Allegretti, C

Armenia, I

Balzaretti, R

Bernardini, G

D'Arrigo, P

Gornati, R

Piubelli, L

Pollegioni, L

Rosini, E

Tessaro, D

Valentino, M

ALLEGRETTI, CHIARA

TESSARO, DAVIDE

D'ARRIGO, PAOLA

Archivio istituzionale della ricerca - Politecnico di Milano

(1)Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Piazza L. Da Vinci 32, Milano, Italy(2)Department of Biotechnology and Life Sciences, Università degli Studi dell’Insubria, via J.H. Dunant 3, Varese, Italy(3)The Protein Factory, Politecnico di Milano and Università degli Studi dell’Insubria, via Mancinelli 7, Milano, Italy(4)ICRM, CNR, Via M. Bianco 9, Milano, ItalyChiara Allegretti(1), Ilaria Armenia(2), Riccardo Balzaretti(2), Luciano Piubelli(2,3), Elena Rosini(2,3), Davide Tessaro(1,3), Mattia Valentino(3,4), Rosalba Gornati(2,3), Giovanni Bernardini(2,3), Loredano Pollegioni(2,3), Paola D'Arrigo(1,3,4)Towards the production of an industrial amino acid oxidase: stabilization of a thermophilic L‐aspartate oxidaseSeptember, 22nd – 23rd Contact: chiara.allegretti@polimi.itAcknowledgment:Pavia.IntroductionImmobilization as CLEAImmobilization on Relizyme™ HA403/SRL‐Aspartate oxidase is a FAD containing enzyme, catalyzing the stereospecific oxidative deamination of L‐aspartic acid to oxalacetate, ammonia and hydrogen peroxide.This flavoenzyme from the thermophilic archea Sulfolobus tokodaii (StLASPO) was efficiently produced as a recombinant protein in E. coli and has been deeply characterized.[1] StLASPO presents very distinctive features such as: high thermostability,  stable activity over a broad range of pH values,  low Km for dioxygen and  tight binding of the FAD cofactor, making it very attractive for biotechnological applications . Immobilization studies of this novel biocatalyst on different solid supports such as resins or in the form of cross‐linked enzyme aggregates (CLEA) were performed.[2]A recently developed approach is represented by the immobilization of a flavooxidase on magnetic Fe3O4 nanoparticles (NP@RgDAAO)[3], which now paves the way to theimmobilization of StLASPO.Immobilization on Fe3O4 NPsEffect of pH ‐ 150 mg of Fe3O4 NP‐ 5 mL APTES (2% v/v) in H2Oleft under stirring for 5 h at 50 °C.Fe3O4NP‐APTES were then separatedfrom unbound APTES by using a magnet .‐ 4 mg of Fe3O4NP@APTES‐ 2 mg of EDC‐ 3 mg of NHS‐ 0.037 U of StLASPOThe reaction was carried out for 2 h at r.t.‐Fe3O4NP@APTES‐StLASPO (0.2699 U on 16 mg NP)‐100 µL of 50 mM L‐Aspartate in water atdifferent pH values (8, 9, 10, 11)‐100 µL of water (adjusted to the desired pHwith NaOH 0.5 M)‐70 °C on a thermomixer (600 rpm)Effect of temperature‐Fe3O4NP@APTES‐StLASPO (0.322 U on 16 mg NP) ‐100 µL of a 50 mM L‐Aspartate solution at atpH 10‐100 µL of water (adjusted at pH 10 with NaOH0.5 M)These reactions were conducted at different temperatures (25, 37, 50, 60, 70, 80 °C) on a thermomixer (600 rpm) .[1] D. Bifulco, L. Pollegioni, D. Tessaro, S. Servi, G. Molla Appl. Microbiol. Biotechnol. 97 (2013) 7285‐7295. [2] P. D'Arrigo, C. Allegretti, A. Fiorati, L. Piubelli, E. Rosini, D. Tessaro, M. Valentino, L. Pollegioni Catal. Sci. Technol. 5 (2015) 1106‐1114.[3] A. Bava, R. Gornati, F. Cappellini, L. Caldinelli, L. Pollegioni, G. Bernardini Nanomed. (Lond.) 11 (2013) 1797‐1806.Conclusions‐ 200 mg of resin HA 403/ S R‐ 10 mL of 0.125% (v/v) glutaraldehydesolution in water‐ 1 mL StLASPO solution (2 U)‐ 0.1 M phosphate buffer, pH 7.5Best Result:Resolution of 50 mM D,L‐Aspartate at pH 10:‐ 0.5 mL of D,L‐Aspartate solution at pH 10‐ 2 U of immobilized StLASPO‐ 70 °C in a thermomixer (600 rpm)Cycle number Time (h) e.e. (%)1 1 > 99.52 1 > 99.53 1 > 99.54 1 872 > 99.55 1 772 > 99.5‐ 4.5 mL (NH4)2SO4 precipitating solution areadded to 0.5 mL of enzyme solution‐ 0.4 % (v/v glutaraldehyde solution/totalprecipitation solution)The mixture was incubated on a rotaryshaker for 2.5 h at room temperatureBest Result: Cycle number Time (h) e.e. (%)1 4 > 99.52 4 > 99.53 4 > 99.54 4 686 > 99.55 4 696 95Resolution of 50 mM D,L‐Aspartate at pH 10:‐ 0.5 mL of D,L‐Aspartate solution at pH 10‐ CLEA‐StLASPO preparation (1.1U)‐ 70 °C in a thermomixer (600 rpm)Immobilization on SEPABEADS® EC‐EP/S‐ 0.5 mL (1 U) StLASPO‐ 60 mg of SEPABEADS ® EC‐EP/S‐ 0.3 mL 1.25 M potassium phosphate buffer at pH 8The mixture was incubated for 18 h at 25  CBest Result:Resolution of 50 mM D,L‐Aspartate at pH 10 :‐ 0.5 mL of D,L Aspartate solution at pH 10‐ 0.5 U of immobilized StLASPO‐ 70 °C in a thermomixer (600 rpm)Cycle number Time (h) e.e. (%)1 4 > 99.52 4 > 99.53 4 > 99.54 4 816 > 99.55 4 4510 > 95 Resin HA 403/SR presents a high yield of immobilization; moreoverthe resulting catalyst can be used for 5 cycles yielding completeconversion in < 2 hours. The CLEA‐StLASPO preparation maintains its activity for 5 cycles, withcomplete conversion in < 8 hours. The immobilization of StLASPO on magnetic NPs presents verypromising results in terms of stability and potential reusability of thebiocatalyst. The recovery of the reaction products and of theimmobilized enzyme NP@StLASPO is very simple. Indeed, the removalof the catalyst from the reaction medium can be easily performed witha magnet; this is a very attracting aspect helpful for the scaling up of thebioconversion.Immobilization yield : 100 % (2 U)The preparation showed an activity of 10 U/g wet matrix.Immobilization yield: 50% (0.5 U)The preparation showed an activity of  8.3 U/g dry matrix.Immobilization yield : 100%The preparation showed an activity of 4 U/g wet matrixImmobilization yield: 95% (0.035 U)

Towards the production of an industrial amino acid oxidase: stabilization of a thermophilic L-aspartate oxidase

https://re.public.polimi.it/retrieve/handle/11311/964539/50905/Allegretti.pdf

Towards the production of an industrial amino acid oxidase: stabilization of a thermophilic L-aspartate oxidase

Abstract

Similar works

Full text

Available Versions

Archivio istituzionale della ricerca - Politecnico di Milano