Partial purification and characterisation of two actinomycete tyrosinases and their application in cross-linking reactions

Actinomycetes are a ubiquitous group of bacteria, and are hypothesised to produce tyrosinases for pro-tection against the potential toxic effect of phenolic compounds and for the production of melanin. In thisstudy, tyrosinase production by Streptomyces pharetrae CZA14T(CZA14Tyr) and Streptomyces polyantibi-oticus SPRT(SPRTyr) was optimised. The enzymes were partially purified and biochemically characterisedto determine their suitability for industrial applications. SPRTyr was stable up to 40◦C and at pH 4.5–10.0,while CZA14Tyr was stable up to 40◦C and at pH 6.5–10.0. The enzymes showed variable stability in thepresence of water-miscible organic solvents and were able to oxidize l-DOPA in the presence of these sol-vents. A limited inhibitory effect was observed with arbutin, EDTA, sodium chloride and sodium dodecylsulphate, while both enzymes were strongly inhibited by the reducing agents used in this study. Inhibi-tion of enzyme activity was observed in the presence of 1 mM Cu2+and 5 mM Co2+for SPRTyr, and 5 mMFe2+and 5 mM Zn2+for CZA14Tyr. When applied in various cross-linking reactions both tyrosinases wereable to cross-link casein and gelatine in the absence of a phenolic compound, showing potential forapplication in the food industry and for the production of biomaterials.National Research Foundation (NRF) of South Africa for project funding [Grant No. 73691] and Cape Peninsula University of Technology (CPUT) University Research Funding.http://www.elsevier.com/locate/molcatb2016-12-31hb201

An unusual feruloyl esterase belonging to family VIII esterases and displaying a broad substrate range

A thermophilic compost metagenomic library constructed in Escherichia coli was functionally screenedfor novel esterases. Of the 110,592 fosmid clones screened, 25 clones demonstrated degradative activ-ity on glyceryl tributyrate (a hit rate of 1:4,423). Four clones displayed ferulic acid esterase activityand were sequenced using 454 Titanium sequencing technology. EstG34, a 410 amino acid protein, wasidentified as having high sequence identity with a number of bacterial -lactamases. EstG34 has theS-X-X-K motif which is conserved in class C -lactamases and family VIII carboxylesterases. Purifiedrecombinant EstG34 had a molecular mass of 42 kDa and displayed hydrolytic activity towards a vari-ety of p-nitrophenyl esters, hydroxycinnamic acid esters and -naphthol acetate. EstG34 represents thefirst family VIII carboxylesterase and -lactamase fold enzyme, able to hydrolyse ferulate and a numberof other hydroxycinnamic acid esters. In addition, EstG34 is the first reported FAE to not adopt the / hydrolase conformation. The sequence similarity and wide substrate utilization capability of this esterasecomplicates its placement within current classification systems, but also draws attention to the enzyme’spotential versatility.The National Research Foundation (NRF) and the Technology Innovation Agency (TIA), South Africa(Project number PB99/08).http://www.elsevier.com/locate/molcatb2016-08-31hb201

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Next generation sequencing improves detection of drug resistance mutations in infants after PMTCT failure.

BackgroundNext generation sequencing (NGS) allows the detection of minor variant HIV drug resistance mutations (DRMs). However data from new NGS platforms after Prevention-of-Mother-to-Child-Transmission (PMTCT) regimen failure are limited.ObjectiveTo compare major and minor variant HIV DRMs with Illumina MiSeq and Life Technologies Ion Personal Genome Machine (PGM) in infants infected despite a PMTCT regimen.Study designWe conducted a cross-sectional study of NGS for detecting DRMs in infants infected despite a zidovudine (AZT) and Nevirapine (NVP) regimen, before initiation of combination antiretroviral therapy. Sequencing was performed on PCR products from plasma samples on PGM and MiSeq platforms. Bioinformatic analyses were undertaken using a codon-aware version of the Smith-Waterman mapping algorithm and a mixture multinomial error filtering statistical model.ResultsOf 15 infants, tested at a median age of 3.4 months after birth, 2 (13%) had non-nucleoside reverse transcriptase inhibitor (NNRTI) DRMs (K103N and Y181C) by bulk sequencing, whereas PGM detected 4 (26%) and MiSeq 5 (30%). NGS enabled the detection of additional minor variant DRMs in the infant with K103N. Coverage and instrument quality scores were higher with MiSeq, increasing the confidence of minor variant calls.ConclusionsNGS followed by bioinformatic analyses detected multiple minor variant DRMs in HIV-1 RT among infants where PMTCT failed. The high coverage of MiSeq and high read quality improved the confidence of identified DRMs and may make this platform ideal for minor variant detection

An unusual feruloyl esterase belonging to family VIII esterases and displaying a broad substrate range

A thermophilic compost metagenomic library constructed in Escherichia coli was functionally screenedfor novel esterases. Of the 110,592 fosmid clones screened, 25 clones demonstrated degradative activ-ity on glyceryl tributyrate (a hit rate of 1:4,423). Four clones displayed ferulic acid esterase activityand were sequenced using 454 Titanium sequencing technology. EstG34, a 410 amino acid protein, wasidentified as having high sequence identity with a number of bacterial -lactamases. EstG34 has theS-X-X-K motif which is conserved in class C -lactamases and family VIII carboxylesterases. Purifiedrecombinant EstG34 had a molecular mass of 42 kDa and displayed hydrolytic activity towards a vari-ety of p-nitrophenyl esters, hydroxycinnamic acid esters and -naphthol acetate. EstG34 represents thefirst family VIII carboxylesterase and -lactamase fold enzyme, able to hydrolyse ferulate and a numberof other hydroxycinnamic acid esters. In addition, EstG34 is the first reported FAE to not adopt the / hydrolase conformation. The sequence similarity and wide substrate utilization capability of this esterasecomplicates its placement within current classification systems, but also draws attention to the enzyme’spotential versatility.The National Research Foundation (NRF) and the Technology Innovation Agency (TIA), South Africa(Project number PB99/08).http://www.elsevier.com/locate/molcatb2016-08-31hb201