Temperature affects the production, activity and stability of ligninolytic enzymes inPleurotus ostreatus andTrametes versicolor (original) (raw)
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Saudi Journal of Biological Sciences, 2021
In recent years, many research on the quantity of lignocellulosic waste have been developed. The production, partial purification, and characterisation of ligninolytic enzymes from various fungi are described in this work. On the 21st day of incubation in Potato Dextrose (PD) broth, Hypsizygus ulmarius developed the most laccase (14.83 Â 10 À6 IU/ml) and manganese peroxidase (24.11 Â 10 À6 IU/ml), while Pleurotus florida produced the most lignin peroxidase (19.56 Â À6 IU/ml). Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), all generated by selected basidiomycetes mushroom fungi, were largely isolated using ammonium sulphate precipitation followed by dialysis. Laccase, lignin peroxidase, and manganese peroxidase purification findings indicated 1.83, 2.13, and 1.77 fold purity enhancements, respectively. Specific activity of purified laccase enzyme preparations ranged from 305.80 to 376.85 IU/mg, purified lignin peroxidase from 258.51 to 336.95 IU/mg, and purified manganese peroxidase from 253.45 to 529.34 IU/mg. H. ulmarius laccase (376.85 IU/mg) with 1.83 fold purification had the highest specific activity of all the ligninolytic enzymes studied, followed by 2.13 fold purification in lignin peroxidase (350.57 IU/mg) and manganese peroxidase (529.34 IU/mg) with 1.77-fold purification. Three notable bands with molecular weights ranging from 43 to 68 kDa and a single prominent band with a molecular weight of 97.4 kDa were identified on a Native PAGE gel from mycelial proteins of selected mushroom fungus. The SDS PAGE profiles of the mycelial proteins from the selected mushroom fungus were similar to the native PAGE. All three partially purified ligninolytic isozymes display three bands in native gel electrophoresis, with only one prominent band in enzyme activity staining. The 43 kDa, 55 kDa, and 68 kDa protein bands correspond to laccase, lignin peroxidase, and manganese peroxidase, respectively.
Influence of the cultivation conditions on ligninolytic enzyme production in Pleurotus pulmonarius
Zbornik Matice Srpske Za Prirodne Nauke, 2007
The highest level of laccase activity (391 Ul -1 ), as well as significant Mn-oxidizing peroxidases production, were found in solid-state culture with grapevine sawdust as the carbon source. After purification of extracellular crude enzyme mixture of Pleurotus pulmonarius, grown in the medium with the best carbon source (grapevine sawdust), three peaks of laccase activity were noted. The results obtained by purification also showed that the levels of phenol red oxidation, in absence of external Mn 2+ , were higher than phenol red oxidation levels in presence of external Mn 2+ . The highest laccase activity was in the medium with grapevine sawdust, as carbon source, and NH 4 Cl at a nitrogen concentration of 30 mM (441 Ul -1 ). The best nitrogen source for Mn-oxidizing peroxidase production was NH 4 NO 3 at nitrogen concentration of 30 mM. The highest laccase activity was found in the presence of 5 mM Cu 2+ , and 5 mM Mn 2+ , respectively. The absence of Cu 2+ and Mn 2+ , as well as their presence at the concentration of 1 mM, led to the peaks of Mn-oxidizing peroxidases activities. Zn 2+ and Fe 2+ caused a decrease, and Se, in all investigated forms, an increase of laccase and peroxidases activities.
Fungal Genetics and Biology, 2014
Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25°C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25°C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn 2+ (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3 > vp2/vp3/ mnp1/mnp2/mnp6 > mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10°C or 37°C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10°C treatment, while many of them were alternatively upregulated (often 6 h after the thermal shock) and downregulated (12 h) at 37°C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion.
Production of ligninolytic enzymes by cultures of white rot fungi
Polish journal of microbiology / Polskie Towarzystwo Mikrobiologów = The Polish Society of Microbiologists, 2014
Some Basidiomycota were chosen for studies of key ligninases synthesis (25°C, 30 days) in modified medium (shaken or not cultures) with added wheat straw. Liquid Czapek medium with straw yielded a higher amount of laccase than peroxidase, ground straw induced enzyme worse than chopped straw. With peroxidase the reverse dependencies were observed. Laccase of Lentinus edodes synthesized two enzyme isoforms (ca 30 and 16 kDa). In T. versicolor culture active laccase protein with highest molecular mass ca 65 kDa was found. P. sajor-caju yielded three different peroxidase isoforms. Ligninase biosynthesis depended on strain, straw fragmentation extent, culture method and growth medium.
Applied and environmental microbiology, 1993
The ligninolytic enzymes synthesized by Phanerochaete chrysosporium BKM-F-1767 immobilized on polyurethane foam were characterized under limiting, sufficient, and excess nutrient conditions. The fungus was grown in a nonimmersed liquid culture system under conditions close to those occurring in nature, with nitrogen concentrations ranging from 2.4 to 60 mM. This nonimmersed liquid culture system consisted of fungal mycelium immobilized on porous pieces of polyurethane foam saturated with liquid medium and highly exposed to gaseous oxygen. Lignin peroxidase (LIP) activity decreased to almost undetectable levels as the initial NH4+ levels were increased over the range from 2.4 to 14 mM and then increased with additional increases in initial NH4+ concentration. At 45 mM NH4+, LIP was overproduced, reaching levels of 800 U/liter. In addition, almost simultaneous secretion of LIP and secretion of manganese-dependent lignin peroxidase were observed on the third day of incubation. Manganes...
Ligninolytic Enzymes of the White Rot Basidiomycete Trametes trogii
Acta Biotechnologica, 2001
The white rot fungus Trametes trogii strain BAFC 463 produced laccase, manganese peroxidase, lignin peroxidase and cellobiose dehydrogenase, as well as two hydrogen peroxide-producing activities: glucose oxidizing activity and glyoxal oxidase. In high-N (40 mM N) cultures, the titres of laccase, MnP and GLOX were 27 (6.55 U/ml), 45 (403.00 mU/ml) and 8 (32,14 mU/ml) fold higher, respectively, than those measured in an N-limited medium. This is consistent with the fact that the ligninolytic system of T. trogii is expressed constitutively. Lower activities of all the enzymes tested were recorded upon decreasing the initial pH of the medium from 6.5 to 4.5. Adding veratryl alcohol improved GLOX production, while laccase activity was stimulated by tryptophan. Supplying Tween 80 strongly reduced the activity of both MnP and GLOX, but increased laccase production. The titre of MnP was affected by the concentration of Mn in the culture medium, the highest levels were obtained with 90 µM Mn (II). LiP activity, as CDH activity, were detected only in the medium supplemented with sawdust. In this medium, laccase production reached a maximum of 4.75 U/ml, MnP 747.60 mU/ml and GLOX 117.11 mU/ml. LiP, MnP and GLOX activities were co-induced, attaining their highest levels at the beginning of secondary metabolism, but while MnP, laccase, GLOX and CDH activities were also present in the primary growth phase, LiP activity appears to be idiophasic. The simultaneous presence of high ligninolytic and hydrogen peroxide producing activities in this fungus makes it an attractive microorganism for future biotechnological applications.
Enzyme and Microbial Technology, 2004
In this study, we show that the wild-type strain Phanerochaete chrysosporium ME-446 is able to effectively produce manganese peroxidase (MnP) and lignin peroxidase (LiP) in submerged cultures under nitrogen and carbon non-limiting conditions, if the lignocellulosic substrate is added to the medium. No activity of LiP or MnP could be detected when the fungus was grown in the basal glucose-peptone-corn steep liquor medium. Only the addition of the lignocellulose-containing substrates, wheat straw or hemp woody core, to the medium led to production of the enzymes. This result strongly indicates that some compounds derived from the lignocellulosic substrates may function as inducers for production of the ligninolytic peroxidases. In the presence of the lignocellulosic substrates high amounts of organic nitrogen source (peptone; 3-4 g l −1 ) did not repress production of the ligninolytic peroxidases, as it commonly occurs with the wild-type strains of P. chrysosporium, but instead stimulated it. A simple medium, which does not require addition of Tween 80 or oxygen purging, was developed for effective production of ligninolytic peroxidases by P. chrysosporium ME-446 in submerged culture. The medium allows simultaneous production of high activities of both MnP and LiP.
Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity
World Journal of Microbiology and Biotechnology, 2006
The effect of various carbon and nitrogen sources on laccase, manganese-dependent peroxidase (MnP), and peroxidase production by two strains of Pleurotus ostreatus was investigated. The maximal laccase yield of P. ostreatus 98 and P. ostreatus 108 varied depending upon the carbon source from 5 to 62 U l )1 and from 55 to 390 U l )1 , respectively. The highest MnP and peroxidase activities were revealed in medium supplemented by xylan. Laccase, MnP, and peroxidase activities of mushrooms decreased with supplementation of defined medium by inorganic nitrogen sources. Peptone followed by casein hydrolysate appeared to be the best nitrogen sources for laccase accumulation by both fungi. However, their positive effects on enzyme accumulation were due to a higher biomass production. The secretion of MnP and peroxidase by P. ostreatus 108 was stimulated with supplementation of casein hydrolysate to the control medium since the specific MnP and peroxidase activities increased 15-fold and 3.5fold, respectively.
Journal of Basic Microbiology, 2001
Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in selected subtropical white rot fungal species from Zimbabwe were determined. The enzyme activities were assayed at varying concentrations of C, N and Mn 2+. Manganese peroxidase and laccase activities were the only expressed activities in the fungi under the culture conditions tested. Trametes species, T. cingulata, T. elegans and T. pocas produced the highest manganese peroxidase activities in a medium containing high carbon and low nitrogen conditions. High nitrogen conditions favoured high manganese peroxidase activity in DSPM95, L. velutinus and Irpex spp. High manganese peroxidase activity was notable for T. versicolor when both carbon and nitrogen in the medium were present at high levels. Laccase production by the isolates was highest under conditions of high nitrogen and those conditions with both nitrogen and carbon at high concentration. Mn 2+ concentrations between 11-25 ppm gave the highest manganese peroxidase activity compared to a concentration of 40 ppm or when there was no Mn 2+ added. Laccase activity was less influenced by Mn 2+ levels. While some laccase activity was produced in the absence of Mn 2+ , the enzyme levels were higher when Mn 2+ was added to the culture medium.
Journal of Biotechnology, 1990
The extracellular enzymes synthesized by Phlebia radiata Fr. 79 (ATCC 64658) under various cultivation conditions were studied in order to find out suitable enzyme combinations for the modification of lignin. The fungus produced lignin peroxidase (LIP, ligninase), manganese-dependent peroxidase (MnP) and laccase (benzenediol : oxygen oxidoreductase, EC 1.10.3.2) in various proportions depending on the cultivation conditions. Addition of veratryl alcohol increased lignin peroxidase and manganese-dependent peroxidase activities both in agitated and nonagitated flask cultures. Laccase production was more enhanced by the addition of benzyl alcohol and veratric acid. However, the highest lignin peroxidase activities were obtained using a non-phenolic dimeric /3-0-4 model compound. Pressure ground wood (PGW), chemithermomechanical pulp (CTMP) or spruce shavings in the bioreactors decreased the amount of lignin peroxidase, whereas laccase and manganese-dependent peroxidase activities increased. Lignin peroxidase and partly manganese-dependent peroxidase was adsorbed on the lignocellulose substrate, but Correspondence to: M.-L. Niku-Paavola, VTT, Biotechnical Laboratory, Tietotie 2, SF-02150 Espoo, Finland. Abbreoiations: LIP1,2,3 = lignin peroxidase isozymes of Phlebia radiata; MnP = manganese-dependent peroxidase of Phlebia radiata; PGW = pressure ground wood; CTMP--chemitliermomechanical pulp; MWL = milled wood lignin; VALC = veratryl alcohol (3,4-dimethoxybenzyl alcohol); VACID = veratric acid (3,4-dimethoxybenzoic acid); BALC == benzyl alcohol; fl-O-4 = non-phenolic fl-O-4 dimer [1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-l,3-diol]; IEF = isoelectric focusing; SDS = sodium dodecyl sulphate; PAGE = polyacrylarnide gel electrophoresis; ABTS = 2,2'-azinodi-3-ethylbenzothiazoline-6-sulphuric acid; LN-ADMS = low-nitrogen asparagine dimethylsuccinate medium. 0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division) 212