In-situ crosslinking of Aspergillus flavus lipase: improvement of activity, stability and properties

The mycelium-bound lipase of Aspergillus flavus was stabilised by cross-linkage using glutaraldehyde or methylglyoxal. Lipase activity was enhanced by up to 48% by treatment with methylglyoxal but not by glutaraldehyde. Cross-linking with methylglyoxal increased the thermal stability of the lipase by 58% at 50°C

Enzymatic hydrolysis of palm olein with mycelium-bound lipase of Aspergillus flavus link

Hydrolysis of palm olein was studied using mycelium-bound lipase of Aspergillus flavus Link. The fatty acid composition, triacylglycerol profile and melting properties of the palm olein before and after 72 h hydrolysis were compared. A slight decrease of palmitic acid and increase in oleic acid and linolenic acid concentrations in palm olein was noted. The relative concentration of triunsaturated triacylglycerol, low melting glycerides, such as trioleoyl glycerol, oleoyl-dilinoleoyl glycerol and dioleoyl-linoleoyl glycerol of modified palm olein was increased while the relative concentration of high melting glycerides e.g. dipalmitoyl-oleoyl glycerol and palmitoyl-oleoyl-steroyl glycerol was decreased except for tripalmitoyl glycerol. The melting range of modified palm olein tends to be broad, that is it starts melting (X1) at -28°C and totally melted (X2) at 45°C

Substrate preference of mycelium-bound lipase from a strain of Aspergillus flavus Link

Aspergillus flavus mycelium-bound lipase demonstrates high preference towards short chain triacylglycerols and discriminates against triunsaturated triacylglycerols e.g. triolein. The great discriminating power of its lipase against triolein was shown in comparison with its ability to catalyse the hydrolysis of shorter chain triacylglycerols e.g. tricaprin and less was shown when hydrolysing tripalmitin. A similar phenomenon was noted when the mycelium-bound lipase was used to catalyse the reaction of coconut oil with palmitic acid or oleic acid in n-hexane. The relative percentages of octanoic acid and decanoic acid of coconut oil remaining after 20 h reaction were much less than those of the medium, long and unsaturated chain fatty acids suggesting that short chain fatty acids are preferred. The lipase hydrolyses coconut oil faster than palm olein followed by corn oil, rapeseed oil, soy bean oil and cottonseed oil. This indicates that A. flavus lipase has preference for oils containing saturated fatty acids rather than unsaturated fatty acids

Physiochemical properties and specificity of mycelium-bound lipase from a locally isolated strain of Aspergillus flavus link

The properties of mycelium-bound lipase of Aspergillus flavus Link obtained after 3 days growth in palm olein were studied. The mycelium-bound lipase has an optimum activity at a temperature range of 50–55°C and once extracted, the activity was optimum at a temperature range of 25–35 °C. The extracted lipase was stable at alkaline pH and heat labile, thus lost 14% of its activity after being exposed to 30°C for 4 h. Ca2+ enhanced the lipase activity while EDTA (1 mM) had no effect. The enzyme hydrolysed coconut oil faster than other vegetable oils and tributyrin was not hydrolysed by the extracted lipase. The apparent Km values obtained for mycelium-bound lipase (11.76 mg/mL) was three times higher than the extracted lipase (3.92 mg/mL) suggesting that there was a diffusion limitation of the substrate in reaching the bound lipase. The extracted lipase displayed 1,3 positional specificity

Mycelium-bound lipase from a locally isolated strain of Aspergillus flavus link: pattern and factors involved in its production

Aspergillus flavus produces a lipase (EC 3.1.1.3) which is partly bound to the mycelium during growth. The production of the mycelium-bound lipase is concomitant with growth, and declines when growth ceases. Maximum productivity of the enzyme is obtained when the culture is incubated at 30°C, an initial culture pH of 6·5 and with 2% (w/v) each of corn oil and yeast extract as carbon and organic nitrogen source. Yeast extract affects not only the production of lipase but also the secretion of proteases into the culture medium. Production of the latter enzymes, which inactivate the free lipase, is enhanced by adding yeast extract (1–2%, w/v) to the culture medium. However, at 5% (w/v) yeast extract concentration, proteolytic activity is not detected and consequently, the activity of free lipase may easily be measured. Free lipase activity can easily be detected when 0·001 mol dm−3 EDTA is added to the culture medium. The presence of the chelating agent enhances the production and maintains the stability of the extracted mycelium-bound lipase

Enzymatic hydrolysis of palm olein with mycelium-bound lipase of Aspergillus flavus Link (Hydrolysis minyak olein menggunakan lipase terikat miselium daripada Aspergillus flavus Link)

Abstrak Hidrolisis minyak olein menggunakan lipase-terikat miselium daripada Aspergillus flavus Link telah dikaji. Komposisi asid lemak, profil triasigliserol dan sifat lebur minyak olein sebelum dan selepas 72 jam tindak balas dibandingkan. Kepekatan asid palmitik didapati menurun sedikit diikuti dengan pertambahan asid oleik dan asid linolenik pada minyak tersebut. Kepekatan bandingan bagi triasigliserol tri-tak tepu, minyak olein terubahsuai yang mempunyai takat lebur rendah, seperti trioleoil gliserol, oleoil-dilinoleiol gliserol dan dioleoil oleoil gliserol, didapati meningkat, manakala kepekatan triasigliserol yang mempunyai takat lebur tinggi seperti dipalmitoil-oleoil gliserol dan palmitoil-oleoil steroil gliserol berkurangan kecuali tripalmitoil gliserol. Julat lebur bagi minyak olein terubah suai selepas tindak balas didapati menjadi lebar, iaitu apabila minyak mula lebur (X 1 ) pada suhu -28 °C dan lebur keseluruhannya (X 2 ) pada suhu 45 °C. Abstract Hydrolysis of palm olein was studied using mycelium-bound lipase of Aspergillus flavus Link. The fatty acid composition, triacylglycerol profile and melting properties of the palm olein before and after 72 h hydrolysis were compared. A slight decrease of palmitic acid and increase in oleic acid and linolenic acid concentrations in palm olein was noted. The relative concentration of triunsaturated triacylglycerol, low melting glycerides, such as trioleoyl glycerol, oleoyl-dilinoleoyl glycerol and dioleoyl-linoleoyl glycerol of modified palm olein was increased while the relative concentration of high melting glycerides e.g. dipalmitoyl-oleoyl glycerol and palmitoyl-oleoyl-steroyl glycerol was decreased except for tripalmitoyl glycerol. The melting range of modified palm olein tends to be broad, that is it starts melting (X 1 ) at -28 °C and totally melted (X 2 ) at 45 °C

Combined point of care nucleic acid and antibody testing for SARS-CoV-2 following emergence of D614G Spike Variant

Rapid COVID-19 diagnosis in hospital is essential, though complicated by 30-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant now dominates the pandemic and it is unclear how serological tests designed to detect anti-Spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95CI 57.8-92.9%) by rapid NAAT alone. Combined point of care antibody test and rapid NAAT is not impacted by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity

Challenges and opportunities for conducting a vaccine trial during the COVID-19 pandemic in the United Kingdom

The COVID-19 pandemic has resulted in unprecedented challenges for healthcare systems worldwide. It has also stimulated research in a wide range of areas including rapid diagnostics, novel therapeutics, use of technology to track patients and vaccine development. Here, we describe our experience of rapidly setting up and delivering a novel COVID-19 vaccine trial, using clinical and research staff and facilities in three National Health Service Trusts in Cambridgeshire, United Kingdom. We encountered and overcame a number of challenges including differences in organisational structures, research facilities available, staff experience and skills, information technology and communications infrastructure, and research training and assessment procedures. We overcame these by setting up a project team that included key members from all three organisations that met at least daily by teleconference. This group together worked to identify the best practices and procedures and to harmonise and cascade these to the wider trial team. This enabled us to set up the trial within 25 days and to recruit and vaccinate the participants within a further 23 days. The lessons learned from our experiences could be used to inform the conduct of clinical trials during a future infectious disease pandemic or public health emergency

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission

Funder: Addenbrooke's Charitable Trust, Cambridge University Hospitals; FundRef: http://dx.doi.org/10.13039/501100002927Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3 week period (April 2020), 1032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in the asymptomatic screening group tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (COVID-19)>7 days prior to testing, most self-isolating, returning well. Clusters of HCW infection were discovered on two independent wards. Viral genome sequencing showed that the majority of HCWs had the dominant lineage B∙1. Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms. This approach will be critical for protecting patients and hospital staff

Complement lectin pathway activation is associated with COVID-19 disease severity, independent of MBL2 genotype subgroups

IntroductionWhile complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood.MethodsWe therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome.ResultsWe show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p<0.0001) and COVID-19 severity. Despite this, genetic variations in MBL2 are not associated with susceptibility to SARS-CoV-2 infection or disease outcomes such as mortality and the development of Long COVID.ConclusionIn conclusion, activation of the MBL-LP only plays a minor role in COVID-19 pathogenesis, since no clinically meaningful, consistent associations with disease outcomes were noted