Dataset Information

Effect of Temperature on Xylanase II from Trichoderma reesei QM 9414: A Calorimetric, Catalytic, and Conformational Study.

ABSTRACT: The secondary structure of xylanase II from Trichoderma reesei is lost in an apparent irreversible cooperative process as temperature is increased with a midpoint transition of 58.8 ± 0.1°C. The shift of the spectral centre of mass above 50°C is also apparently cooperative with midpoint transition of 56.3 ± 0.2°C, but the existence of two isofluorescent points in the fluorescence emission spectra suggests a non-two-state process. Further corroboration comes from differential scanning calorimetry experiments. At protein concentrations ?0.56?mg·mL(-1) the calorimetric transition is reversible and the data were fitted to a non-two-state model and deconvoluted into six transitions, whereas at concentrations greater than 0.56?mg·mL(-1) the calorimetric transition is irreversible with an exothermic contribution to the thermogram. The apparent T m increased linearly with the scan rate according to first order inactivation kinetics. The effect of additives on the calorimetric transition of xylanase is dependent on their nature. The addition of sorbitol transforms reversible transitions into irreversible transitions while stabilizing the protein as the apparent T m increases linearly with sorbitol concentration. d-Glucono-1,5-lactone, a noncompetitive inhibitor in xylanase kinetics, and soluble xylan change irreversible processes into reversible processes at high protein concentration.

SUBMITTER: Lopez G

PROVIDER: S-EPMC4170777 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Effect of Temperature on Xylanase II from Trichoderma reesei QM 9414: A Calorimetric, Catalytic, and Conformational Study.

López Gloria G Estrada Pilar P

Enzyme research 20140907

The secondary structure of xylanase II from Trichoderma reesei is lost in an apparent irreversible cooperative process as temperature is increased with a midpoint transition of 58.8 ± 0.1°C. The shift of the spectral centre of mass above 50°C is also apparently cooperative with midpoint transition of 56.3 ± 0.2°C, but the existence of two isofluorescent points in the fluorescence emission spectra suggests a non-two-state process. Further corroboration comes from differential scanning calorimetry ...[more]

PMID: 25276420

Similar Datasets

Project description:Trichoderma reesei is known for its ability to produce large amounts of extracellular proteins and is one of the most important industrially used filamentous fungus. Xylanase regulator 1 (XYR1) as the master regulator is responsible for the activation of cellulase and hemicellulase gene expression, normally under inducing conditions. It has been reported that strains with point mutations in certain areas of xyr1 bypass the carbon catabolite repression, allowing cellulase and hemicellulase production even in the presence of glucose. These mutations also change the profile of produced proteins, shifting it more towards xylanase production, and increase the overall protein production in inducing conditions. However, how these mutations alter the metabolism and other cellular processes to cause these changes remains unclear. In the present study, our aim was to explore changes caused by a point mutation in xyr1 on transcriptomic and metabolic level to better understand the reasons behind the increased protein production in both repressing glucose and inducing lactose conditions. We observed that the xyr1 mutant strain built more biomass and produced more extracellular proteins during growth on lactose compared to the wild type xyr1 strain. Genes involved in oxidoreductive D-galactose catabolism pathway were upregulated in the xyr1 mutant strain, potentially contributing to the more efficient utilization of lactose. The shift from utilizing lactose to using glucose as carbon source seemed faster in the xyr1 mutant strain. Clustering and enrichment analysis showed over-representation of mitochondria-related Gene Ontology terms in clusters where gene expression was higher in the xyr1 mutant, indicating that mitochondria play a role in the altered metabolic state associated with the xyr1 mutation. Metabolomics revealed that free tyrosine was more abundant in the xyr1 mutant strain in all measured timepoints, whereas multiple fatty acids were less abundant in the mutant strain on glucose. The results contribute to more in-depth knowledge on T. reesei physiology and aid in finding new targets for improved protein production.

Project description:BackgroundFilamentous fungus Trichoderma reesei has been widely used as a workhorse for cellulase and xylanase productions. Xylanase has been reported as the crucial accessory enzyme in the degradation of lignocellulose for higher accessibility of cellulase. In addition, the efficient hydrolysis of xylan needs the co-work of multiple xylanolytic enzymes, which rise an increasing demand for the high yield of xylanase for efficient biomass degradation.ResultsIn this study, a xylanase hyper-producing system in T. reesei was established by tailoring two transcription factors, XYR1 and ACE1, and homologous overexpression of the major endo-xylanase XYNII. The expressed xylanase cocktail contained 5256 U/mL xylanase activity and 9.25 U/mL β-xylosidase (pNPXase) activity. Meanwhile, the transcription level of the xylanolytic genes in the strain with XYR1 overexpressed was upregulated, which was well correlated with the amount of XYR1-binding sites. In addition, the higher expression of associated xylanolytic enzymes would result in more efficient xylan hydrolysis. Besides, 2310-3085 U/mL of xylanase activities were achieved using soluble carbon source, which was more efficient and economical than the traditional strategy of xylan induction. Unexpectedly, deletion of ace1 in C30OExyr1 did not give any improvement, which might be the result of the disturbed function of the complex formed between ACE1 and XYR1. The enzymatic hydrolysis of alkali pretreated corn stover using the crude xylanase cocktails as accessory enzymes resulted in a 36.64% increase in saccharification efficiency with the ratio of xylanase activity vs FPase activity at 500, compared to that using cellulase alone.ConclusionsAn efficient and economical xylanase hyper-producing platform was developed in T. reesei RUT-C30. The novel platform with outstanding ability for crude xylanase cocktail production would greatly fit in biomass degradation and give a new perspective of further engineering in T. reesei for industrial purposes.

Dataset Information

Effect of Temperature on Xylanase II from Trichoderma reesei QM 9414: A Calorimetric, Catalytic, and Conformational Study.

Publications

Effect of Temperature on Xylanase II from Trichoderma reesei QM 9414: A Calorimetric, Catalytic, and Conformational Study.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure