By using the well-established E. coli platform, the recombinant enzyme variants can be produced in large amounts. Further, the expression of the variants in E. coli allows avoiding the use of X. campestris, a potential plant pathogen.

Protection

Pectate lyase with increased thermostability and enzymatic activity (NRC no. 11927).

Contacts

Related Information

Institutes:

• NRC Biotechnology Research Institute