Advancement and comparative profiles in the production technologies using solid-state and submerged fermentation for microbial cellulases

Laccases are blue multicopper oxidases, which catalyze the monoelectronic oxidation of a broad spectrum of substrates, for example, ortho- and para-diphenols, polyphenols, aminophenols, and aromatic or aliphatic amines, coupled with a full, four-electron reduction of O2 to H2O. Hence, they are capable of degrading lignin and are present abundantly in many white-rot fungi. Laccases decolorize and detoxify the industrial effluents and help in wastewater treatment. They act on both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and they can be effectively used in paper and pulp industries, textile industries, xenobiotic degradation, and bioremediation and act as biosensors. Recently, laccase has been applied to nanobiotechnology, which is an increasing research field, and catalyzes electron transfer reactions without additional cofactors. Several techniques have been developed for the immobilization of biomolecule such as micropatterning, self-assembled monolayer, and layer-by-layer techniques, which immobilize laccase and preserve their enzymatic activity. In this review, we describe the fungal source of laccases and their application in environment protection.

https://downloads.hindawi.com/archive/2014/163242.pdf

Fungal Laccases and Their Applications in Bioremediation

Lentinus edodes and Pleurotus species lignocellulolytic enzymes activity in submerged and solid-state fermentation of lignocellulosic wastes of different composition.

Fungal laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet.

Induction and transcriptional regulation of laccases in fungi.

Cellulosic biomass includes agricultural (e.g., corn stover and sugarcane bagasse) and forestry (e.g., sawdust, thinnings, andmill wastes) residues, portions ofmunicipal solid waste (e.g., waste paper), and herbaceous (e.g., switchgrass) and woody (e.g., poplar trees) crops. Such materials are abundant and competitive in price with petroleum, and cellulosic biomass can provide a sustainable resource that is truly unique for making organic products [1]. Furthermore, cellulosic biomass can be produced in many regions of the world that do not have much petroleum, opening up a new route to manufacturing organic fuels and chemicals. The structural portion of cellulosic biomass is a composite of cellulose chains joined together by hydrogen bonding. These long cellulose fibers are in turn held together with hemicellulose and lignin, allowing growth to large aerial plants that canwithstand weather and attack by microorganisms and insects. Traditionally, humans have employed cellulosic biomass for such uses as construction, soil stabilization, animal feed, and paper manufacture. However, the cellulose and hemicellulose portions of biomass, representing about 40–50% and 20–30% by dry weight of plants, respectively, are polysaccharides that can be broken down into sugars and fermented or chemically altered to valuable fuels and chemicals. Sugars from such sources have been used to make ethanol and other products during periods of war by approaches such as the Scholler process during the 1940s and in controlled economies such as the former Soviet Union. Research over the last two decades has advanced biological conversion of cellulosic biomass to the point of becoming economically competitive for production of fuels and chemicals that offer important strategic, environmental, and economic advantages [2]. The challenge is to overcome the risk of commercializing first-of-a-kind technology and to continue to advance hydrolysis processes so that fuels and chemicals from cellulosic resources are competitive without subsidies. This chapter presents a comprehensive overview of the technology and economic status for cellulose and hemicellulose hydrolysis, including a description of important structural features of cellulosic materials, applications, process steps, and stoichiometry for hydrolysis reactions. Included is an overview of the basic chemistry and processes followed by consideration of both chemical and enzymatic approaches to these reactions. The structure and functions of enzymes are described along with pretreatment of biomass to achieve high yields for these systems. The chapter continues with an overview of acid

Hydrolysis of Cellulose and Hemicellulose

The production of lignocellulolytic enzymes by eleven basidiomycetes species isolated from two ecosystems of Georgia was investigated for the first time under submerged (SF) and solid-state fermentation (SSF) of lignocellulosic by-products. Notable intergeneric and intrageneric differences were revealed with regard to the extent of hydrolase and oxidase activity. Several fungi produced laccase along with hydrolases in parallel with growth during the trophophase, showing that the synthesis of this enzyme is not connected with secondary metabolism. The lignocellulosic substrate type had the greatest impact on enzyme secretion. Some of the substrates significantly stimulated lignocellulolytic enzyme synthesis without supplementation of the culture medium with specific inducers. Exceptionally high carboxymethyl cellulase (CMCase, 122 U ml(-1)) and xylanase (195 U ml(-1)) activities were revealed in SF of mandarin peelings by Pseudotremella gibbosa IBB 22 and of residue after ethanol production (REP) by Fomes fomentarius IBB 38, respectively. The SSF of REP by T. pubescens IBB 11 ensured the highest level of laccase activity (24,690 U l(-1)), whereas the SSF of wheat bran and SF of mandarin peels provided the highest manganese peroxidase activity (570-620 U l(-1)) of Trichaptum biforme IBB 117. Moreover, the variation of lignocellulosic growth substrate provides an opportunity to obtain enzyme preparations containing different ratios of individual enzymes.

Lignocellulose-degrading enzyme production by white-rot Basidiomycetes isolated from the forests of Georgia

Three white-rot fungi displayed a wide diversity in their response to supplemented aromatic compounds. Pyrogallol stimulated Cerrena unicolor laccase and manganese peroxidase (MnP) synthesis in synthetic medium 2.5- and 2-fold, respectively, whereas 2,4,6-trinitrotoluene (TNT) brought about a 2.8-fold increase in laccase yield by Trametes versicolor in submerged fermentation of ethanol production residue. No effect of the tested aromatic compounds on enzyme secretion by Ganoderma lucidum in mannitol-containing medium was detected. Nevertheless, G. lucidum is a potent producer of laccase in submerged fermentation of wheat bran and enzyme synthesis can be further increased by supplementation of medium with an appropriate inducer. The structure and the concentration of aromatic compounds play an important role in the regulation of enzyme synthesis. The supplementation of synthetic medium with 0.03-0.3 mM TNT or hydroquinone increased the differential rate of laccase synthesis by C. unicolor from 1,267 to 3,125-8,630 U mg biomass(-1) day(-1). Moreover, the same aromatic compound may function as either an inducer or a repressor, depending on the fungus and enzyme studied. Thus, hydroquinone increased 3-fold T. versicolor laccase activity decreasing 2- and 8-fold the yields of MnP and endoglucanase, respectively.

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Effect of aromatic compounds on the production of laccase and manganese peroxidase by white-rot basidiomycetes

Physiological Regulation of Edible and Medicinal Higher Basidiomycetes Lignocellulolytic Enzyme Activity

Cotton wastes can be converted without substrate pasteurization into value-added products, such as gourmet mushrooms, with biological efficacy up to 90% and lignocellulolytic enzymes. Edible and medicinal mushroom Pleurotus ostreatus carboxymethyl cellulase and laccase activities, extracted from both the surface and inside layers of blocks, increased during primordia and fruiting bodies development and declined rapidly after the harvest. In a study of xylanase production, it has been observed that the activity of the enzyme in inside layers of the substrate gradually increased during the 49 days (second fruiting stage) after inoculation and then gradually declined towards the end of mushroom cultivation. Fluctuations in xylanase activity in the surface layer were observed, so that maximal enzyme activities coincided with the fruiting stage of P. ostreatus development. In contrast to laccase, manganese peroxidase (MnP) activity was high during the colonization stage and declined during the first primordia and fruiting body formation stages. After this, while laccase activity decreased, MnP activity peaked at the mycelia stage in oyster mushroom development. Data on the levels of cellulase, xylanase, laccase, and MnP activity in spent mushroom substrate demonstrate that it can be used as an excellent source of these enzymes.

Lignocellulolytic Enzyme Activity During Growth and Fruiting of the Edible and Medicinal Mushroom Pleurotus ostreatus (Jacq.:Fr.) Kumm. (Agaricomycetideae)

In this study, the effects of several key factors to increase spore production by Bacillus subtilis subsp. KATMIRA 1933 were evaluated in shake flask experiments. In a synthetic medium, glucose concentration played a crucial role in the expression of bacilli sporulation capacity. In particular, maximum spore yield (2.3 × 109 spores/mL) was achieved at low glucose concentration (2 g/L), and further gradual increase of the carbon source content in the medium caused a decrease in sporulation capacity. Substitution of glucose with several inexpensive lignocellulosic materials was found to be a reasonable way to achieve high cell density and sporulation. Of the materials tested, milled mandarin peels at a concentration of 40 g/L served as the best growth substrate. In these conditions, bacilli secreted sufficient levels of glycosyl hydrolases, providing slow hydrolysis of the mandarin peel’s polysaccharides to metabolizable sugars, providing the bacterial culture with an adequate carbon and energy source. Among nitrogen sources tested, peptone was found to favor spore production. Moreover, it was shown that cheese and cottage cheese whey usage, instead of distilled water, significantly increases spore formation. After optimization of the nutrient medium in the shake flask experiments, the technical feasibility of large-scale spore production by B. subtilis KATMIRA 1933 was confirmed in a laboratory fermenter. The spore yield (7 × 1010 spores/mL) obtained using a bioreactor was higher than those previously reported.

Tamar Khardziani

Papers

Lignocellulose-degrading enzyme production by white-rot Basidiomycetes isolated from the forests of Georgia

Effect of aromatic compounds on the production of laccase and manganese peroxidase by white-rot basidiomycetes

Physiological Regulation of Edible and Medicinal Higher Basidiomycetes Lignocellulolytic Enzyme Activity

Lignocellulolytic Enzyme Activity During Growth and Fruiting of the Edible and Medicinal Mushroom Pleurotus ostreatus (Jacq.:Fr.) Kumm. (Agaricomycetideae)

Elucidation of Bacillus subtilis KATMIRA 1933 Potential for Spore Production in Submerged Fermentation of Plant Raw Materials.