1,314 research outputs found
Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures.
BackgroundTrichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60-70°C.ResultsA B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50-70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in Tm) and improved half-life at 60°C (t1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.ConclusionStructure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes
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Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies
The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin
Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies
The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin
Multiple-length-scale elastic instability mimics parametric resonance of nonlinear oscillators
Spatially confined rigid membranes reorganize their morphology in response to
the imposed constraints. A crumpled elastic sheet presents a complex pattern of
random folds focusing the deformation energy while compressing a membrane
resting on a soft foundation creates a regular pattern of sinusoidal wrinkles
with a broad distribution of energy. Here, we study the energy distribution for
highly confined membranes and show the emergence of a new morphological
instability triggered by a period-doubling bifurcation. A periodic
self-organized focalization of the deformation energy is observed provided an
up-down symmetry breaking, induced by the intrinsic nonlinearity of the
elasticity equations, occurs. The physical model, exhibiting an analogy with
parametric resonance in nonlinear oscillator, is a new theoretical toolkit to
understand the morphology of various confined systems, such as coated materials
or living tissues, e.g., wrinkled skin, internal structure of lungs, internal
elastica of an artery, brain convolutions or formation of fingerprints.
Moreover, it opens the way to new kind of microfabrication design of
multiperiodic or chaotic (aperiodic) surface topography via self-organization.Comment: Submitted for publicatio
Theory and Computation of the Spheroidal Wave Functions
In this paper we report on a package, written in the Mathematica computer
algebra system, which has been developed to compute the spheroidal wave
functions of Meixner [J. Meixner and R.W. Schaefke, Mathieusche Funktionen und
Sphaeroidfunktionen, 1954] and is available online
(www.physics.uwa.edu.au/~falloon/spheroidal/spheroidal.html). This package
represents a substantial contribution to the existing software, since it
computes the spheroidal wave functions to arbitrary precision for general
complex parameters mu, nu, gamma and argument z; existing software can only
handle integer mu, nu and does not give arbitrary precision. The package also
incorporates various special cases and computes analytic power series and
asymptotic expansions in the parameter gamma. The spheroidal wave functions of
Flammer [C. Flammer, Spheroidal Wave Functions, 1957] are included as a special
case of Meixner's more general functions. This paper presents a concise review
of the general theory of spheroidal wave functions and a description of the
formulas and algorithms used in their computation, and gives high-precision
numerical examples.Comment: 26 pages, 4 Appendices, 5 Table
MAGIC observations of very high energy gamma-rays from HESS J1813-178
Recently, the HESS collaboration has reported the detection of gamma-ray
emission above a few hundred GeV from eight new sources located close to the
Galactic Plane. The source HESS J1813-178 has sparked particular interest, as
subsequent radio observations imply an association with SNR G12.82-0.02.
Triggered by the detection in VHE gamma-rays, a positionally coincident source
has also been found in INTEGRAL and ASCA data. In this Letter we present MAGIC
observations of HESS J1813-178, resulting in the detection of a differential
gamma-ray flux consistent with a hard-slope power law, described as dN/(dA dt
dE) = (3.3+/-0.5)*10^{-12} (E/TeV)^{-2.1+/-0.2} cm^(-2)s^(-1)TeV^(-1). We
briefly discuss the observational technique used, the procedure implemented for
the data analysis, and put this detection in the perspective of multifrequency
observations.Comment: Accepted by ApJ Letter
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Effects of selected changes in Federal land use on a rural economy
Published March 1968. Facts and recommendations in this publication may no longer be valid. Please look for up-to-date information in the OSU Extension Catalog: http://extension.oregonstate.edu/catalo
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