Optimising biocatalyst design for obtaining high transesterification activity by α-chymotrypsin in non-aqueous media

<p>Abstract</p> <p>Background</p> <p>Enzymes are often used in organic solvents for catalyzing organic synthesis. Two enzyme preparations, EPRP (enzyme precipitated and rinsed with n-propanol) and PCMC (protein coated microcrystals) show much higher activities than lyophilized powders in such systems. Both preparations involve precipitation by an organic solvent. The clear understanding of why these preparations show higher catalytic activity than lyophilized powders in organic solvents is not available.</p> <p>Results</p> <p>It was found that EPRPs of α-chymotrypsin prepared by precipitation with <it>n</it>-propanol in the presence of trehalose contained substantial amount of trehalose (even though trehalose alone at these lower concentrations was not precipitated by <it>n</it>-propanol). The presence of trehalose in these EPRPs resulted in much higher transesterification rates (45.2 nmoles mg^-1min^-1) as compared with EPRPs prepared in the absence of trehalose (16.6 nmoles mg^-1min^-1) in octane. Both kinds of EPRPs gave similar initial transesterification rates in acetonitrile. Use of higher concentrations of trehalose (when trehalose alone also precipitates out), resulted in the formation of PCMCs, which showed higher transesterification rates in both octane and acetonitrile. SEM analysis showed the relative sizes of various preparations. Presence of trehalose resulted in EPRPs of smaller sizes.</p> <p>Conclusion</p> <p>The two different forms of enzymes (EPRP and PCMC) known to show higher activity in organic solvents were found to be different only in the way the low molecular weight additive was present along with the protein. Therefore, the enhancement in the transesterification activity in EPRPs prepared in the presence of trehalose was due to: (a) better retention of essential water layer for catalysis due to the presence of the sugar. This effect disappeared where the reaction media was polar as the polar solvent (acetonitrile) is more effective in stripping off the water from the enzyme; (b) reduction in particle size as revealed by SEM. In the case of PCMC, the enhancement in the initial rates was due to an increase in the surface area of the biocatalyst since protein is coated over the core material (trehalose or salt).</p> <p>It is hoped that the insight gained in this work would help in a better understanding for designing high activity biocatalyst preparation of non-aqueous media.</p

Enhancement of lipase activity in non-aqueous media upon immobilization on multi-walled carbon nanotubes

<p>Abstract</p> <p>Background</p> <p>Immobilization of biologically active proteins on nanosized surfaces is a key process in bionanofabrication. Carbon nanotubes with their high surface areas, as well as useful electronic, thermal and mechanical properties, constitute important building blocks in the fabrication of novel functional materials.</p> <p>Results</p> <p>Lipases from <it>Candida rugosa </it>(CRL) were found to be adsorbed on the multiwalled carbon nanotubes with very high retention of their biological activity (97%). The immobilized biocatalyst showed 2.2- and 14-fold increases in the initial rates of transesterification activity in nearly anhydrous hexane and water immiscible ionic liquid [Bmim] [PF6] respectively, as compared to the lyophilized powdered enzyme. It is presumed that the interaction with the hydrophobic surface of the nanotubes resulted in conformational changes leading to the 'open lid' structure of CRL. The immobilized enzyme was found to give 64% conversion over 24 h (as opposed to 14% with free enzyme) in the formation of butylbutyrate in nearly anhydrous hexane. Similarly, with ionic liquid [Bmim] [PF6], the immobilized enzyme allowed 71% conversion as compared to 16% with the free enzyme. The immobilized lipase also showed high enantioselectivity as determined by kinetic resolution of (±) 1-phenylethanol in [Bmim] [PF6]. While free CRL gave only 5% conversion after 36 h, the immobilized enzyme resulted in 37% conversion with > 99% enantiomeric excess. TEM studies on the immobilized biocatalyst showed that the enzyme is attached to the multiwalled nanotubes.</p> <p>Conclusion</p> <p>Successful immobilization of enzymes on nanosized carriers could pave the way for reduced reactor volumes required for biotransformations, as well as having a use in the construction of miniaturized biosensensor devices.</p

Exploiting open source 3D printer architecture for laboratory robotics to automate high-throughput time-lapse imaging for analytical microbiology

Growth in open-source hardware designs combined with the low-cost of high performance optoelectronic and robotics components has supported a resurgence of in-house custom lab equipment development. We describe a low cost (below USD700), open-source, fully customizable high-throughput imaging system for analytical microbiology applications. The system comprises a Raspberry Pi camera mounted on an aluminium extrusion frame with 3D-printed joints controlled by an Arduino microcontroller running open-source Repetier Host Firmware. The camera position is controlled by simple G-code scripts supplied from a Raspberry Pi singleboard computer and allow customized time-lapse imaging of microdevices over a large imaging area. Open-source OctoPrint software allows remote access and control. This simple yet effective design allows high-throughput microbiology testing in multiple formats including formats for bacterial motility, colony growth, microtitre plates and microfluidic devices termed ‘lab-on-a-comb’ to screen the effects of different culture media components and antibiotics on bacterial growth. The open-source robot design allows customization of the size of the imaging area; the current design has an imaging area of ~420 × 300mm, which allows 29 ‘lab-on-a-comb’ devices to be imaged which is equivalent 3480 individual 1μl samples. The system can also be modified for fluorescence detection using LED and emission filters embedded on the PiCam for more sensitive detection of bacterial growth using fluorescent dyes

18F-Fluciclovine PET/CT performance in biochemical recurrence of prostate cancer: a systematic review

Background A systematic literature review of the performance of 18Fluorine-fluciclovine PET/CT for imaging of men with recurrent prostate cancer was performed. Methods Scientific literature databases (MEDLINE, ScienceDirect and Cochrane Libraries) were searched systematically during Oct 2020 using PRISMA criteria. No limit was put on the date of publication. Prospective studies reporting a patient-level 18F-fluciclovine detection rate (DR) from ≥25 patients with recurrent prostate cancer were sought. Proceedings of relevant meetings held from 2018 through Oct 2020 were searched for abstracts meeting criteria. Results Searches identified 321 unique articles. In total, nine articles (six papers and three conference abstracts), comprising a total of 850 patients met inclusion criteria. Most studies (n = 6) relied on ASTRO-Phoenix Criteria, EAU-ESTRO-SIOG, and/or ASTRO-AUA guidelines to identify patients with biochemical recurrence. Patients’ PSA levels ranged from 0.02–301.7 ng/mL (median level per study, 0.34–4.10 ng/mL [n = 8]). Approximately 64% of patients had undergone prostatectomy, but three studies focused solely on post-prostatectomy patients. Adherence to imaging protocol guidelines was heterogeneous, with variance seen in administered activity, uptake and scan times. Overall patient-level DR varied between studies from 26% to 83%, with 78% of studies reporting a DR > 50%. DR was proportional to PSA, but even at PSA < 0.5 ng/mL DR of up to 53% were reported. Prostate/bed DR (n = 7) ranged from 18% to 78% and extra-prostatic rates (n = 6) from 8% to 72%. Pelvic node and bone lesion DR ranged from 8% to 47% and 0% to 26%, respectively (n = 5). 18F-Fluciclovine PET/CT was shown to impact patient management and outcomes. Two studies reported 59–63% of patients to have a management change post-scan. A further study showed significant increase in failure-free survival following 18F-fluciclovine-guided compared with conventional imaging-guided radiotherapy planning. Conclusions 18F-Fluciclovine PET/CT shows good performance in patients with recurrent prostate cancer leading to measurable clinical benefits. Careful adherence to recommended imaging protocols may help optimize DR

Methods of probing the interactions between small molecules and disordered proteins

It is generally recognized that a large fraction of the human proteome is made up of proteins that remain disordered in their native states. Despite the fact that such proteins play key biological roles and are involved in many major human diseases, they still represent challenging targets for drug discovery. A major bottleneck for the identification of compounds capable of interacting with these proteins and modulating their disease-promoting behaviour is the development of effective techniques to probe such interactions. The difficulties in carrying out binding measurements have resulted in a poor understanding of the mechanisms underlying these interactions. In order to facilitate further methodological advances, here we review the most commonly used techniques to probe three types of interactions involving small molecules: (1) those that disrupt functional interactions between disordered proteins; (2) those that inhibit the aberrant aggregation of disordered proteins, and (3) those that lead to binding disordered proteins in their monomeric states. In discussing these techniques, we also point out directions for future developments.Gabriella T. Heller is supported by the Gates Cambridge Trust Scholarship. Francesco A. Aprile is supported by a Senior Research Fellowship award from the Alzheimer’s Society, UK (grant number 317, AS-SF-16-003)

Approaches in biotechnological applications of natural polymers

Natural polymers, such as gums and mucilage, are biocompatible, cheap, easily available and non-toxic materials of native origin. These polymers are increasingly preferred over synthetic materials for industrial applications due to their intrinsic properties, as well as they are considered alternative sources of raw materials since they present characteristics of sustainability, biodegradability and biosafety. As definition, gums and mucilages are polysaccharides or complex carbohydrates consisting of one or more monosaccharides or their derivatives linked in bewildering variety of linkages and structures. Natural gums are considered polysaccharides naturally occurring in varieties of plant seeds and exudates, tree or shrub exudates, seaweed extracts, fungi, bacteria, and animal sources. Water-soluble gums, also known as hydrocolloids, are considered exudates and are pathological products; therefore, they do not form a part of cell wall. On the other hand, mucilages are part of cell and physiological products. It is important to highlight that gums represent the largest amounts of polymer materials derived from plants. Gums have enormously large and broad applications in both food and non-food industries, being commonly used as thickening, binding, emulsifying, suspending, stabilizing agents and matrices for drug release in pharmaceutical and cosmetic industries. In the food industry, their gelling properties and the ability to mold edible films and coatings are extensively studied. The use of gums depends on the intrinsic properties that they provide, often at costs below those of synthetic polymers. For upgrading the value of gums, they are being processed into various forms, including the most recent nanomaterials, for various biotechnological applications. Thus, the main natural polymers including galactomannans, cellulose, chitin, agar, carrageenan, alginate, cashew gum, pectin and starch, in addition to the current researches about them are reviewed in this article.. }To the Conselho Nacional de Desenvolvimento Cientfíico e Tecnológico (CNPq) for fellowships (LCBBC and MGCC) and the Coordenação de Aperfeiçoamento de Pessoal de Nvíel Superior (CAPES) (PBSA). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and COMPETE 2020 (POCI-01-0145-FEDER-006684) (JAT)

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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