Engineering of aniline dioxygenase for bioremediation and industrial applications

Ph.DNUS-UIUC JOINT PH.D. PROGRAMM

A coupled chlorinase-fluorinase system with high efficiency of trans-halogenation and a shared substrate tolerance

Enzymatic trans-halogenation enables radiolabeling under mild and aqueous conditions, but rapid reactions are desired. We discovered two new S-adenosyl-L-methionine (SAM)-dependent chlorinases from soil bacteria and developed a coupled chlorinase-fluorinase system for highly improved trans-halogenation reactions. The chlorinase was for the first time demonstrated to tolerate the modification at the C-2 position of the adenine ring and act cooperatively with the fluorinase to accelerate the trans-halogenation of 5’-chlorodeoxy-2-ethynyladenosine (5’-ClDEA) to 5’-fluorodeoxy-2-ethynyladenosine (5’-FDEA). The acetylene group will enable the linkage with an azide tethered peptide via a “click” reaction. The coupled chlorinase-fluorinase system offers the prospect of developing rapid radiolabeling protocols under mild and aqueous conditions. Please click Additional Files below to see the full abstract

Directed evolution and predictive modelling of galactose oxidase towards bulky benzylic and unactivated secondary alcohols

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Precision in medicinal chemistry: Harnessing enzymes for advanced halogenation

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Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Engineering of Aniline Dioxygenase for Bioremediation and Industrial Applications

202 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007.Another round of saturation mutagenesis on active site residues with 1-K31 as parent followed by random mutagenesis yielded the mutant 3-R21. Whole cell activity of 3-R21 for AN, 24DMA and 2IPA were 27.7, 9.8, and 12 nmol/min/mg protein respectively. The activities of 3-R21 for AN, 24DMA and 2IPA were improved by 8.9, 98.0, and 2.0-fold respectively over its parent 1-K31. In particular, the activity of the final mutant 3-R21 was improved by 3.5-fold over the WT AtdA enzyme. Overall, mutant 3-R21 had three mutations---V205A, 1248L, and S404C.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

A Spirobicyclo[3.1.0] Terpene from the Investigation of Sesquiter-pene Synthases from Lactarius deliciosus

Milk cap mushrooms in the genus Lactarius are known to produce a wide variety of terpene natural products. However, their repertoire of terpene biosynthetic enzymes has not been fully explored. In this study, several candidate sesquiterpene synthases were identified from the genome of the saffron milk cap mushroom L. deliciosus, and expressed in a sesquiterpene-overproducing Escherichia coli strain. In addition to enzymes that produce several known terpenes, we identified an enzyme belonging to a previously unknown clade of sesquiterpene synthases that produces a terpene with a unique spirotricyclic scaffold. These findings further add to the rich diversity of terpene scaffolds and mushroom terpene synthases and are valuable for biotechnological applications in producing these terpenoids