Biotechnological uses of enzymes from psychrophiles.

The bulk of the Earth's biosphere is cold (e.g. 90% of the ocean's waters are ≤ 5°C), sustaining a broad diversity of microbial life. The permanently cold environments vary from the deep ocean to alpine reaches and to polar regions. Commensurate with the extent and diversity of the ecosystems that harbour psychrophilic life, the functional capacity of the microorganisms that inhabitat the cold biosphere are equally diverse. As a result, indigenous psychrophilic microorganisms provide an enormous natural resource of enzymes that function effectively in the cold, and these cold-adapted enzymes have been targeted for their biotechnological potential. In this review we describe the main properties of enzymes from psychrophiles and describe some of their known biotechnological applications and ways to potentially improve their value for biotechnology. The review also covers the use of metagenomics for enzyme screening, the development of psychrophilic gene expression systems and the use of enzymes for cleaning.

Archery performance level and repeatability of event-related EMG.

The purpose of the current study was to compare the repeatability of electromyographic linear envelopes (LE) of archery groups. Surface electromyography (EMG) signals of musculus flexor digitorum superficialis (MFDS) and extensor digitorum (MED) of 23 participants (seven skilled, six beginner archers and ten non-archers) were recorded during archery shooting. Two-second periods (clicker falls at first second) of 12 shots' EMG data were recorded, full-wave rectified and filtered (60 ms moving-average filter) for each participant's drawing arm. Repeatability was investigated by using a statistical criterion, variance ratio (VR). Archers' performances were evaluated in terms of FITA scores. The results showed that FITA scores were significantly correlated to the VRs of MFDS and MED. EMG LEs were more repeatable among archers than non-archers. Therefore, we inferred that VRs of MFDS and MED might be important variables for (a) assessing shooting techniques, (b) evaluation of archers' progress, and (c) selection of talented archers.

Quantification the relationship between FITA scores and EMG skill indexes in archery.

Forearm electromyographic (EMG) data are assumed to be an effective method in estimating performance level in archery. The aim of the current study was to establish archery skill indexes based on EMG data. Elite (n=7, FITA score=1303.4+/-26.2), beginner (n=6, FITA score=1152+/-9.0) and non-archers (n=10, assumed FITA score=250+/-0), were involved in the study. EMG activity of Muscle flexor digitorum superficialis and Muscle extensor digitorum were quantified. Two-second periods--1 s before and 1 s after the fall of the clicker--were used to obtain averaged and rectified EMG data. The averaged and rectified EMG data were filtered by averaging finite impulse response filter with 40 ms time window and then normalized with respect to maximum voluntary contraction. To estimate FITA scores from EMG data, the following skill indexes that based on mean area under some parts of processed EMG waveforms was offered for archery. These were the pre-clicker archery skill index (PreCASI), post-clicker archery skill index (PostCASI), archery skill index (ASI) and post-clicker archery skill index 2 (PostCASI2). The correlations between rank of FITA scores and natural logarithms of archery skill indexes were significant for log(PreCASI): r=-0.66, p<0.0008; for log(PostCASI): r=-0.70, p<0.0003; for log(ASI): r=-0.74, p<0.0001; log(PostCASI2): r=-0.63, p<0.002. It is concluded that EMG skill indexes may be useful for: (a) assessing shooting techniques, (b) evaluation of archers' progress and (c) selection of talented archers.

Activation patterns in forearm muscles during archery shooting.

A contraction and relaxation strategy with regard to forearm muscles during the release of the bowstring has often been observed during archery, but has not well been described. The purpose of this study was to analyze this strategy in archers with different levels of expertise; elite, beginner and non-archers. Electromyography (EMG) activity of the M. flexor digitorum superficialis and the M. extensor digitorum were recorded at a sampling frequency of 500 Hz, together with a pulse synchronized with the clicker snap, for twelve shots by each subject. Raw EMG records, 1-s before and after the clicker pulse, were rectified, integrated and normalized. The data was then averaged for successive shots of each subject and later for each group. All subjects including non-archers developed an active contraction of the M. extensor digitorum and a gradual relaxation of the M. flexor digitorum superficialis with the fall of the clicker. In elite archers release started about 100 ms after the fall of the clicker, whereas in beginners and non-archers release started after about 200 and 300 ms, respectively. Non-archers displayed a preparation phase involving extensive extensor activity before the release of the bowstring, which was not observed in elite and beginner archers. In conclusion, archers released the bowstring by active contraction of the forearm extensors, whereas a clear relaxation of the forearm flexors affecting the release movement was not observed.

Stabilization of Escherichia coli penicillin G acylase against thermal inactivation by cross-linking with dextran dialdehyde polymers.

The thermostabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11,105 by cross-linking with dextran dialdehyde molecules, at a molecular mass of 11,500, 37,700 and 71,000 Da, was studied. The thermal inactivation mechanisms of the native and modified PGA were both considered to obey first-order inactivation kinetics during prolonged heat treatment, forming fully active but temperature-sensitive transient states. The highest enhancement to the thermostability of PGA was obtained using dextran-71000-dialdehyde modification, as a nearly ninefold increase at temperatures above 50 degrees C. The modification of PGA by dextran-11500-dialdehyde resulted in a considerable reduction of the Vm and Km parameters of the enzyme. However, other dextran dialdehyde derivatives used for modification did not cause a meaningful change in either Vm and Km. Modification by dextran dialdehyde derivatives did not result in significant change to either the optimal temperature or the activation energy of PGA. All modified PGA preparations showed lower inactivation rate constants but higher half-lives for inactivation than those of the native PGA at all temperatures studied. As indicated by the half-life times and Ki values, dextran 71000-dialdehyde was found to be more effective at cross-linking in the thermo-stabilization of PGA than any other agent studied in this work.

Some properties of glutamate dehydrogenase, glutamine synthetase and glutamate synthase from Corynebacterium callunae.

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GO-GAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP+ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for alpha-ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP(+)-GDH activity (deamination). The results showed that NADPH-GDH and NADP(+)-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn+2 while the biosynthetic activity of the enzyme was dependent on Mg2+ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the Km values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5 x 10(-3) mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required alpha-ketoglutarate and glutamine as substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae.

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent Km values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent Km values were 0.1 mM for alpha-ketoglutarate and 0.22 mM for glutamine.