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Research Project: Technologies for Improving Industrial Biorefineries that Produce Marketable Biobased Products

Location: Bioproducts Research

Title: Chromohalobacter salixigens Uronate Dehydrogenase: directed evolution for improved thermal stability and mutant

item Wagschal, Kurt
item Chan, Victor
item PEREIRA, JOSE - Joint Bioenergy Institute (JBEI)
item ZWART, PETER - Lawrence Berkeley National Laboratory
item SANKARAN, BANUMATHI - Lawrence Berkeley National Laboratory

Submitted to: Process Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/13/2020
Publication Date: 2/14/2020
Citation: Wagschal, K.C., Chan, V.J., Pereira, J.H., Zwart, P.H., Sankaran, B. 2020. Chromohalobacter salixigens Uronate Dehydrogenase: directed evolution for improved thermal stability and mutant. Process Biochemistry.

Interpretive Summary: The world-wide yearly production of orange juice (~1.7 x 106 metric tons/year) and the pulp waste steam from sugar beet refining together generate significant food processing waste that can be a disposal problem, which could be lessened by generating new products from this under-utilized waste stream that contains ~25% uronic acid (GalUA), the main sugar acid component of pectin. Likewise, alginate from algae biomass is composed largely of uronic acids. The enzyme uronate dehydrogenase can be an environmentally benign, cost effective method to convert uronic acids to dicarboxylic aldaric acids, which are a DOE top 10 platform chemical from biomass that can be used for both fiber and bulk fine chemical applications such as detergents. Important for cost-effective industrial application of the enzyme is thermal stability. We used enzyme engineering techniques to improve the thermal stability by 18 'C, and determined the atomic structure of the enzyme.

Technical Abstract: Chromohalobacter salixigens contains a uronate dehydrogenase termed CsUDH that can convert uronic acids to their corresponding C1,C6-dicarboxy aldaric acids, an important enzyme reaction applicable for biotechnological use of sugar acids. To increase the thermal stability of this enzyme for biotechnological processes, directed evolution using gene family shuffling was applied, and the hits selected from 2-tier screening of a shuffled gene family library contained in total 16 mutations, only some of which when examined individually appreciably increased thermal stability. Most mutations, while having minimal or no effect on thermal stability when tested in isolation, were found to exhibit synergy when combined; CsUDH-inc containing all 16 mutations had 'Kt0.5 +18 'C, such that kcat was unaffected by incubation for 1 hr at ~70 'C. X-ray crystal structure of CsUDH-inc showed tight packing of the mutated residue side-chains, and comparison of rescaled B-values showed no obvious differences between wild type and mutant structures. Activity of CsUDH-inc was severely depressed on glucuronic and galacturonic acids. Combining select combinations of only three mutations resulted in good or comparable activity on these uronic acids, while maintaining some improved thermostability with 'Kt0.5 ~+ 10 'C, indicating potential to further thermally optimize CsUDH for hyperthermophilic reaction environments.