RESUMEN
Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.
Asunto(s)
Biomasa , Proteínas Fúngicas , Genoma Fúngico , Lignina , Saccharomycetales , Sordariales , Lignina/metabolismo , Sordariales/genética , Sordariales/enzimología , Sordariales/metabolismo , Hidrólisis , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deshidrogenasas de Carbohidratos/metabolismo , Deshidrogenasas de Carbohidratos/genética , Celulosa/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Celulasa/metabolismo , Celulasa/genéticaRESUMEN
The current study is the first to describe the temporal and differential transcriptional expression of two lytic polysaccharide monooxygenase (LPMO) genes of Rasamsonia emersonii in response to various carbon sources. The mass spectrometry based secretome analysis of carbohydrate active enzymes (CAZymes) expression in response to different carbon sources showed varying levels of LPMOs (AA9), AA3, AA7, catalase, and superoxide dismutase enzymes pointing toward the redox-interplay between the LPMOs and auxiliary enzymes. Moreover, it was observed that cello-oligosaccharides have a negative impact on the expression of LPMOs, which has not been highlighted in previous reports. The LPMO1 (30 kDa) and LPMO2 (47 kDa), cloned and expressed in Pichia pastoris, were catalytically active with (kcat/Km) of 6.6×10-2 mg-1 ml min-1 and 1.8×10-2 mg-1 ml min-1 against Avicel, respectively. The mass spectrometry of hydrolysis products of Avicel/carboxy methyl cellulose (CMC) showed presence of C1/C4 oxidized oligosaccharides indicating them to be Type 3 LPMOs. The 3D structural analysis of LPMO1 and LPMO2 revealed distinct arrangements of conserved catalytic residues at their active site. The developed enzyme cocktails consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant LPMO1/LPMO2 resulted in significantly enhanced saccharification of steam/acid pretreated unwashed rice straw slurry from PRAJ industries (Pune, India). The current work indicates that LPMO1 and LPMO2 are catalytically efficient and have a high degree of thermostability, emphasizing their usefulness in improving benchmark enzyme cocktail performance. KEY POINTS: ⢠Mass spectrometry depicts subtle interactions between LPMOs and auxiliary enzymes. ⢠Cello-oligosaccharides strongly downregulated the LPMO1 expression. ⢠Developed LPMO cocktails showed superior hydrolysis in comparison to CellicCTec3.
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Oxigenasas de Función Mixta , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/química , Polisacáridos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Hidrólisis , Celulosa/metabolismo , Regulación Fúngica de la Expresión Génica , Oligosacáridos/metabolismo , Clonación MolecularRESUMEN
The present study reports a highly thermostable ß-glucosidase (GH3) from Rasamsonia emersonii that was heterologously expressed in Pichia pastoris. Extracellular ß-glucosidase was purified to homogeneity using single step affinity chromatography with molecular weight of ~ 110 kDa. Intriguingly, the purified enzyme displayed high tolerance to inhibitors mainly acetic acid, formic acid, ferulic acid, vanillin and 5-hydroxymethyl furfural at concentrations exceeding those present in acid steam pretreated rice straw slurry used for hydrolysis and subsequent fermentation in 2G ethanol plants. Characteristics of purified ß-glucosidase revealed the optimal activity at 80 °C, pH 5.0 and displayed high thermostability over broad range of temperature 50-70 °C with maximum half-life of ~ 60 h at 50 °C, pH 5.0. The putative transglycosylation activity of ß-glucosidase was appreciably enhanced in the presence of methanol as an acceptor. Using the transglycosylation ability of ß-glucosidase, the generated low cost mixed glucose disaccharides resulted in the increased induction of R. emersonii cellulase under submerged fermentation. Scaling up the recombinant protein production at fermenter level using temporal feeding approach resulted in maximal ß-glucosidase titres of 134,660 units/L. Furthermore, a developed custom made enzyme cocktail consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant ß-glucosidase resulted in significantly enhanced hydrolysis of pretreated rice straw slurry from IOCL industries (India). Our results suggest multi-faceted ß-glucosidase from R. emersonii can overcome obstacles mainly high cost associated enzyme production, inhibitors that impair the sugar yields and thermal inactivation of enzyme.
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Eurotiales , beta-Glucosidasa , Hidrólisis , beta-Glucosidasa/química , BiomasaRESUMEN
BACKGROUND: Increase in the number of infections caused by Gram-negative bacteria in neutropenic cancer patients has prompted the search for novel therapeutic agents having dual anticancer and antimicrobial properties. Bacteriocins are cationic proteins of prokaryotic origin that have emerged as one of the most promising alternative antimicrobial agents with applications as food preservatives and therapeutic agents. Apart from their antimicrobial activities, bacteriocins are also being explored for their anticancer potential. RESULTS: In this study, a broad-spectrum, cell membrane-permeabilizing enterocin with a molecular weight of 65 kDa was purified and characterized from the culture supernatant of vaginal Enterococcus faecium 12a. Enterocin 12a inhibited multidrug-resistant strains of various Gram-negative pathogens such as Salmonella enterica, Shigella flexneri, Vibrio cholerae, Escherichia coli and Gram-positive, Listeria monocytogenes, but had no activities against different strains of gut lactobacilli. The mass spectrometric analysis showed that the enterocin 12a shared partial homology with 4Fe-4S domain-containing redox protein of E. faecalis R712. Further, enterocin 12a selectively inhibited the proliferation of various human cancer cell lines in a dose-dependent manner but not that of normal human peripheral blood mononuclear cells. Enterocin 12a-treated cancer cells showed apoptosis-like morphological changes. CONCLUSION: Enterocin 12a is a novel bacteriocin that has anticancer properties against human cell lines and negligible activity towards non-malignant cells. Therefore, it should be further evaluated for its anticancer potential in animal models.
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Antibacterianos/farmacología , Anticarcinógenos/farmacología , Proliferación Celular/efectos de los fármacos , Enterococcus faecium/química , Apoptosis/efectos de los fármacos , Hidrocarburos Aromáticos con Puentes/química , Hidrocarburos Aromáticos con Puentes/aislamiento & purificación , Hidrocarburos Aromáticos con Puentes/farmacología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Enterococcus faecium/metabolismo , Femenino , Humanos , Pruebas de Sensibilidad Microbiana , Vagina/microbiologíaRESUMEN
Endoglucanase (EG) from Aspergillus niger HO was sequentially purified through ultrafiltration, ion exchange and size exclusion chromatography to homogeneity, with an overall recovery of 18 %. The purified EG was a monomeric protein with a molecular weight of about 55 kDa. The enzyme was optimally active at pH 3.5 and 70 °C with a half life (t1/2) of 3 h and Km value of 2.5 mg/ml. Metal ions, such as Ca(2+) and Co(2+) helped in enzyme induction, while Hg(2+) and Cu(2+) strongly inhibited the enzyme activity. Peptide mass fingerprinting results revealed that the purified EG is a novel enzyme that belongs to family 12 of glycoside hydrolase (GH12). Molecular docking studies indicated the presence of Glu116 and Glu204 as important determinant residues for the functional interaction with carboxymethylcellulose and showed hydrogen bonding with Asp99, Glu116, Glu204 and hydrophobic interactions with Trp22, Val58, Tyr61, Phe101, Met118, Trp120, Pro129, Ile130, Thr160 and Phe206. Hydrolysis of 2 % CMC with purified acidothermophilic EG at its optimum temperature and pH resulted in complete hydrolysis within 2 h yielding 18 % cellotriose, 72 % cellobiose and 10 % glucose as evident from HPLC analysis. In comparison to most of the EGs reported in literature, EG from A. niger HO exhibited higher thermostability. The acidothermophilic nature of this enzyme makes it potentially useful for industrial applications.
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Aspergillus niger/enzimología , Celulasa/aislamiento & purificación , Celulasa/metabolismo , Carboximetilcelulosa de Sodio/metabolismo , Celulasa/química , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Activadores de Enzimas/análisis , Inhibidores Enzimáticos/análisis , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Simulación del Acoplamiento Molecular , Peso Molecular , Temperatura , UltrafiltraciónRESUMEN
Fungi isolated from partially decayed wood log samples showing characteristic diversity for spore colour, colony morphology and arrangement of spores were assessed for cellulolytic enzyme production. Isolates showing a cellulolytic index of ≥2.0 were assayed for filter paper (FP) cellulase and ß-glucosidase (BGL) production. Molecular characterization confirmed the identity of the selected cellulolytic isolate as a strain of Aspergillus niger (A. niger HN-2). Addition of 2 % (w/v) urea enhanced FP and BGL activity by about 20 and 60 %, respectively. Validation studies conducted at parameters (29 °C, pH 5.4, moisture content 72 % and 66 h) optimized through response surface methodology in a solid-state static tray fermentation resulted in FP, BGL, cellobiohydrolase I (CBHI), endoglucanase (EG), xylanase activity and protein content of 25.3 FPU/g ds, 750 IU/g ds, 13.2 IU/g ds, 190 IU/g ds, 2890 IU/g ds and 0.9 mg/ml, respectively. In comparison, A. niger N402 which is a model organism for growth and development studies, produced significantly lower FP, BGL, CBHI, EG, xylanase activity and protein content of 10.0 FPU/g ds, 100 IU/g ds, 2.3 IU/g ds, 50 IU/g ds, 500 IU/g ds and 0.75 mg/ml, respectively under the same process conditions as were used for A. niger HN-2. Process optimization led to nearly 1.8- and 2.2-fold increase in FP and BGL activity, respectively showing promise for cellulase production by A. niger HN-2 at a higher scale of operation. Zymogram analysis revealed two isoforms each for EG and cellobiohydrolase and three isoforms for BGL. Crude cellulase complex produced by A. niger HN-2 exhibited thermostability under acidic conditions showing potential for use in biofuel industry.
Asunto(s)
Aspergillus niger/enzimología , Aspergillus niger/aislamiento & purificación , Celulasa/metabolismo , Proteínas Fúngicas/metabolismo , Madera/microbiología , Aspergillus niger/genética , Aspergillus niger/crecimiento & desarrollo , Celulasa/química , Celulasa/genética , Celulosa/metabolismo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Estabilidad de Enzimas , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Filogenia , Esporas Fúngicas/química , Esporas Fúngicas/enzimología , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Madera/metabolismoRESUMEN
The lignocellulolytic accessory enzyme, Feruloyl esterase C (FE_5DR), encoded in the genome of thermotolerant Myceliophthora verrucosa was successfully cloned and heterologously expressed in Pichia pastoris. The expressed FE_5DR was purified using UNOsphere™ Q anion exchange chromatography column, exhibiting a homogeneous band of ~ 39 kDa. Its optimum temperature was determined to be 60 °C, with an optimal pH of 6.0. Additionally, the enzyme activity of FE_5DR was significantly enhanced by preincubation in a buffer containing Mg2+, Cu2+ and Ca2 metal ions. Enzyme kinetic parameters, computed from double reciprocal Lineweaver-Burk plots, yielded observed Vmax and Km values of 0.758 U/mg and 0.439 mM, respectively. Furthermore, the potential of custom-made cocktails comprising FE_5DR and benchmark cellulase derived from the developed mutant strain of Aspergillus allahabadii MAN 40, as well as the biorefinery-relevant lignocellulolytic enzyme Cellic CTec 3, resulted in improved saccharification of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw when compared to benchmark MAN 40 and Cellic CTec 3. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04013-7.
RESUMEN
The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable ß-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg-1) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion.
Asunto(s)
Geobacillus , Xilosidasas , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Xilosidasas/química , Xilanos/metabolismo , Especificidad por SustratoRESUMEN
BACKGROUND: The filamentous fungus Rasamsonia emersonii has immense potential to produce biorefinery relevant thermostable cellulase and hemicellulase enzymes using lignocellulosic biomass. Previously in our lab, a hyper-cellulase producing strain of R. emersonii was developed through classical breeding and system biology approaches. ACE1, a pivotal transcription factor in fungi, plays a crucial role in negatively regulating the expression of cellulase genes. In order to identify the role of ACE1 in cellulase production and to further improve the lignocellulolytic enzyme production in R. emersonii, CRISPR/Cas9 mediated disruption of ACE1 gene was employed. RESULTS: A gene-edited ∆ACE1 strain (GN11) was created, that showed 21.97, 20.70 and 24.63, 9.42, 18.12%, improved endoglucanase, cellobiohydrolase (CBHI), ß-glucosidase, FPase, and xylanase, activities, respectively, as compared to parental strain M36. The transcriptional profiling showed that the expression of global regulator (XlnR) and different CAZymes genes including endoglucanases, cellobiohydrolase, ß-xylosidase, xylanase, ß-glucosidase and lytic polysaccharide mono-oxygenases (LPMOs) were significantly enhanced, suggesting critical roles of ACE1 in negatively regulating the expression of various key genes associated with cellulase production in R. emersonii. Whereas, the disruption of ACE1 significantly down-regulated the expression of CreA repressor gene as also evidenced by 2-deoxyglucose (2-DG) resistance phenotype exhibited by edited strain GN11 as well as appreciably higher constitutive production of cellulases in the presence of glucose and mixture of glucose and disaccharide (MGDs) both in batch and flask fed batch mode of culturing. Furthermore, ∆ACE1 strains were evaluated for the hydrolysis of biorefinery relevant steam/acid pretreated unwashed rice straw slurry (Praj Industries Ltd; 15% substrate loading rate) and were found to be significantly superior when compared to the benchmark enzymes produced by parent strain M36 and Cellic Ctec3. CONCLUSIONS: Current work uncovers the crucial role of ACE1 in regulating the expression of the various cellulase genes and carbon catabolite repression mechanism in R. emersonii. This study represents the first successful report of utilizing CRISPR/Cas9 genome editing technology to disrupt the ACE1 gene in the thermophlic fungus R. emersonii. The improved methodologies presented in this work might be applied to other commercially important fungal strains for which genetic manipulation tools are limited.
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The global cannabis (Cannabis sativa) market was 17.7 billion in 2019 and is expected to reach up to 40.6 billion by 2024. Canada is the 2nd nation to legalize cannabis with a massive sale of $246.9 million in the year 2021. Waste cannabis biomass is managed using disposal strategies (i.e., incineration, aerobic/anaerobic digestion, composting, and shredding) that are not good enough for long-term environmental sustainability. On the other hand, greenhouse gas emissions and the rising demand for petroleum-based fuels pose a severe threat to the environment and the circular economy. Cannabis biomass can be used as a feedstock to produce various biofuels and biochemicals. Various research groups have reported production of ethanol 9.2-20.2 g/L, hydrogen 13.5 mmol/L, lipids 53.3%, biogas 12%, and biochar 34.6% from cannabis biomass. This review summarizes its legal and market status (production and consumption), the recent advancements in the lignocellulosic biomass (LCB) pre-treatment (deep eutectic solvents (DES), and ionic liquids (ILs) known as "green solvents") followed by enzymatic hydrolysis using glycosyl hydrolases (GHs) for the efficient conversion efficiency of pre-treated biomass. Recent advances in the bioconversion of hemp into oleochemicals, their challenges, and future perspectives are outlined. A comprehensive insight is provided on the trends and developments of metabolic engineering strategies to improve product yield. The thermochemical processing of disposed-off hemp lignin into bio-oil, bio-char, synthesis gas, and phenol is also discussed. Despite some progress, barricades still need to be met to commercialize advanced biofuels and compete with traditional fuels.
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Two lignocellulolytic accessory enzymes, feruloyl esterase D (FAED_SCYTH) and ß-xylosidase (XYL43B_SCYTH) were cloned and produced in the Pichia pastoris X33 as host. The molecular weight of recombinant enzymes FAED_SCYTH and XYL43B_SCYTH were ~ 31 and 40 kDa, respectively. FAED_SCYTH showed optimal activity at pH 6.0, 60 °C; and XYL43B_SCYTH at pH 7.0, 50 °C. FAED_SCYTH and XYL43B_SCYTH exhibited t1/2: 4 and 0.5 h, respectively (50 °C, pH 5.0). The ß-xylosidase was bi-functional with pronounced activity against pNP-α-arabinofuranoside besides being highly xylose tolerant (retaining ~ 97% activity in the presence of 700 mM xylose). Cocktails prepared using these enzymes along with AA9 protein (PMO9D_SCYTH) and commercial cellulase CellicCTec2, showed improved hydrolysis of the pre-treated lignocellulosic biomass. Priming of pre-treated lignocellulosic biomass with these accessory enzymes was found to further enhance the hydrolytic potential of CellicCTec2 promising to reduce the enzyme load and cost required for obtaining sugars from biorefinery relevant pre-treated substrates.
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Hongos , Hidrolasas de Éster Carboxílico , Hidrólisis , Lignina , Saccharomycetales , Especificidad por Sustrato , XilosidasasRESUMEN
Two Lytic polysaccharide Mono-Oxygenases (LPMOs), non-modular (PMO_08942) and modular (PMO_07920), from thermotolerant fungus Aspergillus terreus 9DR cloned and expressed in Pichia pastoris X33 and purified to homogeneity using ion-exchange chromatography were found to be of ~29 and ~40 kDa, respectively. Both LPMOs were optimally active at 50 °C; PMO_08942 was active under acidic condition (pH 5.0) and PMO_07920 at pH 7.0. Modular LPMO (PMO_07920) tethered to CBM-1 was found to be versatile as it showed appreciable activity on complex polysaccharide (both cellulose and xylans) as compared to non-modular (PMO_08942). The t1/2 of PMO_08942 (~192 h, pH 5.0) and PMO_0792 (~192 h, pH 7.0) at 50 °C, suggests highly stable nature of these LPMOs. Fluorescently tagged modular AA9 was studied microscopically to understand interaction with pretreated biomass. Priming of biomass for up to 6 h with LPMOs prior to initiating hydrolysis with core cellulase enzyme resulted in significantly higher saccharification.
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Etanol , Oxigenasas , Lignina , Oxigenasas de Función Mixta , PolisacáridosRESUMEN
In this study, two novel thermostable lytic polysaccharide monooxygenases (LPMOs) were cloned from thermophilic fungus Scytalidium thermophilum (PMO9D_SCYTH) and Malbranchea cinnamomea (PMO9D_MALCI) and expressed in the methylotrophic yeast Pichia pastoris X33. The purified PMO9D_SCYTH was active at 60 °C (t1/2 = 60.58 h, pH 7.0), whereas, PMO9D_MALCI was optimally active at 50 °C (t1/2 = 144 h, pH 7.0). The respective catalytic efficiency (kcat/Km) of PMO9D_SCYTH and PMO9D_MALCI determined against avicel in presence of H2O2 was (6.58 × 10-3 and 1.79 × 10-3 mg-1 ml min-1) and carboxy-methylcellulose (CMC) (1.52 × 10-1 and 2.62 × 10-2 mg-1 ml min-1). The HRMS analysis of products obtained after hydrolysis of avicel and CMC showed the presence of both C1 and C4 oxidized oligosaccharides, in addition to phylogenetic tree constructed with other characterized type 1 and 3 LPMOs demonstrated that both LPMOs belongs to type-3 family of AA9s. The release of sugars during saccharification of acid/alkali pretreated sugarcane bagasse and rice straw was enhanced upon replacing one part of commercial enzyme Cellic CTec2 with these LPMOs.
Asunto(s)
Hongos/enzimología , Hongos/metabolismo , Lignina/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Polisacáridos/metabolismo , Carboximetilcelulosa de Sodio , Celulosa/química , Clonación Molecular , Estabilidad de Enzimas , Proteínas Fúngicas/química , Hongos/genética , Regulación Fúngica de la Expresión Génica , Peróxido de Hidrógeno , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Oxigenasas de Función Mixta/clasificación , Onygenales/enzimología , Onygenales/genética , Onygenales/metabolismo , Filogenia , Saccharomycetales/enzimología , Especificidad por Sustrato , TemperaturaRESUMEN
The enantioselectivity of the enzymes responsible for reduction of prochiral compound 3-phenylthiopropan-2-one was dependent on the concentration of yeast extract and glucose in the growth medium. Low concentrations of yeast extract (0.1-0.9% w/v) favored the formation of S-enantiomer (62% ee at 0.1% w/v yeast extract) of 3-phenylthiopropan-2-ol. However, R-enantiomer of the reduced product was formed when MSM was supplemented with yeast extract at a concentration of 1% (w/v) or more with a maximum ee of 85% at 2.0% (w/v) yeast extract supplement in the growth medium.
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Bacillus/metabolismo , Cetonas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Biotransformación , Cetonas/química , Conformación Molecular , Estructura Molecular , Oxidación-Reducción , Compuestos de Sulfhidrilo/químicaRESUMEN
Mycothermus thermophilus (Syn. Scytalidium thermophilum/Humicola insolens), a thermophilic fungus, is being reported to produce appreciable titers of cellulases and hemicellulases during shake flask culturing on cellulose/wheat-bran/rice straw based production medium. The sequential and differential expression profile of endoglucanases, ß-glucosidases, cellobiohydrolases and xylanases using zymography was studied. Mass spectrometry analysis of secretome (Q-TOF LC/MS) revealed a total of 240 proteins with 92 CAZymes of which 62 glycosyl hydrolases belonging to 30 different families were present. Cellobiohydrolase I (17.42%), ß glucosidase (8.69%), endoglucanase (6.2%), xylanase (4.16%) and AA9 (3.95%) were the major proteins in the secretome. In addition, carbohydrate esterases, polysaccharide lyases, auxiliary activity and a variety of carbohydrate binding modules (CBM) were identified using genomic database of the culture indicating to an elaborate genetic potential of this strain for hydrolysis of lignocellulosics. The cellulases from the strain hydrolyzed alkali treated rice straw and bagasse into fermentable sugars efficiently.
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Ascomicetos/enzimología , Celulasas/metabolismo , Glicósido Hidrolasas/metabolismo , Celulasa/metabolismo , Celulosa/química , Celulosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Medios de Cultivo/química , Fibras de la Dieta , Esterasas/metabolismo , Hidrólisis , Espectrometría de Masas , Oryza/química , Oryza/metabolismo , Polisacárido Liasas/metabolismo , beta-Glucosidasa/metabolismoRESUMEN
This study reports thermophilic fungus Malbranchea flava as a potent source of xylanase and xylan-debranching accessory enzymes. M. flava produced high levels of xylanase on sorghum straw containing solidified culture medium. The optimization of culture conditions for production of hemicellulases was carried out using one factor at a time approach and Box-Behnken design of experiments with casein (%), inoculum age (h) and inoculum level (ml) as process variables and xylanase, ß-xylosidase, acetyl esterases and arabinofuranosidase as response variables. The results showed that casein concentration between 3.0 and 3.5 %, inoculum age (56-60 h) and inoculum level (2-2.5 ml) resulted in production of 16,978, 10.0, 67.7 and 3.8 (U/gds) of xylanase, ß-xylosidase, acetyl esterase and α-L-arabinofuranosidase, respectively. Under optimized conditions M. flava produced eight functionally diverse xylanases with distinct substrate specificity against different xylan types. The peptide mass fingerprinting of 2-D gel electrophoresis resolved proteins indicated to the presence of cellobiose dehydrogenase and glycosyl hydrolases suggesting the potential of this strain in oxidative and classical cellulase-mediated hydrolysis of lignocellulosics. Addition of xylanase (300 U/g substrate) during saccharification (at 15 % substrate loading) of different pretreated (acid/alkali) substrates (cotton stalks, wheat straw, rice straw, carrot grass) by commercial cellulase (NS28066) resulted in 9-36 % increase in saccharification and subsequent fermentation to ethanol when compared to experiment with commercial enzyme only. High ethanol level 46 (g/l) was achieved with acid pretreated cotton stalk when M. flava xylanase was supplemented as compared to 39 (g/l) with xylanase without xylanase addition.
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The decolorization potential of the consortium HM-4 constituted by mixing four laboratory isolates identified as Bacillus cereus (BN-7), Pseudomonas putida (BN-4), Pseudomonas fluorescens (BN-5) and Stenotrophomonas acidaminiphila (BN-3) was compared with that of individual isolates. Six different azo dyes viz., C.I. Acid Red 88 (AR-88), C.I. Acid Red 119 (AR-119), C.I. Acid Red 97 (AR-97), C.I. Reactive Red 120 (RR-120), C.I. Acid Blue 113 (AB-113) and C.I. Acid Brown 100 (AB-100) were used in this study. The individual bacterial isolates were not able to completely decolorize these dyes, except for dyes AR-119 and AB-113. The consortium HM-4 was able to decolorize all the dyes used at an initial dye concentration of 20 mg L(-1) at a significantly higher rate as compared to that achieved by individual isolates.
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Compuestos Azo/metabolismo , Bacterias/metabolismo , Colorantes/metabolismo , Purificación del Agua/métodos , Bacterias/clasificación , Bacterias/aislamiento & purificación , Biodegradación Ambiental , Color , Residuos Industriales , Eliminación de Residuos LíquidosRESUMEN
Cellulolytic enzyme production by newly isolated Aspergillus niger HN-1 was statistically optimized using Plackett-Burman and central composite design (CCD). Optimum concentrations of 2, 0.40, 0.01, and 0.60 g L (-1) for KH2PO4, urea, trace elements solution, and CaCl2·2H2O, respectively, were suggested by Design-Expert software. The two-stage optimization process led to a 3- and 2-fold increases in the filter paper cellulase (FP) and ß-glucosidase activities, respectively. FP, ß-glucosidase, endoglucanase, exopolygalaturonase, cellobiohydrolase, xylanase, α-l-arabinofuranosidase, ß-xylosidase, and xylan esterase activities of 36.7 ± 1.54 FPU gds(-1), 252.3 ± 7.4 IU gds(-1), 416.3 ± 22.8 IU gds(-1), 111.2 ± 5.4 IU gds(-1), 8.9 ± 0.50 IU gds(-1), 2593.5 ± 78.9 IU gds(-1), 79.4 ± 4.3 IU gds(-1), 180.8 ± 9.3 IU gds(-1), and 288.7 ± 11.8 IU gds(-1), respectively, were obtained through solid-state fermentation during the validation studies. Hydrolysis of alkali-treated rice straw with crude cellulases resulted in about 84% glucan to glucose, 89% xylan to xylose, and 91% arabinan to arabinose conversions, indicating potential for biomass hydrolysis by the crude cellulase consortium obtained in this study.
Asunto(s)
Aspergillus niger/enzimología , Técnicas de Cultivo Celular por Lotes/métodos , Celulasas/biosíntesis , Proteínas Fúngicas/biosíntesis , Oryza/química , Aspergillus niger/clasificación , Aspergillus niger/crecimiento & desarrollo , Aspergillus niger/aislamiento & purificación , Técnicas de Cultivo Celular por Lotes/instrumentación , Biocatálisis , Celulasas/metabolismo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Fermentación , Proteínas Fúngicas/metabolismo , Hidrólisis , Datos de Secuencia Molecular , Filogenia , Tallos de la Planta/metabolismo , Tallos de la Planta/microbiología , Eliminación de Residuos , Microbiología del SueloRESUMEN
A thermotolerant Aspergillus fumigatus strain isolated from composting pile of mixed industrial waste was found to produce a spectrum of cellulase and hemicellulases when cultured on rice straw solidified substrate. The two-dimensional electrophoresis (2DE) resolved the secretome into 57 distinct protein spots. The zymograms developed against 2DE gels identified the presence of three ß-glucosidases and five CBHI/EGI isoforms in the secretome. The peptide mass fingerprinting of 17 protein spots by liquid chromatography mass spectrometry characterized the secretome into different glycosyl hydrolase families. The enzyme cocktail produced by A. fumigatus was capable of efficient hydrolysis of alkali pretreated rice straw (at 7% and 10% w/v) resulting in 95% and 91% saccharification, respectively.
Asunto(s)
Álcalis/química , Aspergillus fumigatus/enzimología , Glicósido Hidrolasas/metabolismo , Oryza/química , Celulasas/metabolismo , Electroforesis en Gel Bidimensional , Hidrólisis , Isoformas de Proteínas/metabolismoRESUMEN
Two xylanases, MFX I and MFX II, from the thermophilic fungus Malbranchea flava MTCC 4889 with molecular masses of 25.2 and 30kDa and pIs of 4.5 and 3.7, respectively were purified to homogeneity. The xylanases were optimally active at pH 9.0 and at 60 degrees C, exhibited a half-life of 4h at 60 degrees C, and showed distinct mode of action and product profiles when applied to birchwood, oat spelt, and larchwood xylan, and to wheat and rye arabinoxylan. The xylanases were most active on larchwood xylan with K(m) values of 1.25 and 3.7mg/ml. K(cat)/K(m) values suggested that the xylanases preferentially hydrolyzed rye arabinoxylan. LC-MS/MS (liquid chromatography/mass spectrometry) analysis of tryptic digests of MFX I and MFX II revealed similarity with known fungal xylanases and suggests that that they belonged to the GH 11 and 10 glycosyl hydrolase super families, respectively. These xylanases can potentially be used in enzyme-assisted bleaching of the pulp derived from agro-residues, as well as production of xylooligosaccharides for pre-biotic functional food applications.