Fong, Wing Ping

Tsang, Paul Wai Kei

Wong, Kin Sing

English

VTC Institutional Repository (Vocational Training Council)

POSTER PRESENTATION Open AccessEffects of an extra Trp113Tyr substitution onyeast D-amino acid oxidase variantKin-sing Wong1, Wing-ping Fong2, Paul Wai-kei Tsang3*From Practical Social and Industrial Research (PSIR) Symposium 2015Wan Chai, Hong Kong. 27 November 2015Backgroundb-Lactam antibiotics (cephalosporins and penicillins) areused in clinical practice to combat microbial infections.Commercial cephalosporins are all semi-synthetic andchemically derived from 7-aminocephalosporanic acid(7-ACA), a cephem nucleus that is traditionally producedby multi-step chemical reactions from cephalosporinC (CPC). However, the procedures are environmentallydamaging, costly, and low yield [1]. The increasing annualdemand for semi-synthetic cephalosporins and the con-cept of environmental sustainability have created a direneed to develop a ‘greener’ production of 7-ACA. 7-ACAcan be enzymatically produced from CPC by a two-stepprocess that involves D-amino acid oxidase (DAAO) andglutaryl-7-aminocephalosporanic acid (GL-7-ACA)acylase. Recently, we characterised the highly active andthermostable Trigonopsis variabilis DAAO (TvDAAO)variant F54Y for oxidative deamination of CPC. A singleamino acid substitution at the 54th position of TvDAAOfrom phenylalanine (F) to tyrosine (Y) resulted in a 6-foldimprovement in catalytic activity towards CPC and inthermostability after heat treatment at 55°C [2]. Thepotential application of the TvDAAO F54Y variant in theantibiotics industry as a simple and efficient biocatalystwas evaluated [3]. To further elucidate the structural basisof the catalytic activity and thermostability of TvDAAOF54Y, we investigated the role of the 113th amino acidresidue (tryptophan, W113) that locates near the substratebinding cleft.MethodsThe W113 (tryptophan) residue of the TvDAAO F54Yvariant was converted to tyrosine by the overlappingpolymerase chain reaction using mutagenic primers. Themutations in the resultant TvDAAO variant, TvDAAOF54Y/W113Y, were confirmed by automated DNAsequencing. The variant was expressed in Escherichia coliBL21(DE3)pLysS. E. coli cells were harvested by centrifu-gation (2,400 g, 15 min) at 4°C and lysed in ice-cold20 mM sodium phosphate (pH 7.0) supplemented with 1mM phenylmethylsulphonyl fluoride by sonication (tencycles, 15 sec each, at 15 sec intervals). The cell-freeextract was clarified by centrifugation (13,000 g, 30 min)at 4°C, filtered through a 0.20 μm nylon membrane, andpurified by a diethylaminoethyl-cellulose column and aHiLoad 16/60 Superdex 200 prep grade gel filtrationcolumn. Purity of the enzyme was analysed by sodiumdodecyl sulphate-polyacrylamide gel electrophoresis, andits concentration was determined by Bradford assay. Thecatalytic activity of the purified enzyme was determined bymeasuring the formation of GL-7-ACA at 22°C with agita-tion [3]. One mL of the reaction mixture contained 20 μgTvDAAO F54Y/W113Y variant, 5 mM cephalosporin C,and 50 mM sodium phosphate (pH 7.5). The amount ofGL-7-ACA liberated was quantified by high performanceliquid chromatography equipped with a C-18 reverse phasecolumn, using 6% acetonitrile in 50 mM potassium phos-phate (pH 7.5) as the mobile phase. Thermostability of thepurified enzyme was evaluated by measuring the residualcatalytic activity after heat treatment at 55°C for 30 min.ResultsThe TvDAAO F54Y/W113Y variant was prepared andpurified to homogeneity. Catalytic activity was increasedby ~10% with respect to the original TvDAAO F54Yvariant, but was deactivated by heat treatment.ConclusionsThe W113 of TvDAAO plays a pivotal role in thermaldeactivation. W113Y substitution may alter the local* Correspondence: paulwktsang@vtc.edu.hk3School of General Education and Languages, Technological and HigherEducation Institute of Hong Kong, Hong KongFull list of author information is available at the end of the articleWong et al. SpringerPlus 2015, 4(Suppl 2):P6http://www.springerplus.com/content/4/S2/P6a SpringerOpen Journal© 2015 Wong et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.spatial arrangement of the substrate binding cleft, andhence increase the vulnerability of the nearby oxidative-sensitive C108 [4], promoting deactivation of theenzyme at elevated temperatures.Authors’ details1Racing Laboratory, The Hong Kong Jockey Club, Hong Kong. 2School of LifeSciences, The Chinese University of Hong Kong, Hong Kong. 3School ofGeneral Education and Languages, Technological and Higher EducationInstitute of Hong Kong, Hong Kong.Published: 27 November 2015References1. Hamed RB, Gomez-Castellanos JR, Henry L, Ducho C, McDonough MA,Schofield CJ: The enzymes of β-lactam biosynthesis. Nat Prod Rep 2013,30(1):21-107.2. Wong KS, Fong WP, Tsang PW: A single Phe54Tyr substitution improvesthe catalytic activity and thermostability of Trigonopsis variabilis D-aminoacid oxidase. N Biotechnol 2010, 27(1):78-84.3. Wong KS, Fong WP, Tsang PWK: Entrapment of a Trigonopsis variabilis D-amino acid oxidase variant F54Y for oxidative deamination ofcephalosporin C. Eng Life Sci 2011, 11(5):491-495.4. Dib I, Slavica A, Riethorst W, Nidetzky B: Thermal inactivation of D-aminoacid oxidase from Trigonopsis variabilis occurs via three parallel paths ofirreversible denaturation. Biotechnol Bioeng 2006, 94(4):645-654.doi:10.1186/2193-1801-4-S2-P6Cite this article as: Wong et al.: Effects of an extra Trp113Tyrsubstitution on yeast D-amino acid oxidase variant. SpringerPlus 2015 4(Suppl 2):P6.Submit your manuscript to a journal and benefi t from:7 Convenient online submission7 Rigorous peer review7 Immediate publication on acceptance7 Open access: articles freely available online7 High visibility within the fi eld7 Retaining the copyright to your article    Submit your next manuscript at 7 springeropen.comWong et al. SpringerPlus 2015, 4(Suppl 2):P6http://www.springerplus.com/content/4/S2/P6Page 2 of 2

Effects of an extra Trp113Tyr substitution on yeast D-amino acid oxidase variant

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Effects of an extra Trp113Tyr substitution on yeast D-amino acid oxidase variant

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