ATP measurement for cellulase production control

Summary Plotting intracellular ATP concentrations as a function of time during cellulase production byTrichoderma reesei, provides a good evaluation of fungal growth and specific rate of cellulose consumption. ATP content evaluation is of great help for running fed-batch and semi-continuous fermentations.     

A kinetic model for methanogenesis of acetic acid in a multireactor system

Bioconversion of acetic acid to methane by a crude culture of methanogens in a continuous multireactor system was investigated. Culture of methanogens was drawn from an active cow-dung digester (12 days) and was grown in a semisynthetic medium (pH 6.3, 37 degrees C) with acetic acid as the sole carbon source. The solubilities of CO(2), HCO(3) (-), and CO(3) (2-) increased with the rise in pH and exercised considerable influence on the gas composition. Various mechanisms for methanogenesis of acetic acid based on the available pathways were considered. Experimental data were compared with these mechanisms, the best fit was determined, and the corresponding rate expression was identified. This mechanism predicted that, of the total methane produced, 72%;comes from acetic acid directly and 28%;via the CO(2) reduction route.     

Method To Estimate Growth of Trichoderma reesei and Aspergillus wentii in Mixed Culture on Cellulosic Substrates

A simple differential method based on measurement of an intracellular pigment of Aspergillus wentii was developed for estimation of the individual growths of two fungi, Trichoderma reesei and A. wentii, in mixed-culture fermentation of an insoluble cellulosic substrate.     

Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism

Oxygen influences behaviour in many organisms, with low levels (hypoxia) having devastating consequences for neuron survival. How neurons respond physiologically to counter the effects of hypoxia is not fully understood. Here, we show that hypoxia regulates the trafficking of the glutamate receptor GLR-1 in C. elegans neurons. Either hypoxia or mutations in egl-9, a prolyl hydroxylase cellular oxygen sensor, result in the internalization of GLR-1, the reduction of glutamate-activated currents, and the depression of GLR-1-mediated behaviours. Surprisingly, hypoxia-inducible factor (HIF)-1, the canonical substrate of EGL-9, is not required for this effect. Instead, EGL-9 interacts with the Mint orthologue LIN-10, a mediator of GLR-1 membrane recycling, to promote LIN-10 subcellular localization in an oxygen-dependent manner. The observed effects of hypoxia and egl-9 mutations require the activity of the proline-directed CDK-5 kinase and the CDK-5 phosphorylation sites on LIN-10, suggesting that EGL-9 and CDK-5 compete in an oxygen-dependent manner to regulate LIN-10 activity and thus GLR-1 trafficking. Our findings demonstrate a novel mechanism by which neurons sense and respond to hypoxia.     

Glowworm swarm optimization for simultaneous capture of multiple local optima of multimodal functions

This paper presents glowworm swarm optimization (GSO), a novel algorithm for the simultaneous computation of multiple optima of multimodal functions. The algorithm shares a few features with some better known swarm intelligence based optimization algorithms, such as ant colony optimization and particle swarm optimization, but with several significant differences. The agents in GSO are thought of as glowworms that carry a luminescence quantity called luciferin along with them. The glowworms encode the fitness of their current locations, evaluated using the objective function, into a luciferin value that they broadcast to their neighbors. The glowworm identifies its neighbors and computes its movements by exploiting an adaptive neighborhood, which is bounded above by its sensor range. Each glowworm selects, using a probabilistic mechanism, a neighbor that has a luciferin value higher than its own and moves toward it. These movements—based only on local information and selective neighbor interactions—enable the swarm of glowworms to partition into disjoint subgroups that converge on multiple optima of a given multimodal function. We provide some theoretical results related to the luciferin update mechanism in order to prove the bounded nature and convergence of luciferin levels of the glowworms. Experimental results demonstrate the efficacy of the proposed glowworm based algorithm in capturing multiple optima of a series of standard multimodal test functions and more complex ones, such as stair-case and multiple-plateau functions. We also report the results of tests in higher dimensional spaces with a large number of peaks. We address the parameter selection problem by conducting experiments to show that only two parameters need to be selected by the user. Finally, we provide some comparisons of GSO with PSO and an experimental comparison with Niche-PSO, a PSO variant that is designed for the simultaneous computation of multiple optima. This work is partially supported by a project grant from the Ministry of Human Resources Development, India and by DRDO-IISc Mathematical Engineering Program.     

Defining process design space for a hydrophobic interaction chromatography (HIC) purification step: Application of quality by design (QbD) principles

The concept of design space has been taking root under the quality by design paradigm as a foundation of in‐process control strategies for biopharmaceutical manufacturing processes. This paper outlines the development of a design space for a hydrophobic interaction chromatography (HIC) process step. The design space included the impact of raw material lot‐to‐lot variability and variations in the feed stream from cell culture. A failure modes and effects analysis was employed as the basis for the process characterization exercise. During mapping of the process design space, the multi‐dimensional combination of operational variables were studied to quantify the impact on process performance in terms of yield and product quality. Variability in resin hydrophobicity was found to have a significant influence on step yield and high‐molecular weight aggregate clearance through the HIC step. A robust operating window was identified for this process step that enabled a higher step yield while ensuring acceptable product quality. Biotechnol. Bioeng. 2010;107: 985–997. © 2010 Wiley Periodicals, Inc.     

The effect of column verticality on separation efficiency in expanded bed adsorption

The effect of column verticality on liquid dispersion and separation efficiency in expanded bed adsorption columns was investigated using 1 and 5 cm diameter columns. Column misalignment of only 0.15° resulted in the reduction of the Bodenstein number from 140 to 50 for the 1 cm dia. column and from 75 to 45 for the 5 cm dia. column. This degree of misalignment was not detectable by visual assessment of adsorbent particle movement within the column. Depending on the relative importance of transport limitations, kinetic limitations and dispersion to any specific separation, this increase in dispersion with column alignment can significantly affect separation efficiency. Pure protein breakthrough profiles resulting from the application of bovine serum albumin onto STREAMLINE Q XL demonstrated that, at 10% breakthrough, 7.8% more protein could be applied to a vertical 1 cm dia. column compared to the same column misaligned by 0.15°. When an unclarified yeast homogenate was applied to a 1 cm dia. vertical column packed with STREAMLINE DEAE, 10% breakthrough of glucose-6-phosphate dehydrogenase (G6PDH) corresponded to a load 55% greater compared to the same column aligned 0.185° off-vertical. The G6PDH breakthrough curves for vertical and 0.15° off-vertical runs performed using a 5 cm column were essentially indistinguishable.     

Backbone dynamics of the N-terminal domain in E. coli DnaJ determined by 15N- and 13CO-relaxation measurements.

The backbone dynamics of the N-terminal domain of the chaperone protein Escherichia coli DnaJ have been investigated using steady-state 1H-15N NOEs, 15N T1, T2, and T1 rho relaxation times, steady-state 13C alpha-13CO NOEs, and 13CO T1 relaxation times. Two recombinant constructs of the N-terminal domain of DnaJ have been studied. One, DnaJ(1-78), contains the most conserved "J-domain" of DnaJ, and the other, DnaJ(1-104), includes a glycine/phenylalanine rich region ("G/F" region) in addition to the "J-domain". DnaJ(1-78) is not capable of stimulating ATP hydrolysis by DnaK, despite the fact that all currently identified sites responsible for DnaJ-DnaK interaction are located in this region. DnaJ(1-104), on the other hand, retains nearly the full ATPase stimulatory activity of full length DnaJ. Recently, a structural analysis of these two molecules was presented in an effort to elucidate the origin of their functional differences [Huang, K., Flanagan, J. M., and Prestegard, J. H. (1999) Protein Science 8, 203-214]. Herein, an analysis of dynamic properties is presented in a similar effort. A generalized model-free approach with a full treatment of the anisotropic overall rotation of the proteins is used in the analysis of measured relaxation parameters. Our results show that internal motions on pico- to nanosecond time scales in the backbone of DnaJ(1-78) are reduced on the inclusion of the "G/F" region, while conformational exchange on micro- to millisecond time scales increases. We speculate that the enhanced flexibility of residues on the slow time scale upon the inclusion of the "G/F" region could be relevant to the ATPase stimulatory activity of DnaJ if an "induced-fit" mechanism applies to DnaJ-DnaK interactions.     

Probing Slow Backbone Dynamics in Proteins Using TROSY-Based Experiments to Detect Cross-Correlated Time-Modulation of Isotropic Chemical Shifts

The difference in the relaxation rates of zero-quantum (ZQ) and double-quantum (DQ) coherences is the result of three principal mechanisms. These include the cross-correlation between the chemical shift anisotropies of the two participating nuclei, dipolar interactions with remote protons as well as interference effects due to the time-modulation of their isotropic chemical shifts as a consequence of slow micros-ms dynamics. The last effect when present, dominates the others resulting in large differences between the relaxation rates of ZQ and DQ coherences. We present here four sets of TROSY-based (Salzmann et al., 1998) experiments that measure this effect for several pairs of backbone nuclei including (15)N, (13)C(alpha) and (13)C'. These experiments allow the detection of the presence of slow dynamic processes in the protein backbone including correlated motion over two and three bonds. Further, we define a new parameter chi which represents the extent of correlated motion on the slow (micros-ms) timescale. This methodology has been applied to (15)N,(13)C,REDPRO-(2)H-labeled (Shekhtman et al., 2002) human ubiquitin. The ubiquitin backbone is seen to exhibit extensive dynamics on the slow timescale. This is most pronounced in several residues in the N-terminal region of the alpha-helix and in the loop connecting the strands beta(4) and beta(5). These residues which include Glu24, Asn25, Glu51 and Asp 52 form a continuous surface. As an additional benefit, the measured rates confirm the dependence of the (13)C(alpha) chemical shift tensor on local secondary structure of the protein backbone.