scholarly journals Stable and functionally diverse versatile peroxidases by computational design directly from sequence

2021 ◽  
Author(s):  
Shiran Barber-Zucker ◽  
Vladimir Mindel ◽  
Eva Garcia-Ruiz ◽  
Jonathan Jacob Weinstein ◽  
Miguel Alcalde ◽  
...  
Keyword(s):  
Structure Prediction ◽  
White Rot Fungi ◽  
Computational Design ◽  
Optimization Methods ◽  
Functional Expression ◽  
White Rot ◽  
Wild Type ◽  
Natural Enzyme ◽  
Design Calculations ◽  
Enzyme Optimization

White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use, but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel, but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wild type enzymes are functionally expressed in yeast whereas their wild type parents are not. Three of these designs exhibit substantial and useful diversity in reactivity profile and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families.

scholarly journals Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System

2020 ◽  
Vol 21 (2) ◽  
pp. 416
Author(s):  
Angel De La Cruz Pech-Canul ◽  
Javier Carrillo-Campos ◽  
María de Lourdes Ballinas-Casarrubias ◽  
Rosa Lidia Solis-Oviedo ◽  
Selena Karina Hernández-Rascón ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Protein Purification ◽  
Phanerochaete Chrysosporium ◽  
Manganese Peroxidase ◽  
Expression System ◽  
White Rot Fungi ◽  
Functional Expression ◽  
White Rot ◽  
E Coli ◽  
One Step

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

Evaluation of two wild types of Pleurotus ostreatus (MCC07 and MCC20) isolated from nature for their ability to decolorize Benazol Black ZN textile dye in comparison to some commercial types of white rot fungi: Pleurotus ostreatus, Pleurotus djamor, and Pleurotus citrinopileatus

2008 ◽  
Vol 54 (5) ◽  
pp. 366-370 ◽  
Author(s):  
Erbil Kalmış ◽  
Nuri Azbar ◽  
Fatih Kalyoncu
Keyword(s):  
Pleurotus Ostreatus ◽  
White Rot Fungi ◽  
White Rot ◽  
Textile Dye ◽  
Wild Type ◽  
Pleurotus Djamor ◽  
Decolorization Efficiency ◽  
Pleurotus Citrinopileatus

Biological decolorization of Benazol Black ZN, a reactive azo-type textile dyestuff, was comparatively studied using 3 different commercial-type white rot fungi strains ( Pleurotus ostreatus , Pleurotus cornucopiae var. citrinopileatus, Pleurotus djamor , and 2 wild types of P. ostreatus (MCC07 and MCC20) isolated from the nature. The initial dye concentrations in the medium were 500 and 1000 mg·L–1. All the organisms studied decolorized Benazol Black ZN to varying degrees. At low dye concentration, both commercial and wild type of P. ostreatus resulted in the best decolorization, conversely, wild-type P. ostreatus (MCC07) was found to be much more robust against increasing dye concentration and provided the best decolorization efficiency at high dye concentration.

OPTIMIZATION OF BIOMASS AND ENDO-B-1,4-GLUCANASE BY WHITE ROT FUNGI NATIVE FROM ARGENTINA

2017 ◽  
Vol 16 (11) ◽  
pp. 2581-2588
Author(s):  
Ernesto M. Giorgio ◽  
Maria I. Fonseca ◽  
Andrea L. Morales ◽  
Pedro D. Zapata ◽  
Laura L. Villalba
Keyword(s):  
White Rot Fungi ◽  
White Rot

The Onset of Lignin-Modifying Enzymes, Decrease of AOX and Color Removal by White-Rot Fungi Grown on Bleach Plant Effluents

1991 ◽  
Vol 24 (3-4) ◽  
pp. 189-198 ◽  
Author(s):  
V. P. Lankinen ◽  
M. M. Inkeröinen ◽  
J. Pellinen ◽  
A. I. Hatakka
Keyword(s):  
Lignin Peroxidase ◽  
Active Form ◽  
White Rot Fungi ◽  
Pulp Mill ◽  
Color Removal ◽  
Effluent Treatment ◽  
White Rot ◽  
White Rot Fungus ◽  
Lignin Peroxidases ◽  
Pulp Mill Effluents

Decrease of adsorbable organic chlorine (AOX) is becoming the most important criterion for the efficiency of pulp mill effluent treatment in the 1990s. Two methods, designated MYCOR and MYCOPOR which utilize the white-rot fungus Phanerochaete chrysosporium have earlier been developed for the color removal of pulp mill effluents, but the processes have also a capacity to decrease the amount of chlorinated organic compounds. Lignin peroxidases (ligninases) produced by P. chrvsosporium may dechlorinate chlorinated phenols. In this work possibilities to use selected white-rot fungi in the treatment of E1-stage bleach plant effluent were studied. Phlebia radiata. Phanerochaete chrvsosporium and Merulius (Phlebia) tremellosus were compared in shake flasks for their ability to produce laccase, lignin peroxidase(s) and manganese-dependent peroxidase(s) and to remove color from a medium containing effluent. Softwood bleaching effluents were treated by carrier-immobilized P. radiata in 2 1 bioreactors and a 10 1 BiostatR -fermentor. Dechlorination was followed using Cl ion and AOX determinations. All fungi removed the color of the effluent. In P. radiata cultivations AOX decrease was ca. 4 mg l−1 in one day. Apparent lignin peroxidase activities as determined by veratryl alcohol oxidation method were negligible or zero in a medium with AOX content of ca. 60 mg l−1, prepared using about 20 % (v/v) of softwood effluent. However, the purification of extracellular enzymes implied that large amounts of lignin peroxidases were present in the medium and, after the purification, in active form. Enzyme proteins were separated using anion exchange chromatography, and they were further characterized by electrophoresis (SDS-PAGE) to reveal the kind of enzymes that were present during AOX decrease and color removal. The most characteristic lignin peroxidase isoenzymes in effluent media were LiP2 and LiP3.

Revalorizing Lignocellulose for the Production of Natural Pharmaceuticals and Other High Value Bioproducts

2019 ◽  
Vol 26 (14) ◽  
pp. 2475-2484 ◽  
Author(s):  
Congqiang Zhang ◽  
Heng-Phon Too
Keyword(s):  
Clostridium Thermocellum ◽  
White Rot Fungi ◽  
Brown Rot ◽  
Cost Effective ◽  
White Rot ◽  
Chemical Pretreatment ◽  
Significant Challenge ◽  
Drug Molecules ◽  
Natural Medicine ◽  
Renewable Natural Resource

Lignocellulose is the most abundant renewable natural resource on earth and has been successfully used for the production of biofuels. A significant challenge is to develop cost-effective, environmentally friendly and efficient processes for the conversion of lignocellulose materials into suitable substrates for biotransformation. A number of approaches have been explored to convert lignocellulose into sugars, e.g. combining chemical pretreatment and enzymatic hydrolysis. In nature, there are organisms that can transform the complex lignocellulose efficiently, such as wood-degrading fungi (brown rot and white rot fungi), bacteria (e.g. Clostridium thermocellum), arthropods (e.g. termite) and certain animals (e.g. ruminant). Here, we highlight recent case studies of the natural degraders and the mechanisms involved, providing new utilities in biotechnology. The sugars produced from such biotransformations can be used in metabolic engineering and synthetic biology for the complete biosynthesis of natural medicine. The unique opportunities in using lignocellulose directly to produce natural drug molecules with either using mushroom and/or ‘industrial workhorse’ organisms (Escherichia coli and Saccharomyces cerevisiae) will be discussed.

scholarly journals Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

1989 ◽  
Vol 9 (4) ◽  
pp. 1507-1512 ◽  
Author(s):  
H Zhu ◽  
H Conrad-Webb ◽  
X S Liao ◽  
P S Perlman ◽  
R A Butow
Keyword(s):  
Genetic Analysis ◽  
Rna Processing ◽  
Fold Increase ◽  
Functional Expression ◽  
Rrna Gene ◽  
Yeast Mitochondria ◽  
Wild Type ◽  
Site Specific ◽  
Var1 Gene ◽  
A Site

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

Bacterial Community Coexisting with White-Rot Fungi in Decayed Wood in Nature

2021 ◽  
Author(s):  
Yosuke Iimura ◽  
Hisashi Abe ◽  
Yuichiro Otsuka ◽  
Yuya Sato ◽  
Hiroshi Habe
Keyword(s):  
Bacterial Community ◽  
White Rot Fungi ◽  
White Rot ◽  
Decayed Wood

scholarly journals Evaluation of Basidiomycetes Wild Strains Grown in Agro-Industrial Residues for Their Anti-Tyrosinase and Antioxidant Potential and for the Production of Biocatalysts

Fermentation ◽  
2021 ◽  
Vol 7 (1) ◽  
pp. 19
Author(s):  
Anastasia Zerva ◽  
Nikolaos Tsafantakis ◽  
Evangelos Topakas
Keyword(s):  
Biomass Conversion ◽  
White Rot Fungi ◽  
Ligninolytic Enzymes ◽  
Inhibitory Effect ◽  
Biologically Active ◽  
White Rot ◽  
Oxidative Enzymes ◽  
Industrial Residues ◽  
Pure Compounds ◽  
Wild Strains

White-rot basidiomycetes are the only microorganisms with the ability to produce both hydrolytic (cellulases and hemicellulases) and oxidative (ligninolytic) enzymes for degrading cellulose/hemicellulose and lignin. In addition, they produce biologically active natural products with important application in cosmetic formulations, either as pure compounds or as standardized extracts. In the present work, three wild strains of Basidiomycetes fungi (Pleurotus citrinopileatus, Abortiporus biennis and Ganoderma resinaceum) from Greek habitats were grown in agro-industrial residues (oil mill wastewater, and corn cob) and evaluated for their anti-tyrosinase and antioxidant activity and for the production of biotechnologically relevant enzymes. P. citrinopileatus showed the most interesting tyrosinase inhibitory activity, while A. biennis showed the highest DPPH(2,2-diphenyl-1-picryl-hydrazyl) scavenging potential. Corn cobs were the most appropriate carbon source for maximizing the inhibitory effect of fungal biomasses on both activities, while the use of oil mill wastewater selectively increased the anti-tyrosinase potential of P. citrinopileatus culture filtrate. All strains were found to be preferential lignin degraders, similarly to most white-rot fungi. Bioinformatic analyses were performed on the proteome of the strains P. citrinopileatus and A. biennis, focusing on CAZymes with biotechnological relevance, and the results were compared with the enzyme activities of culture supernatants. Overall, all three strains showed strong production of oxidative enzymes for biomass conversion applications.

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