Immobilization-stabilization of a complex multimeric sucrose synthase from Nitrosomonas europaea. Synthesis of UDP-glucose

Orrego, Alejandro H.; Trobo-Maseda, Lara; Rocha-Martín, Javier; Guisán, José M.

doi:10.1016/j.enzmictec.2017.06.008

Cited by 19 publications

(15 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Based on such thermal stability studies, we selected the Tt27-HBDH immobilised on AG-G for 3 h as the optimal heterogeneous biocatalyst for further studies. This heterogeneous biocatalyst was 476 times more stable than its soluble counterpart under high temperature and acidic pH, suggesting a rigidification of the enzyme tertiary structure as observed for other multimeric enzymes immobilized on AG-G. 22,57,58 Unfortunately, this immobilization protocol failed to stabilize the hexameric structure of Tt27-HBDH as some subunits were still released after incubating the immobilised enzyme under denaturing conditions (Fig. S6 †).…”

Section: Stability Of Immobilised Tt27-hbdh Preparationsmentioning

confidence: 86%

Self-sufficient asymmetric reduction of β-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH

Orrego

Andrés-Sanz

Velasco‐Lozano

et al. 2021

Catal. Sci. Technol.

Self Cite

View full text Add to dashboard Cite

show abstract

Section: Stability Of Immobilised Tt27-hbdh Preparationsmentioning

confidence: 86%

Self-sufficient asymmetric reduction of β-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH

Orrego

Andrés-Sanz

Velasco‐Lozano

et al. 2021

Catal. Sci. Technol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…The immobilization of glycosyltransferases has been achieved by attachment onto solid supports [78,128,258,259,260], entrapment inside a porous carrier [252,261], or cross-linking in larger aggregates (CLEA) [262], as is shown in Figure 14. Furthermore, after immobilization the reusability, thermal, pH, and operational stability of the enzymes was often increased [128,262,263]. In particular cases, enzyme immobilization even created a more favorable micro-environment for enzyme activity [264] and selectivity [265,266].…”

Section: Reactor Engineering For (Non)-leloir Glycosyltransferasesmentioning

confidence: 99%

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

Mestrom

Przypis

Kowalczykiewicz

et al. 2019

IJMS

View full text Add to dashboard Cite

Enzymes are nature's catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio-and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations.hydroxynitrile lyases catalyzed the enzymatic hydrolysis of the glycoside amygdalin [3]. Moving almost two centuries forward, the largest volumetric biocatalytic industrial process is the application of glucose isomerase for the production of high fructose syrup for food and drink applications, producing fructose from glucose at 10 7 tons per year [4]. The secret of the success of enzymes in the production or treatment of carbohydrates and glycosides is their exquisite stereo-and regioselectivity. The excellent selectivity of enzymes is required due to the diversity of structural features of carbohydrates [5], comprising d-and l-epimers, ring size, anomeric configuration, linkages, branching, and oxidation state(s). Since drug targets often exhibit specificity for all of these structural features, the production process should not contain any side-products to prevent undesired side-effects [6].The challenge in the synthesis of carbohydrates is their wide variety of functionalities and stereochemistry ( Figure 1). (Poly)hydroxyaldehydes containing a terminal aldehyde are referred to as aldoses and (poly)hydroxyketones are defined as ketoses. In aqueous solutions, monosaccharides form equilibrium mixtures of linear open-chain and ring-closed 5-or 6-membered furanoses or pyranoses, respectively. For aldoses, the asymmetric ring forms at C-1. For ketoses, it closes at C-2 as an axial (α) or equatorial (β) hemiacetal or hemiketal, respectively (commonly defined as the anomeric center). A glycosidic linkage is a covalent O-, S-, N-, or C-bond connecting a monosaccharide to another residue resulting in a glycoside, while glucoside is specific for a glucose moiety. The equatorial or axial position of the glycosidic bond is referred to as α-(axial) or β-linkage (equatorial). The number of carbohydrates linked via glycosidic bonds can be subdivided into oligosaccharides with two to ten linked carbohydrates, while polysaccharides (glycans) contain more than ten glycosidic bonds. A glycan either con...

show abstract

“…Immobilization is an important method from the general applied bio‐catalysis toolbox for reaction development at the interface with process engineering. [14] Besides occasional reports scattered across glycosyltransferase types (e. g., sialyltransferase, [15a] galactosyltransferase, [15b] antibiotic glycosyltrans‐ferase, [15c] sucrose synthase [15d,e] ), enzyme immobili‐zation for (semi)continuous synthesis with reuse of solid catalyst is not well developed for the Leloir glycosyltransferases. In particular, broadly applicable technology for glycosyltransferase co‐immobilization to establish glycosylation cascades on an insoluble support is lacking.…”

Section: Introductionmentioning

confidence: 99%

Glycosyltransferase Co‐Immobilization for Natural Product Glycosylation: Cascade Biosynthesis of the C‐Glucoside Nothofagin with Efficient Reuse of Enzymes

2021

View full text Add to dashboard Cite

Sugar nucleotide‐dependent (Leloir) glycosyltransferases are synthetically important for oligosaccharides and small molecule glycosides. Their practical use involves one‐pot cascade reactions to regenerate the sugar nucleotide substrate. Glycosyltransferase co‐immobilization is vital to advance multi‐enzyme glycosylation systems on solid support. Here, we show glycosyltransferase chimeras with the cationic binding module Zbasic2 for efficient and well‐controllable two‐enzyme co‐immobilization on anionic (ReliSorb SP400) carrier material. We use the C‐glycosyltransferase from rice (Oryza sativa; OsCGT) and the sucrose synthase from soybean (Glycine max; GmSuSy) to synthesize nothofagin, the natural 3’‐C‐β‐d‐glucoside of the dihydrochalcone phloretin, with regeneration of uridine 5’‐diphosphate (UDP) glucose from sucrose and UDP. Exploiting enzyme surface tethering via Zbasic2, we achieve programmable loading of the glycosyltransferases (∼18 mg/g carrier; 60%–70% yield; ∼80% effectiveness) in an activity ratio (OsCGT:GmSuSy=∼1.2) optimal for the overall reaction rate (∼0.2 mmol h−1 g−1 catalyst; 30 °C, pH 7.5). Using phloretin solubilized at 120 mM as inclusion complex with 2‐hydroxypropyl‐β‐cyclodextrin, we demonstrate complete substrate conversion into nothofagin (∼52 g/L; 21.8 mg product h−1 g−1 catalyst) at 4% mass loading of the catalyst. The UDP‐glucose was recycled 240 times. The solid catalyst showed excellent reusability, retaining ∼40% of initial activity after 15 cycles of phloretin conversion (60 mM) with a catalyst turnover number of ∼273 g nothofagin/g protein used. Our study presents important progress towards applied bio‐catalysis with immobilized glycosyltransferase cascades.

show abstract

Immobilization-stabilization of a complex multimeric sucrose synthase from Nitrosomonas europaea. Synthesis of UDP-glucose

Cited by 19 publications

References 36 publications

Self-sufficient asymmetric reduction of β-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH

Self-sufficient asymmetric reduction of β-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

Glycosyltransferase Co‐Immobilization for Natural Product Glycosylation: Cascade Biosynthesis of the C‐Glucoside Nothofagin with Efficient Reuse of Enzymes

Contact Info

Product

Resources

About