Deciphering the signaling mechanisms of the plant cell wall degradation machinery in Aspergillus oryzae

published_or_final_versio

Probing of Carbohydrate-Protein Interactions Using Galactonoamidine Inhibitors

Glycoside hydrolases are ubiquitous and one of the most catalytically proficient enzymes known, and thus understanding their mechanisms are crucial. Most research has focused on the interaction of the glycon of substrates and their inhibitors within the active site of glycoside hydrolases. The inhibitors employed to probe these interactions generally had small aglycons (i.e. a hydrogen atom, amidines, small aliphatic groups, or benzyl groups). Here, the interactions of the aglycon with glycoside hydrolases are examined by probing the active sites with a library of 25 galactonoamidines. The studies described in this dissertation aim to increase the understanding of stabilization of the transition state by glycoside hydrolases, which allows for the acceleration of substrate hydrolysis by the enzymes up to 1017 over non-enzymatic hydrolysis. To understand this stabilization, the active sites of beta-galactosidases from Aspergillus oryzae, bovine liver, and Escherichia coli were evaluated using spectroscopic, molecular docking, and modeling analyses to determine transition state analogs (TSAs) and how the TSAs interact within the active site of glycoside hydrolases. The probing with the galactonoamidine library revealed hydrophobic interactions, pi-pi interactions, and CH-pi interactions within the active sites to varying extent. Further, three TSAs were found for the hydrolysis of substrates by beta-galactosidase (A. oryzae), and two TSAs for the beta-galactosidases from bovine liver and E. coli. Upon TSA binding to the three beta-galactosidases, conformational changes occurred to stabilize the galactonoamidines within the active sites, which did not occur when fortuitous binders interacted with the enzyme. The conformational changes within the active sites of beta-galactosidases from bovine liver and E. coli closes off the active site via a loop movement resulting in a substantially higher binding affinity than those observed with beta-galactosidase (A. oryzae). A subsequent evaluation of galactonoamidine specificity in the presence of other proteins revealed an increase of inhibitory activity two orders of magnitude more than a purified beta-galactosidase (E. coli)

Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus, Alternaria solani

Citation: Chandrasekaran, M., Chandrasekar, R., Sa, T., & Sathiyabama, M. (2014). Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus, Alternaria solani. Retrieved from http://krex.ksu.eduSerine proteases generally share a relatively high degree of sequence identity and play a major role in the diversity of biological processes. Here we focus on three-dimensional molecular architecture of serine proteases from Alernaria solani. The difference in flexibility of active binding pockets and electrostatic surface potential distribution of serine proteases in comparison with other fungal species is reported in this study. In this study we have purified a serine protease from the early blight pathogen, Alernaria solani. MALDI-TOF-MS/MS analysis revealed that protease produced by A. solani belongs to alkaline serine proteases. AsP is made up of 403 amino acid residues with molecular weight of 42.1kDa (Isoelectric point (pI)-6.51) and molecular formula C[subscript 1859]H[subscript 2930]N[subscript 516]O[subscript 595]S[subscript 4]. The follow-up research on the molecular structure prediction is used for assessing the quality of A. solani Protease (AsP). The AsP protein structure model was built based on its comparative homology with serine protease using the program, MODELER. AsP had 16 β-sheets and 10 α-helices, with Ser[superscript 350] (G347-G357), Asp[superscript 158] (D158-H169) and His[superscript 193] (H193-G203) in separate turn/coil structures. Biological metal binding region situated near the 6th-helix and His[superscript 193] residue is responsible for metal binding site. In addition, the calcium ion is coordinated by the carboxyl groups of Lys[superscript 84], Ile[superscript 85], Lys[superscript 86], Asp[superscript 87], Phe[superscript 88], Ala[superscript 89], Ala[superscript 90] (K84-A90) for first calcium (Ca[superscript 2+]) binding site and carbonyl oxygen atom of Lys[superscript 244], Gly[superscript 245], Arg[superscript 246], Thr[superscript 247], Lys[superscript 248], Lys[superscript 249], and Ala[superscript 250] (K244–A250), for second Ca[superscript 2+] binding site. Moreover, Ramachandran plot analysis of protein residues falling into most favored secondary structures were determined (83.3%). The predicted molecular 3D structural model was further verified using PROCHECK, ERRAT and VADAR servers to confirm the geometry and stereo-chemical parameters of the molecular structural design. The functional analysis of AsP 3D molecular structure predictions familiar in the current study may provide a new perspective in the understanding and identification of antifungal protease inhibitor designing

A Review of natural and engineered enzymes involved in bioethanol production

Alternative petroleum-derived fuels, such as biofuels, is another form to decrease the dependence of non-renewable energy. The most promising alternative energy is cellulosic ethanol because of the abundance of cellulose and the overall lack of concern for the food-versus-fuel dilemma. In order to produce ethanol from cellulosic materials, pretreatment is required to “open” the lignocellulosic matrix and make cellulose more susceptible to enzymatic degradation. Enzymatic hydrolysis of lignocellulose is an important area of research due to the absence of negative effects in downstream processes in comparison with acid hydrolysis. Both natural enzymes and engineered enzymes can be used in the process of ethanol production. Natural enzymes are found either individually or as a part of a complex known as cellulosome. Such complexes are the focus of many studies due to the efficiency in the degradation of cellulose. Research in enzymatic engineering is being done in order to mimic these natural systems. Engineered individual enzymes are also used to improve the properties of the enzymes found in nature. Enzymes can be engineered by rational design or directed evolution. Directed evolution is the most efficient technology, since it only requires the knowledge of protein sequences. However, this approach also possesses some limitations. A combination of both methods or a “semi-rational” approach is perhaps the best option to develop higher performance lignocellulolytic enzymes. Many advances regarding engineering of lignocellulolytic enzymes have been made in the last past years. Further research, however, is required in the development of enzymes systems and enzyme industrial testing to establish cellulosic bioethanol as main substitute for petroleum-derived fuel energy

Membrane transporters in the bioproduction of organic acids: state of the art and future perspectives for industrial applications

Organic acids such as monocarboxylic acids, dicarboxylic acids or even more complex molecules such as sugar acids, have displayed great applicability in the industry as these compounds are used as platform chemicals for polymer, food, agricultural and pharmaceutical sectors. Chemical synthesis of these compounds from petroleum derivatives is currently their major source of production. However, increasing environmental concerns have prompted the production of organic acids by microorganisms. The current trend is the exploitation of industrial biowastes to sustain microbial cell growth and valorize biomass conversion into organic acids. One of the major bottlenecks for the efficient and cost-effective bioproduction is the export of organic acids through the microbial plasma membrane. Membrane transporter proteins are crucial elements for the optimization of substrate import and final product export. Several transporters have been expressed in organic acid-producing species, resulting in increased final product titers in the extracellular medium and higher productivity levels. In this review, the state of the art of plasma membrane transport of organic acids is presented, along with the implications for industrial biotechnology.This work was supported by the strategic programme UID/BIA/04050/2019 funded by Portuguese fundsthrough the FCT I.P., and the projects: PTDC/BIAMIC/5184/2014, funded by national funds through the Fundacao para a Ciencia e Tecnologia (FCT) I.P. and by the European Regional Development Fund (ERDF) through the COMPETE 2020-Programa Operacional Competitividade e Internacionalizacao (POCI), and EcoAgriFood: Innovative green products and processes to promote AgriFood BioEconomy (operacao NORTE-01-0145-FEDER-000009), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). DR acknowledges FCT for the SFRH/BD/96166/2013 PhD grant. MSS acknowledges the Norte2020 for the UMINHO/BD/25/2016 PhD grant with the reference NORTE-08-5369-FSE-000060. TR acknowledges Yeastdoc European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 764927

Recent advances in β-galactosidase and fructosyltransferase immobilization technology

The highly demanding conditions of industrial processes may lower the stability and affect the activity of enzymes used as biocatalysts. Enzyme immobilization emerged as an approach to promote stabilization and easy removal of enzymes for their reusability. The aim of this review is to go through the principal immobilization strategies addressed to achieve optimal industrial processes with special care on those reported for two types of enzymes: β-galactosidases and fructosyltransferases. The main methods used to immobilize these two enzymes are adsorption, entrapment, covalent coupling and cross-linking or aggregation (no support is used), all of them having pros and cons. Regarding the support, it should be cost-effective, assure the reusability and an easy recovery of the enzyme, increasing its stability and durability. The discussion provided showed that the type of enzyme, its origin, its purity, together with the type of immobilization method and the support will affect the performance during the enzymatic synthesis. Enzymes’ immobilization involves interdisciplinary knowledge including enzymology, nanotechnology, molecular dynamics, cellular physiology and process design. The increasing availability of facilities has opened a variety of possibilities to define strategies to optimize the activity and re-usability of β-galactosidases and fructosyltransferases, but there is still great place for innovative developments.Centro de Investigación y Desarrollo en Criotecnología de Alimento

Reaction control and protein engineering of bacillus lehensis G1 maltogenic amylase for higher malto-oligosaccharide synthesis

A multi-functional maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 exhibited remarkable hydrolysis and transglycosylation activity to produce malto-oligosaccharides of various lengths. MAG1 demonstrated hydrolysis activity over wide range of substrates. Kinetic analysis revealed that the enzyme hydrolyzed small substrate more efficiently than the larger substrate. This was shown by lower Michaelis constant (Km) value and higher turnover number (kcat) and second order rate constant (kcat/Km) values for β-cyclodextrin compared to that of soluble starch. Malto-oligosaccharide synthesis by transglycosylation activity of MAG1 faces problem of product re-hydrolyzation due to the hydrolysis activity of the enzyme. An equilibrium-control reaction approach has been successfully employed to improve malto-oligosaccharides production by decreasing hydrolysis activity. A yield of 38% transglycosylation products was obtained with the presence of malto-oligosaccharides longer than maltoheptaose. The addition of organic solvents demonstrated an increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15. The transglycosylation activity of MAG1 was also successfully enhanced by using structure-guided protein engineering approach. A molecular modeling and substrate docking was performed to study the structure-function relationship for rational design. A unique subsite structure which has not been reported in other maltogenic amylases was revealed and the information was used to design mutants that have active sites with reduced steric interference and higher hydrophobicity properties to increase the transglycosylation activity. Mutations decreased the hydrolysis activity of the enzyme and caused various modulations in its transglycosylation property. W359F, Y377F and M375I mutations caused reductions in steric interference and alteration of subsite occupation. In addition, the mutations increased internal flexibility to accommodate longer donor/acceptor molecule for transglycosylation, resulted in increased transglycosylation to hydrolysis ratio of up to 4.0-fold. The increase of the active site hydrophobicity from W359F and M375I mutations reduced concentration of maltotriose used as donor/acceptor for transglycosylation to 100 mM and 50 mM, respectively compared to 200 mM of the wild-type. The improvement of the transglycosylation to hydrolysis ratio by 4.3-fold was also demonstrated by both mutants. Interestingly, reductions of both steric interference and hydrolysis by Y377F and W359F mutations caused a synergistic effect to produce malto-oligosaccharides with higher degree of polymerization than the wild-type. These findings showed that the transglycosylation activity of MAG1 was successfully improved by controlling water activity and modification of the active site structure. The high transglycosylation activity of MAG1 and mutants offers a great advantage for synthesizing malto-oligosaccharides and rare carbohydrates