Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6–12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d /l -, α/β-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.     

A New Stereospecific Synthesis of 1,2,4-Trideoxy-1,4-Imino-D-Erythro-Pentitol

ABSTRACT

1,2,4-Trideoxy-1,4-imino-D-erythro-pentitol [(2R,3S)-3-hydroxy-2-hydroxyme-thylpyrrolidine] (4) was synthesised from 2,5-di-O-tosyl-D-ribono-1,4-lactone in 42% overall yield. The key steps were deoxygenation at C-2 and a stereospecific inversion of the configuration at C-4. Compound 4 inhibited α-D-glucosidase (K_i = 25 μM) and β-D-glucosidase (K_i = 80 μM).     

Validating state-dependent queueing models for uninterrupted traffic flows using simulation

In this paper, the effectiveness of state-dependent queueing models for analyzing traffic flows is tested by comparing the speeds generated by the queueing models with the ones obtained by simulation. Simulation is thus used to evaluate speeds generated by the different queueing models. Different state-dependency functions are described and their performance is assessed. An M/G/1 queueing model with Gaussian state-dependency outperforms all other state-dependent queueing models. Different test results and insights are provided. Received: July 2004, Revised: September 2005 AMS classification: 60K25, 60K30     

Analysis and Evaluation of a Voice-Training Program in Future Professional Voice Users

The goal of this study is to analyze and evaluate the effectiveness of a voice-training program. Twenty-three professional voice users received voice training for 2 years and vocal hygiene education for 1 year. The voice-training program consisted of lectures, technical workshops, and vocal coaching. The European Laryngological Society (ELS) protocol, including the Dysphonia Severity Index (DSI) and the Voice Handicap Index (VHI), was applied before and after, respectively, 9 and 18 months of voice training. A questionnaire on daily habits was presented at study onset and after 18 months. The DSI improvement is more significant after 9 months (P=0.005) than it is after 18 months (P=0.2). On the other hand, the perceptual evaluation remained unchanged after 9 months, whereas it improved significantly after 18 months. The results of the daily habit questionnaire are disturbing: the prevalence of smoking, vocal abuse, stress, and late meals were not influenced by the lectures and remained high. This study emphasizes the need for a well-organized voice-training program that is most effective after 9 months. Regarding the low effectiveness of the vocal hygiene program, the concept needs revision.     

Onsite Enzyme Production During Bioethanol Production from Biomass: Screening for Suitable Fungal Strains

Cellulosic ethanol production from biomass raw materials involves process steps such as pre-treatment, enzymatic hydrolysis, fermentation, and distillation. Use of streams within cellulosic ethanol production was explored for onsite enzyme production as part of a biorefinery concept. Sixty-four fungal isolates were in plate assays screened for lignocellulolytic activities to discover the most suitable fungal strain with efficient hydrolytic enzymes for lignocellulose conversion. Twenty-five were selected for further enzyme activity studies using a stream derived from the bioethanol process. The filter cake left after hydrolysis and fermentation was chosen as substrate for enzyme production. Five of the 25 isolates were further selected for synergy studies with commercial enzymes, Celluclast 1.5L and Novozym 188. Finally, IBT25747 (Aspergillus niger) and strain AP (CBS 127449, Aspergillus saccharolyticus) were found as promising candidates for onsite enzyme production where the filter cake was inoculated with the respective fungus and in combination with Celluclast 1.5L used for hydrolysis of pre-treated biomass.     

Normative Voice Range Profiles of Male and Female Professional Voice Users

Normative voice range profiles (VRP) are calculated for a group of male and female teachers, based on 43 and 46 recordings, respectively. All individuals had healthy voices. These normative VRPs contain 95% prediction intervals for both frequency and intensity. They are based on a series of mathematical transformations of the original individual VRPs in order to maintain in the normative VRPs the typical oval VRP shape, including the dip between modal and falsetto register. The normative VRPs presented are directly applicable in the clinical practice of otolaryngologists and speech-language pathologists.     

Test-retest study of the GRBAS scale: Influence of experience and professional background on perceptual rating of voice quality

Perceptual evaluation was performed by means of the GRBAS scalefor a series of nine pathological voice samples that were presented twice to a group of 23 judges, consisting of experienced and inexperienced otolaryngologists (ENTs) and speech-language pathologists (S/LPs). The time interval between ratings was 14 days. Test-retest reliability was moderate. The best agreement between the observers was obtained for the G (grade) parameter and the worst for the S (strained) parameter. Considering the medians of the GRBAS ratings, no significant influence was measured for level of experience or professional background. Yet, based on means, the results show that professional background has a greater impact on perceptual rating than experience.