Ing, Nicole; Deng, Kai; Chen, Yan; Aulitto, Martina; Gin, Jennifer W.; Pham, Thanh L.; Petzold, Christopher J.; Singer, Steve W.; Bowen, Benjamin; Sale, Kenneth L.; Simmons, Blake A.; Singh, Anup K.; Adams, Paul D.; Northen, Trent R.
Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.
Laccases are oxidative enzymes containing 4 conserved copper heteroatoms. Laccases catalyze cleavage of bonds in lignin using radical chemistry, yet their exact specificity for bonds (such as the β-O-4 or C-C) in lignin remains unknown and may vary with the diversity of laccases across fungi, plants and bacteria. Bond specificity may perhaps even vary for the same enzyme across different reaction conditions. Determining these differences has been difficult due to the fact that heterologous expression of soluble, active laccases has proven difficult. Here we describe the successful heterologous expression of functional laccases in two strains of Saccharomyces cerevisiae, including one we genetically modified with CRISPR. We phylogenically map the enzymes that we successfully expressed, compared to those that did not express. We also describe differences protein sequence differences and pH and temperature profiles and their ability to functionally express, leading to a potential future screening platform for directed evolution of laccases and other ligninolytic enzymes such as peroxidases.
We are working to generate fundamental understanding of enzymatic depolymerization of lignin and using this understanding to engineer mixtures of enzymes that catalyze the reactions necessary to efficiently depolymerize lignin into defined fragments. Over the years the enzymes involved in these processes have been difficult to study, because 1) the enzymes thought to be most important, fungal laccases and peroxidases, are very difficult to express in soluble, active form; 2) the full complement of required enzymes and whether or not they act synergistically is not known; 3) analysis of bond cleavage events is difficult due to the lack of analytical tools for measuring bond cleavage events in either polymeric lignin or model lignin-like compounds.
Walker, Johnnie A.; Takasuka, Taichi E.; Deng, Kai; Bianchetti, Christopher M.; Udell, Hannah S.; Prom, Ben M.; Kim, Hyunkee; Adams, Paul D.; Northen, Trent R.; Fox, Brian G.
Background: Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe-0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. Results: CelEcc-CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc-CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc-CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. Conclusion: We have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.