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

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DOI: 10.1002/adsc.202001549

Glycosyltransferase Co-Immobilization for Natural Product

Glycosylation: Cascade Biosynthesis of the C-Glucoside

Nothofagin with Efficient Reuse of Enzymes

Hui Liu,^aGregor Tegl,^aand Bernd Nidetzky^{a, b,}*

a

Institute of Biotechnology and Biochemical Engineering,

Graz University of Technology, NAWI Graz,

Petersgasse 12, 8010 Graz, Austria

Austrian Centre of Industrial Biotechnology (acib),

b

Petersgasse 14, 8010 Graz, Austria

Fax: (+43)-316-873-8434

Phone: (+43)-316-873-8400

E-mail: bernd.nidetzky@tugraz.at

Manuscript received: December 10, 2020; Revised manuscript received: February 9, 2021;

Version of record online: March 2, 2021

Supporting information for this article is available on the WWW under https://doi.org/10.1002/adsc.202001549

terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided

the original work is properly cited.

Abstract: Sugar nucleotide-dependent (Leloir) glycosyltransferases are synthetically important for oligosac-

charides 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 Z_basic2for 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 Z_basic2, 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 mmolh^{À 1}g^{À 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^{À 1}g^{À 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.

Keywords: Leloir glycosyltransferase; sugar nucleotide regeneration; co-immobilization; cascade bio-catalysis;

C-glycosylation; nothofagin

Introduction

often represents the key central step of synthetic routes

towards

oligosaccharides,^[1b,2]

and glycoconjugates,

natural

product

including

Sugar

nucleotide-dependent

(Leloir) glycosides^[3]

glycosyltransferases are generally regarded as efficient glycoproteins.^[4]These are important product classes

and practically useful catalysts of glycosylation with promising applications in different commercial

reactions.^[1]In carbohydrate chemistry, glycosylation sectors, including medical healthcare in particular. The

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use of glycosyltransferases in applied bio-catalysis a recyclable catalyst. Thus, the practical use of

must involve an integrated concept of reaction glycosyltransferases in glycoside synthesis could be-

development.^[1a]Like other enzymatic reactions that come substantially more operative. There has been

require (co)-substrates (e.g., nicotinamide coenzymes high interest recently in the identification, character-

for oxidoreductions;^[5]nucleoside triphosphates for ization

and

engineering

of

Leloir

C-

phosphorylation^[6]or nucleotide transfer;^[7]S- adenosyl- glycosyltransferases^[1a,3d,12]as well as in the use of these

methionine for methylation^[8]) which for reasons of enzymes as catalysts of synthetically important C-

atom economy and costs cannot be used stoichiometri- glycosylation reaction.^[13]The idea of this study, to

cally, the overall glycosyltransferase reaction typically develop a generic process technology for the applica-

consists of a multistep cascade transformation in which tion of immobilized glycosyltransferase cascades, is

the immediate enzymatic glycosylation is supplied thus strongly supported. Advances made in the (semi)

in situ with the corresponding sugar nucleotide donor continuous synthesis of nothofagin, according to

substrate.^[1,9]Besides synthesis from free monosacchar- Scheme 1 and using co-immobilized enzymes, would

ide via phosphorylation and nucleotide transfer seem to be largely transferrable to other C-glycosyl-

cascades,^[9a]sugar nucleotides are often prepared transferase-catalyzed glycosylation reactions from

through a second glycosyltransferase reaction, run in UDP-glucose of synthetic importance.^[1a,13]

reverse direction and operated in parallel to the

Immobilization is an important method from the

synthetic reaction.^[1a,3,9b]A suitably activated glycoside general applied bio-catalysis toolbox for reaction

serves as the donor for in situ sugar nucleotide syn- development at the interface with process engineering.

[14]

thesis. A synthetically important example is the supply

Besides occasional reports scattered across glyco-

of uridine 5’-diphosphate (UDP) glucose (UDP-glu- syltransferase

types (e.g.,

sialyltransferase,^[15a]

cose) from sucrose and UDP, catalyzed by sucrose galactosyltransferase,^[15b]antibiotic glycosyltrans-

synthase (SuSy).^[9b,10]Scheme 1 shows the glycosyl- ferase,^[15c]sucrose synthase^[15d,e]), enzyme immobili-

transferase cascade reaction of this study, involving the zation for (semi)continuous synthesis with reuse of

3’-C-β-d-glycosylation of the dihydrochalcone phlor- solid catalyst is not well developed for the Leloir

etin from UDP-glucose that is continuously supplied glycosyltransferases. In particular, broadly applicable

and regenerated from UDP and sucrose. Nothofagin technology for glycosyltransferase co-immobilization

production according to Scheme 1 was used in our to establish glycosylation cascades on an insoluble

earlier studies^[11]to demonstrate a systematic approach support is lacking. Generally, co-immobilization (the

to process intensification for a glycosyltransferase colocalizing immobilization of multiple enzymes on

cascade reaction, performed in batch conversion and in the same solid carrier) can benefit the efficiency of

homogeneous solution. The current inquiry was set out heterogeneously catalyzed enzymatic cascade reactions

to implement the same glycosyltransferase process on by exploiting the effects of spatial proximity.^[14c,16]

the surface of a solid support, with the aim of Glycosyltransferase co-immobilization can be achieved

fundamental improvement in reaction efficiency due to in principle via immobilizing each enzyme according

Scheme 1. Coupled glycosyltransferase reaction for nothofagin synthesis via 3’-C-β-glycosylation of phloretin from UDP-glucose

which in turn is prepared from sucrose and UDP. The Z_basic2fusions of the rice (Oryza sativa) C-glycosyltransferase (OsCGT) and

the soybean (Glycine max) sucrose synthase (GmSuSy) are used as co-immobilized enzyme preparation on ReliSorb SP400.

Catalytic amounts of UDP are used.

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to its own, enzyme-specific strategy. Guisan, Rocha- conversion of phloretin and sucrose into nothofagin.

Martin and coworkers have recently presented an The solid catalyst was easily reusable, retaining ~40%

important example of such “individualized” glycosyl- of initial activity after 15 cycles of phloretin conver-

transferase co-immobilization, applied to a natural sion (60 mM) with a catalyst turnover number of

product glycosyltransferase and a sucrose synthase, as ~273 g nothofagin/g protein used. Our study presents

discussed later in context of the evidence from the important progress towards applied bio-catalysis with

current study.^[17]Alternatively, glycosyltransferase co- immobilized glycosyltransferase cascades.

immobilization can be uniform in the strategy used for

tethering the enzymes to the solid carrier surface. It is

this latter strategy, realized in the form of glycosyl- Results and Discussion

transferase fusions with the cationic binding module

Glycosyltransferase Fusions with Z_basic2

Z_basic2, that we have pursued here with the idea of a

facile and efficient enzyme co-immobilization usable N-terminal fusions of OsCGT and GmSuSy with the

for nothofagin synthesis. Z_basic2module were constructed and the resulting

Z_basic2is a small protein of 58 amino acids and chimeric proteins obtained by expression in E. coli.

~7 kilodalton size.^[18]It is an engineered binding The purified enzymes showed the expected mass for

module, originally developed from the Z-domain of the respective full-length protein fused to Z_basic2(Fig-

staphylococcal protein A. Placed within fusion pro- ure S1). The specific activity of Z-OsCGT (2.06 U/mg)

teins, the Z_basic2module can fold autonomously to adopt and Z-GmSuSy (0.77 U/mg) was decreased to, respec-

its stable three α-helical bundle structure.^[18]Driven by tively, 67% and 37% of the corresponding N-termi-

electrostatic interactions from multiple arginine resi- nally Strep-tagged enzyme. Comparison of specific

dues exposed on one of its sides,^[18]the functional activities in purified enzyme and in cell lysate

Z_basic2will bind with considerable affinity to negatively (Table S1) suggested that expression of Z_basic2protein

charged surfaces, such as those of cation exchange was to a level of ~20–30% of total intracellular

resins^[19a]and silica materials.^[19b]We have demon- protein.

strated fusion to Z_basic2for enzyme immobilization,^[19]

The Z-glycosyltransferases were conveniently re-

and recently also for multiple enzyme co- covered from the cell lysate (Figure S1). Moreover, as

immobilization,^[20]with excellent control, and modu- shown later, the Z-glycosyltransferases could be

larity. Important elements of control are enzyme immobilized directly from cell lysate, without require-

orientation on the solid surface, especially for co- ment for further enrichment and purification of the

immobilization, and relative amount of enzyme loaded enzymes. The specific activity of OsCGT and GmSuSy

onto the solid carrier. Moreover, enzymes immobilized was decreased by ~40–60% in the presence of

via Z_basic2are homogeneously distributed in porous 250 mM NaCl. The effect was distinct for the Z-

carriers,^[19a,e]which is important to enable the co- fusions of the glycosyltransferases. The Strep-tagged

localization of multiple enzymes. Cello-oligosacchar- enzymes, by contrast, were unaffected by 250 mM

ide synthesis from sucrose and glucose by three NaCl (Table S1). The addition of 250 mM NaCl was

glycoside phosphorylases shows practical use of Z_basic2

based enzyme co-immobilization applied to a hetero- immobilization^[19]have shown that this can enhance

geneously catalyzed cascade transformation.^[19a]

the selectivity of the interaction of the Z_basic2module

-

considered because earlier studies of Z-enzyme

Here, we present an efficient and flexible strategy with the used ReliSorb SP400 carrier.

for the co-immobilization of the rice (Oryza sativa) C-

glycosyltransferase (OsCGT) and the soybean (Glycine

max) sucrose synthase (GmSuSy). Building on surface

Single Enzyme Immobilization

tethering via the Z_basic2module, this strategy is novel to The Z-glycosyltransferases were immobilized individ-

the class of Leloir glycosyltransferases; and it opens ually on ReliSorb SP400 resin (Figure S2 and Ta-

up new important opportunities for development of a ble S2). ReliSorb SP400 are polymethacrylate particles

glycosyltransferase-based process technology for gly- of spherical shape (75–200 μm diameter; 120 μm mean

coside synthesis. Fusion to the Z_basic2module was diameter) and involving a network of macro-pores

reasonably tolerated as regards the enzyme specific with a size of ~100 nm. The ReliSorb SP400 material

activity (�40% of the native glycosyltransferase). offers sulfonate surface groups as negatively charged

Single-enzyme immobilization as well as two-enzyme interaction sites for oriented binding of the fusion

co-immobilization were demonstrated on ReliSorb protein via the cationic Z_basic2module.

SP400 carrier in good yield, 50–100% depending on

Previous studies have shown excellent combination

the enzyme loading. The immobilized enzymes showed of efficiency and selectivity of Z-enzyme immobiliza-

high catalytic effectiveness (�50%). Programmable tion on ReliSorb SP400,^[19a,20]encouraging our efforts

loading of the two glycosyltransferases gave an here to directly immobilize the Z-glycosyltransferases

activity ratio (OsCGT:GmSuSy= ~1.2) optimal for from the cell lysate. Results in Figure S2 show

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selective adsorption of both Z-OsCGT and Z-GmSuSy expected for the immobilized enzyme based on the

to the ReliSorb SP400 carrier, leading to near-complete activity yield. For each enzyme, the observable activity

depletion of the target protein from cell lysate. increased dependent on the activity loaded, enabling an

Although the immobilized Z-glycosyltransferases were approximate range of 8–15 U/g for the active solid

tethered non-covalently, their binding appeared to be catalyst. The yield and the catalytic effectiveness both

strong: washing the carrier (~60 mg) two times with decreased with increasing enzyme loading, from a near

1 mL of 50 mM HEPES buffer (pH 7.5; 250 mM 100% at the lowest loading to around 50–60% at the

NaCl) did not cause elution of enzyme activity.

highest loading. The catalytic effectiveness of ~100%

As shown in Figure 1, we analyzed the individual implies that no activity was lost as result of the Z-

immobilization of Z-OsCGT and Z-GmSuSy on enzyme attachment to the solid carrier. The finding is

ReliSorb SP400 by monitoring, at different loadings of as expected for a perfectly oriented immobilization of

enzyme activity on the unit mass of carrier, the activity the Z-glycosyltransferase where the chimeric enzyme

yield (% of the offered activity bound to the carrier) is tethered solely by its Z_basic2module and the function

and the catalytic effectiveness (% activity retention) of of the “catalytic module” is unaffected by the solid

the immobilized enzyme. The catalytic effectiveness is surface (for general discussion, see reference 14c). The

the ratio (×100, %) between the observable activity of decrease in catalytic effectiveness at higher enzyme

the immobilized enzyme (U/g) and the activity loading might arise due to effects of enzyme crowding

or aggregation on the solid surface. Pore clogging by

immobilized enzyme might be another reason. It seems

unlikely that at the enzyme loading used, the carrier

was internally saturated with protein.^[19a]In terms of

protein immobilized, up to ~30 mg/g and ~42 mg/g

were bound for Z-OsCGT and Z-GmSuSy, respec-

tively. Differences in protein binding may be due to

the oligomer structure of the enzymes which differs for

Z-OsCGT (monomer^[11a]) and Z-GmSuSy (homo-

tetramer^[10a]). Immobilization of Z-GmSuSy might thus

benefit from multivalent binding via two or more of its

Z_basic2modules. Results of co-immobilized enzyme

reuse experiments to be reported later show, however,

that the Z-GmSuSy was bound on the carrier not much

more strongly than the monomeric Z-OsCGT. The

structure of Arabidopsis thaliana SuSy^[21]shows that

the N-termini of neighboring protein subunits point to

opposite sides of the enzyme tetramer. Structural

constraints would therefore seem to limit the degree of

multivalent binding in GmSuSy immobilization to

maximally two Z_basic2modules being involved simulta-

neously in tethering the enzyme tetramer to the solid

surface. We presume at this stage, therefore, that multi-

valency effects in Z-GmSuSy immobilization were

small. The relatively similar behavior of the two Z-

glycosyltransferases in terms of catalytic effectiveness,

and dependence thereof on enzyme loading, is worth

noting.

Glycosyltransferase Co-Immobilization

Recent study suggests that immobilization of O-

glycosyltransferase (from apple) and sucrose synthase

Figure 1. Immobilization of (A) Z-OsCGT and (B) Z-GmSuSy

on ReliSorb SP400. Enzymes were immobilized directly from

(from Acidithiobacillus caldus) to co-localize the two

enzyme activities on the same porous carrier benefits

the synthetic efficiency of the bi-enzymatic glycosyla-

tion cascade.^[17]The rate of 5-O-β-glycosylation of

piceid (3-O-β-d-glucoside of resveratrol) was en-

hanced ~2.4-fold compared to reaction of the two

enzymes immobilized individually on separate

the E. coli cell lysates. A 50 mM HEPES buffer (pH 7.5;

250 mM NaCl) was used for enzyme loading and washing. The

carrier loading was 60 mg/ml. The buffer pH was unchanged

during incubation. Immobilized yield (squares, black), observ-

able activity (circles, red) and catalytic effectiveness (triangles,

blue) are shown. Activities of soluble and immobilized enzymes

were determined as described in the Methods.

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carriers.^[17]Therefore, to perform the 3-C-β-glycosyla-

tion of phloretin efficiently, we considered co-immobi-

lization of OsCGT and GmSuSy to place the enzymes

closely together under the confinement of the solid

surface. This would set particle radius of the carrier as

the upper limit for the maximum diffusional distance

in the reaction. Proximity effects could thus be

exploited to maximize the overall flux through the

two-step cascade reaction on solid support (for general

discussion, see reference 16). We immobilized Z-

OsCGT and Z-GmSuSy in an activity ratio for the

enzyme loaded that varied between 10.8 and 0.1. The

maximum activity loaded was 43.2 U/g for Z-OsCGT

(activity ratio 10.8) and 36 U/g for Z-GmSuSy (activity

ratio 0.1). For each activity ratio used, we measured

the observable activity (rate of nothofagin synthesis) of

the solid catalyst and the product yield from the

coupled reaction. Figure 2A shows that both parame-

ters passed through a maximum at an activity ratio of

1.2 and dropped sharply at lower and higher activity

ratios. The optimum synthesis rate was ~13 U/g.

Analysis with SDS PAGE (Figure 2B) reveals, for each

activity ratio used, the protein distribution for Z-

OsCGT and Z-GmSuSy on the solid carrier. The

semiquantitative picture from the polyacrylamide gel

(Figure 2B) suggests that the activity ratio as loaded

was largely retained in the solid catalyst. Using the

optimum ratio of 1.2, we determined the observable

activity for the individual enzymes (Z-OsCGT: 10.9 U/

g; Z-GmSuSy: 10.4 U/g) in the co-immobilized prepa-

ration. The Z-OsCGT and the Z-GmSuSy were

immobilized in a yield of ~60–70% and show an

effectiveness of ~80%, both in good accordance with

the results of single enzyme immobilization studies.

These findings suggest that in Z-enzyme co-immobili-

zation, the immobilization of one enzyme did not

affect negatively the immobilization of the other.

Generally, this is important as it enables programmable

enzyme co-immobilization based on evidence from

single enzyme immobilization experiments.

Figure 2. Co-immobilization of Z-OsCGT and Z-GmSuSy on

ReliSorb SP400. (A) Observable activity of nothofagin syn-

thesis (squares, black) and nothofagin yield from the coupled

reaction (circles, red) dependent on the activity ratio of Z-

OsCGT and Z-GmSuSy loaded. The co-immobilization was

from E. coli lysates mixed in 50 mM HEPES buffer (pH 7.5;

250 mM NaCl). The activity measurements used the following

conditions in 50 mM HEPES buffer (pH 7.5): 1 mM phloretin,

500 mM sucrose, 0.5 mM UDP, 13 mM MgCl₂, 50 mM KCl,

°

20% (by volume) DMSO, 1.3 mg/ml BSA; 30 C and 1000 rpm

agitation rate. (B) SDS polyacrylamide gel showing the proteins

bound on ReliSorb SP400 dependent on the activity ratios

loaded. The protein band corresponding to Z-OsCGT (red

arrow) and Z-GmSuSy (green arrow) is indicated. The activity

ratio (Z-OsCGT/Z-GmSuSy) was 10.8 (a), 2.8 (b), 1.2 (c), 0.5

(d), and 0.1 (e).

Characterization of the Co-Immobilized Glycosyl-

transferases

The pH dependence of activity for soluble and

immobilized enzyme preparations was determined at

°

30 C. As shown in Figure 3A, the activity of the

soluble Z-OsCGT dropped sharply at high pH whereas

the immobilized enzyme retained its maximum activity

of pH 7.5 up to pH 10.5. For Z-GmSuSy, the pH activity at high pH was attenuated compared to the

dependence of activity was relatively narrow, with soluble enzymes. Considering the behavior of the

optimum activity at pH of 6.5. Loss of activity at high single enzyme immobilized preparations (Fig-

pH was less pronounced in the immobilized enzyme ure 3A,B), the decrease in activity at high pH for the

(Figure 3B). Interestingly, an opposite trend was found co-immobilized preparation appears to be dominated

at low pH where immobilized Z-GmSuSy lost activity by the pH dependence of the Z-GmSuSy activity. The

more dramatically than the soluble enzyme. In the co- optimum pH for the co-immobilized enzyme prepara-

immobilized enzyme preparation (Figure 3C), loss of tion was ~7.5.

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Figure 3. Effect of pH on the activity of the soluble (full squares, black) and the (co)-immobilized enzyme preparations (full circles,

red). (A) Z-OsCGT, (B) Z-GmSuSy, and (C) both enzymes used together in cascade reaction. The reaction buffers used were

25 mM sodium citrate/phosphate (pH 3.5–6.5), 50 mM HEPES (pH 7.5–8.5), and 50 mM CHES (pH 9.5.–10.5). The pH shown is

the final pH of the suspension containing the carrier particles. Enzyme activities were measured as described in the Methods. The

observable activity at the optimum pH (100%) was 14 U/g for immobilized Z-OsCGT, 13.5 U/g for immobilized Z-GmSuSy and

13 U/g for the co-immobilized enzymes.

The temperature dependence of enzyme stability enzyme as result of the immobilization. An operational

°

(30 min incubation) was analyzed at pH 7.5 by temperature range between 30 C and 40 C for the bi-

comparing soluble and immobilized enzyme prepara- enzymatic synthesis of nothofagin was suggested from

tions similarly as done for the pH-activity profiles. the data in Figure 4.

Results in Figure 4 (% activity loss dependent on

temperature) reveal that except for Z-GmSuSy that was

Synthetic Activity and Recyclability of the Co-

Immobilized Glycosyltransferases

°

more stable at 50 C due to immobilization, the soluble

and (co)-immobilized enzymes exhibited comparable

stabilities. The idea of oriented immobilization via the The co-immobilized enzyme preparation (activity ratio

Z_basic2module is that the characteristics of the enzyme 1.2) was used to examine its synthetic applicability and

module are largely unaffected by tethering of the Z- recyclability. Initially, to provide a proof of concept, a

enzyme to the solid surface. One would, therefore, not relatively low concentration of the phloretin substrate

expect a large stabilization of the activity of the Z- (1 mM) was used. The time course of nothofagin

Figure 4. Thermostability of soluble (dark gray) and immobilized enzyme preparations (black). (A) Z-OsCGT, (B) Z-GmSuSy, and

(C) both enzymes used together in cascade reaction. Enzyme activities were measured as described in the Methods. Enzyme

activities were measured as described in the Methods. The observable activity at the optimum pH (100%) was 14 U/g for

immobilized Z-OsCGT, 13.5 U/g for immobilized Z-GmSuSy and 13 U/g for the co-immobilized enzymes.

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production is shown in Figure 5. The used phloretin min) was even higher than in reactions using the non-

was converted fully within ~15 min. The solid catalyst complexed phloretin (~0.1 mM/min; Figure 5B, Fig-

was recovered and the reaction was repeated 15 times. ure S3). A space-time yield of ~5 mM/h (2.2 gL^{À 1}h^{À 1})

Figure 5B shows the nothofagin yield in each reaction was calculated for the reaction run to completion. We

of 15 min. The yield decreased gradually to ~60% in increased the phloretin concentration to 120 mM and

the last reaction. The solid catalyst was analyzed by show its full conversion into nothofagin (Figure 6B).

SDS PAGE at reaction start and after the last reaction The time required for production was longer (60 h),

(Figure 5A). Comparison of the two samples reveals due to a decreased initial conversion rate (~0.1 mM/

clear decrease in protein band intensity for both min) and a pronounced slowing down of the reaction

enzymes after repeated use of the catalyst. Despite the already at low degrees of conversion (~50%). The

semiquantitative nature of the image, the loss in band space-time yield of ~2 mM/h (0.87 gL^{À 1}h^{À 1}) was also

intensity (Figure 5A), indicating partial elution of the decreased compared to the 60 mM reaction. Fluid

non-covalently bound enzyme, might well account for viscosity which is increased strongly at high concen-

the observed decrease in synthetic activity (Figure 5C). tration of the phloretin inclusion complex with 2-

hydroxypropyl-β-cyclodextrin^[11c]could be a relevant

factor. In a comparison at equivalent volumetric

activity of the enzymes used, the solid catalyst was

only about half as efficient in terms of space-time yield

Nothofagin Synthesis with Re-Use of the Co-Immo-

bilized Glycosyltransferases

Due to the excellent recyclability of the co-immobi- as the soluble enzymes. Viscosity effects are likely to

lized enzyme preparation, we were encouraged to be stronger in the heterogeneously catalyzed reaction

explore strategies of reaction intensification applicable and thus provide a possible explanation.

to the solid catalyst. In particular, phloretin solubility

To examine re-use of the solid catalyst, we

should be enhanced to increase the product output. performed a total of 15 consecutive reactions with

Using 20% DMSO co-solvent, the phloretin can be 60 mM phloretin, whereby each reaction lasted 12 h

dissolved to ~10 mM and we show its full conversion and involved recovery of the immobilized enzyme for

from 5 mM or 10 mM initial concentration in Fig- the next reaction. As shown in Figure 7, the nothofagin

ure S3. To further increase the soluble phloretin yield decreased from a near 100% in the first reaction

concentration, we used inclusion complexation with 2- to ~40% in the last reaction. As in the re-use experi-

hydroxypropyl-β-cyclodextrin, as used by us previ- ment in Figure 5, partial elution of enzyme from the

ously for nothofagin production with the soluble solid catalysts (data not shown) seemed to be respon-

enzymes.^[11c]We show in Figure 6A that phloretin sible for the gradual loss of catalyst efficiency over the

solubilized at 60 mM was converted very efficiently number of reactions performed. We can calculate from

(�98%) within ~12 h. The conversion rate (~0.2 mM/ the total nothofagin produced in the 15 reactions

Figure 5. Synthetic activity and recyclability of co-immobilized Z-OsCGT and Z-GmSuSy. (A) SDS polyacrylamide gel showing

the protein on the ReliSorb SP400 carrier at reaction start (Int) and after the last reaction (Fin). (B) Reaction time course. (C)

Repeated reaction as in panel B with reuse of the solid catalyst. Each reaction lasted 15 min. Conditions: 1 mM phloretin, 500 mM

°

sucrose, 0.5 mM UDP, 50 mM KCl, 13 mM MgCl₂, 1.3 mg/ml BSA, 20% DMSO, 30 C, 50 mM HEPES buffer (pH 7.5). The

starting activity of immobilized Z-OsCGT and Z-GmSuSy was 0.23 U/ml and 0.21 U/ml, respectively. The solid catalyst was used

at 20 mg/ml. The loaded activity ratio was 1.2.

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Figure 7. Re-use of the co-immobilized enzyme preparation for

nothofagin production. Reaction conditions: 60 mM phloretin as

inclusion complex with 2-hydroxypropyl-β-cyclodextrin,

500 mM sucrose, 0.5 mM UDP, 50 mM KCl, 13 mM MgCl₂,

°

1.3 mg/ml BSA, 50 mM HEPES buffer (pH 7.5) and 30 C. The

starting activity of immobilized Z-OsCGT and Z-GmSuSy was

0.47 U/ml and 0.42 U/ml, respectively. The solid catalyst was

used at 40 mg/ml. The loaded activity ratio was 1.2. Each

reaction cycle lasted 12 h.

strated at 100 g scale in an earlier study from this

laboratory.^[11c]The isolated yield was �65% and the

purity was �95%. Considering the product down-

stream processing to have been established in prior

research, we did not pursue isolation of nothofagin in

the current study.

Figure 6. Nothofagin production (circles, red) from phloretin

(squares, black) solubilized as inclusion complex with 2-

hydroxypropyl-β-cyclodextrin (A, 60 mM; B, 120 mM) using

co-immobilized Z-OsCGT and Z-GmSuSy. Reaction conditions:

500 mM sucrose, 0.5 mM UDP, 50 mM KCl, 13 mM MgCl₂,

°

1.3 mg/ml BSA, 50 mM HEPES buffer (pH 7.5) and 30 C. The

The Advantage of Enzyme Co-Immobilization for

Efficient Glycosylation

starting activity of immobilized Z- OsCGT and Z-GmSuSy was

0.47 U/ml and 0.42 U/ml, respectively. The solid catalyst was

used at 40 mg/ml. The loaded activity ratio was 1.2.

Fusion to Z_basic2can introduce modular design to

enzyme immobilization.^[14c,19a,e]Unlike unstructured

peptide tags, Z_basic2represents a structurally independ-

(278 mg, 15 ml) and the amount of immobilized Z- ent and functionally distinct unit (module) of the fusion

OsCGT present on the solid catalyst (0.30 mg, protein. It can fold separately and provides a program-

recycled) that the C-glycosyltransferase reached a mable function according to a uniform mechanistic

mass-based turnover number of 927 (= 278/0.30) g/g. principle of surface binding.^[18]Due to its independent

With a M_rof 436 for nothofagin and 57,800 for Z- function in the modularly organized fusion protein,

OsCGT, the corresponding mol-based turnover number Z_basic2hardly interferes with the activity of its partner

is 1.2×10⁵mol/mol. Considering the Z-GmSuSy module,

as

shown

for

different

enzymes

(0.72 mg, recycled) that is additionally required, the previously^[19,20,22]and here for the first time for

mass-based turnover number becomes 273 (=278/ representatives of the Leloir glycosyltransferase class.

1.02). These are excellent turnover numbers for a Thus, immobilization via Z_basic2is advantageous in

Leloir glycosyltransferase^[1a,11a]and highlight the effi- particular when multiple enzymes in cascade bio-

ciency of the synthetic reaction when the solid catalyst catalysis should be tethered as a protein ensemble on a

is reused. It may be noted that nothofagin recovery solid surface, to establish multistep reaction sequences

from an enzymatic reaction mixture comparable to the on a single insoluble carrier. Proof of principle was

ones obtained in Figure 6 and Figure 7 was demon- shown from the co-immobilization of the Z-fusions of

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three glycoside phosphorylases and the application of

the solid catalyst thus obtained for cello-oligosacchar-

ide synthesis.^[20a]However, decision about enzyme co-

immobilization on the same carrier, as opposed to

single enzyme immobilization on different carriers,

must balance gain in catalyst efficiency with increased

complexity of catalyst preparation when co-immobi-

lized enzymes are used.^[16]We therefore set out experi-

ments to compare co-immobilized preparation of Z-

OsCGT and Z-GmSuSy with a mixture of the individ-

ually immobilized enzymes for nothofagin synthesis

under otherwise exactly comparable conditions. Time

courses of conversion of 60 mM phloretin inclusion

complex with 2-hydroxypropyl-β-cyclodextrin are

shown in Figure 8. In both reactions, the phloretin was

converted fully. In terms of initial reaction rate,

reaction of the co-immobilized enzyme (34 mM/h) was

~2.5-fold faster than reaction of the individually

immobilized enzymes. The space-time yield for full

conversion (�98%) was ~4-fold higher for the co-

immobilized enzyme than for individually immobilized

Z-OsCGT and Z-GmSuSy. These results quantify the

intensification of nothofagin production due to prox-

imity effects of enzyme co-immobilization. The rate

enhancement for the co-immobilized enzyme prepara-

tion can probably be ascribed to shortened diffusion

paths when enzymes are co-localized on the porous

surface of the solid carrier. Our results are consistent

with study of Rocha-Martin and colleagues who

showed 2.4-fold benefit of glycosyltransferase co-

immobilization, compared to individual enzyme immo-

bilization on separate carrier particles, on piceid Figure 8. Synthesis of nothofagin using co-immobilized (A) or

glycosylation from sucrose-derived UDP-glucose.^[17]

The effect was dedicated to an enhanced efficiency of

the UDP/UDP-glucose shuttle when enzymes are co-

localized on the solid carrier.

individually immobilized (B) enzyme preparations. Phloretin

(squares, black) and nothofagin (circles, red) are shown.

Reaction conditions: 60 mM phloretin as inclusion complex

with 2-hydroxypropyl-β-cyclodextrin, 500 mM sucrose, 0.5 mM

UDP, 50 mM KCl, 13 mM MgCl₂, 1.3 mg/ml BSA, 50 mM

°

HEPES buffer (pH 7.5) and 30 C. The starting activity of

Co-Immobilization of Z-OsCGT and Z-GmSuSy in

Context

immobilized Z-OsCGT and Z-GmSuSy was 1.17 U/ml and

1.05 U/ml, respectively. The solid catalyst was used at 100 mg/

ml. The loaded activity ratio was 1.2.

Leloir glycosyltransferases are widely recognized for

their synthetic importance.^[1–4]They are considered

“precision catalysts” of glycosylation with high poten-

tial for applied bio-catalysis. There have been limited enzyme for up to 10 cycles was shown. Important

efforts, however, to develop immobilized glycosyl- advance made with the Z_basic2fusion approach reported

transferases for heterogeneous catalysis applications here is fourfold. First, the immobilization no longer

(for reviews, see references 1a, 2a). Recently, Guisan, requires purified enzyme and is conveniently done

Rocha-Martin and their co-workers have shown from the cell lysate. Second, it builds on modular

immobilization of bacterial sucrose synthases on design. A proven engineering principle of enzyme

suitably activated agarose carriers.^[15d]They also tethering to the solid surface is flexibly applied to the

demonstrated glycosyltransferase co-immobilization on (co)-immobilization of different glycosyltransferases.

agarose,^[17]as already discussed. Their studies showed Efficiency parameters of the (co)-immobilization are

glycosyltransferase immobilization in good loading good to excellent when applying standard immobiliza-

(several mg protein/g carrier), high yield (�50%) and tion conditions without enzyme-specific optimization.

typically good effectiveness (�44%). Stabilization of Third, a standard (anionic) carrier material can be used

tetrameric sucrose synthase required post-immobiliza- for immobilization without requirement for chemical

tion crosslinking.^[15d]Re-use of the immobilized derivatization/activation of the solid surface. Fourth,

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materials used are described in the Supporting Information.

Authentic standard of nothofagin (phloretin 3’-C-β-D-gluco-

side; �99%) was obtained from recent study using enzymatic

synthesis.^[11c]

enzyme co-localization on the solid carrier does not

require specific adjustment and finely tuned control of

the conditions for single-enzyme immobilization. Im-

portantly, the co-immobilization study of Trobo-

Maseda et al.^[17]shows that unless proper control for

enzyme co-localization is applied to the immobiliza-

tion of the single enzymes, the effect of enzyme co-

immobilization can be lost completely (~50-fold).

The lifetime of our co-immobilized glycosyltrans-

ferase catalyst is sufficient for multiple cycles of

reaction. However, further stabilization of Z-enzyme

binding to the solid surface will be important to

enhance the turnover number of this catalyst. Post-

immobilization techniques of protein crosslinking, as

applied to sucrose synthase in the study of Trobo-

Z-Enzymes

OsCGT (GenBank id: FM179712) and GmSuSy (GenBank id:

AF030231) were used. Enzyme chimeras harboring the Z_basic2

module fused to the N-terminus were constructed from the

plasmid vector pT7_Z_P450 BM3^[20c]using overlap extension

PCR. The oligonucleotide primers used (Table S1) and the PCR

conditions applied are found in the Supporting Information. The

used expression vectors are referred to pT7_Z_OsCGT and

pT7_Z_GmSuSy. The relevant protein sequences and the

methods used for protein expression in E. coli BL21(DE3) and

Maseda et al.,^[15d]may be useful. However, the enzyme purification by cation exchange chromatography are detailed in

the Supporting Information. Protein purification was monitored

by SDS PAGE and measurement of specific enzyme activity.

Enzymes harboring N-terminal Strep-tag II have previously

been reported^[11a]and are used as reference. Protein concen-

trations were measured with ROTI Quant assay (Carl Roth,

Karlsruhe, Germany) referenced against BSA. Z-OsCGT (~

15 mg/ml) and Z-GmSuSy (~20 mg/ml) were stored in 50 mM

turnover number from the current work (~273 g/g)

shows Leloir glycosyltransferases to be already on par

with other enzymes (e.g., glycoside hydrolases,^[23]

glycoside phosphorylases^[20a,24]perceived as more

robust for application in glycosylation.

°

HEPES buffer (pH 7.5) at À 20 C. Enzyme preparations

Conclusion

retained full activity for at least 2 weeks.

Modular bioengineering is a powerful approach for

accelerated development in biotechnology.^[25]Fusion to

the Z_basic2module is shown here for glycosyltransferase

co-immobilization to implement sugar nucleotide-

dependent glycosylation cascade reactions on solid

support. The example of Z-OsCGT and Z-GmSuSy

demonstrates convenient production of the Z-glycosyl-

transferases in E. coli and programmable enzyme co-

immobilization in a facile, high-yielding procedure

directly from the cell lysates. The solid catalyst with

Z-OsCGT and Z-GmSuSy loaded in optimum ratio

shows excellent overall activity for nothofagin syn-

thesis from sucrose and phloretin, and it can be

recycled conveniently from the reaction. Output

parameters, such as yield, product concentration,

space-time yield, and recycling of UDP as catalytic

reagent, are similar for batch syntheses by soluble and

co-immobilized enzymes. Recycling of the co-immobi-

lized enzymes enhances the enzyme turnover number

by another �10-fold. Collectively, this study empha-

sizes enzyme co-immobilization as an important

element of a comprehensive process intensification

strategy for glycosylation reactions catalyzed by

coupled Leloir glycosyltransferases.

The cell lysates used for immobilization were prepared by ultra-

sonication of E. coli cells suspended in a 2-fold volume of 50

mM HEPES buffer (pH 7.5). A Sonic Dismembrator Model 505

(Fisher Scientific, Vienna, Austria) was used and the sonication

protocol (6 min in total) involved alternating 3 s pulse on and

9 s pulse off at 60% amplitude and cooling on ice. Cell lysate

°

was recovered by centrifugation (15,000 rpm, 4 C, 40 min;

Centrifuge 5424R, Eppendorf, Vienna, Austria).

Enzyme Assays

°

Reactions were performed in 1 ml total volume at 30 C in 2 ml

plastic tubes using agitation at 400 rpm (soluble enzymes) or

1000 rpm (immobilized enzymes) in an Eppendorf Thermo-

Mixer C instrument (Vienna, Austria). Samples (20 μl) were

withdrawn after 5, 10 and 20 min and reactions quenched with

the same volume of acetonitrile. Solid material (precipitated

protein, immobilization carrier) was centrifuged off

°

(13200 rpm, 4 C, 20 min; Centrifuge 5424R, Eppendorf) and

the supernatant analyzed by ion-pairing reversed-phase

HPLC.^[26]A Shimadzu model UFLC HPLC system equipped

with a Kinetex^®5 μm EVO C18 LC column (100 Å, 150×

4.6 mm; Phenomenex, Merck, Vienna, Austria) was used. The

°

column was equilibrated at 25 C in 20 mM potassium

phosphate buffer (pH 5.9) containing 40 mM tetrabutylammo-

nium bromide. Elution was with acetonitrile. UV detection was

used.

Experimental Section

Z-OsCGT. The reaction mixture contained in 50 mM HEPES

buffer (pH 7.5), 1 mM phloretin, 2 mM UDP-glucose, 13 mM

MgCl₂, 50 mM KCl, 4% DMSO, and 1.3 mg/ml BSA. NaCl

(250 mM) was added optionally. HPLC analysis used an

acetonitrile gradient (20%–50%) at 1 ml/min and detection at

282 nm. Rates were determined from [nothofagin]/time. One

Materials

ReliSorb SP400 carrier was from Resindion (Binasco, Italy). 2-

Hydroxypropyl-β-cyclodextrin (>98%), phloretin (>98%),

UDP (97%) and UDP-glucose (>98%) were from Carbosynth

(Compton, UK). Other chemicals were of reagent grade. Further

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asc.wiley-vch.de

unit (U) of activity is the enzyme amount releasing 1 μmol

nothofagin/min under the conditions used.

Immobilization yield. This is expressed as ΔA/A₀(×100, %).

Observable activity. This is the activity of the solid catalyst

Z-GmSuSy. The reaction mixture contained in 50 mM BisTris

buffer (pH 6.5), 500 mM sucrose, 2 mM UDP, 13 mM MgCl₂,

50 mM KCl and 1.3 mg/ml BSA. NaCl (250 mM) was added

optionally. HPLC analysis used isocratic acetonitrile conditions

(12.5%) at 2 ml/min and detection at 262 nm. Rates were

determined from [UDP-glucose]/time. One U is the enzyme

amount releasing 1 μmol UDP-glucose/min under the conditions

used.

directly measured in an activity assay. It is expressed as U/g.

Catalytic effectiveness. This is the ratio of observable

activity and bound activity (×100, %). Immobilizations were

performed in triplicates and the mean value with standard error

is reported.

Immobilized Enzyme Characterization

Coupled reaction. The reaction mixture contained in 50 mM

HEPES buffer (pH 7.5), 1 mM phloretin, 500 mM sucrose,

0.5 mM UDP, 13 mM MgCl₂, 50 mM KCl, 20% DMSO and

1.3 mg/ml BSA. HPLC analysis of the Z-OsCGT assay was

used. Rates were determined from [nothofagin]/time. One U is

the enzyme amount releasing 1 μmol nothofagin/min under the

conditions used.

The pH-activity profile of soluble and (co)-immobilized

°

enzymes was recorded at 30 C in the pH range 3.5–10.5. The

buffers used were 25 mM sodium citrate/phosphate (pH 3.5–

6.5), 50 mM HEPES (pH 7.5–8.5), and 50 mM CHES (pH 9.5–

10.5). The other reaction conditions and the analytical

procedures used were the ones of the enzyme assays described

above. The pH values given are from the suspension of carrier

°

particles. Enzyme stability at different temperatures in 10 C

Immobilization

°

interval between 20 C and 60 C were recorded in 50 mM

HEPES buffer (pH 7.5) and using incubation for 30 min.

ReliSorb SP400 carrier (60 mg dry material) was weighed into

2 ml Eppendorf tubes, washed three times with water and

afterwards two times with HEPES buffer (50 mM, 250 mM

NaCl, pH 7.5). The pH of the carrier suspension was verified as

7.5. For single enzyme immobilization, E. coli cell lysate (1 ml;

2–15 mg total protein/ml) was added and incubated at room

temperature at 40 rpm on an end-over-end rotator for 2 h.

Supernatant was removed and carrier washed twice with 50 mM

HEPES (pH 7.5; Z-OsCGT) or 50 mM Bistris (pH 6.5; Z-

GmSuSy) buffer, each containing 250 mM NaCl. Supernatant

and washing solutions were collected for protein and activity

measurements. The pH was always checked and was constant at

~7.5. The carrier was also recovered and used for activity

measurement. For enzyme co-immobilization, E. coli cell

lysates containing Z-OsCGT and Z-GmSuSy were mixed in

variable volume ratio (10.8–0.1) to give 1 ml of total lysate to

be added to 60 mg dry carrier. The mixture was incubated

(50 mM HEPES buffer, pH 7.5; 250 mM NaCl) and processed

as described above. The individual activities of Z-OsCGT and

Z-GmSuSy as well as the combined (coupled) activity of the

two enzymes were measured using the assays already stated.

Unless mentioned, in the co-immobilization experiment activity

refers to the coupled reaction.

°

Residual activity was then recorded at 30 C using the standard

activity assays. In pH and temperature studies, purified

preparations of the soluble enzymes were used. The soluble

enzyme concentration used was typically ~0.1 mg/ml, equiv-

alent to ~0.2 U/ml. The volumetric concentration of the

immobilized enzyme, as prepared from loading cell lysate on

ReliSorb SP400, was ~0.25 U/ml. The co-immobilized enzyme

preparation involved a ~1.2 activity ratio of Z-OsCGT and Z-

GmSuSy loaded on the carrier.

Enzymatic Synthesis of Nothofagin

Formation of the phloretin inclusion complex. 2-Hydrox-

ypropyl-β-cyclodextrin (4.2 g; 3 mmol) was dissolved to

~300 mM in ~4 ml of deionized water in a 50 ml tube.

Microwave heating at 750 W was used. The total heating time

was ~20 s and several steps of heating and mixing by inversion

were used. Phloretin (0.66 g; 2.4 mmol; ~240 mM) was added

and dissolved as before. Not all phloretin could be dissolved

with the procedure used. The final volume was set to 10 ml.

The mixture was equilibrated in a drying chamber at 70 C for

1 h and inverted every 15 min. If required, the solution was

stored overnight at 4 C and microwave-heated shortly prior to

use. Insoluble phloretin was centrifuged off (5000 rpm, 5 min,

room temperature). In each conversion experiment, the actual

phloretin concentration in solution was determined by HPLC.

°

Parameters used to evaluate the immobilization were the

following.

Loaded activity. This is the enzyme activity loaded on the

carrier and is expressed as U/g. Cell lysates used for

immobilization had a known volumetric activity (U/ml) and

protein concentration (mg/ml). The activity loaded was calcu-

lated from the protein loaded in the experiment.

Enzymatic conversion. Reactions were performed using non-

complexed phloretin at low concentrations (1.0–10 mM; 20%

DMSO cosolvent) or phloretin inclusion complex (60 mM,

120 mM; no cosolvent). For 1 mM phloretin (20% DMSO

cosolvent), the reaction was performed in 50 mM HEPES buffer

(pH 7.5) containing 20 mg/ml solid catalyst, 500 mM sucrose,

0.5 mM UDP, 50 mM KCl, 13 mM MgCl₂and 1.3 mg/ml BSA.

A total liquid volume of 500 μl was used. For 60 mM phloretin

inclusion complex, the reaction was performed in 50 mM

HEPES buffer (pH 7.5) containing 40 mg/ml solid catalyst,

500 mM sucrose, 0.5 mM UDP, 50 mM KCl, 13 mM MgCl₂

and 1.3 mg/ml BSA. A total liquid volume of 1 ml was used.

Bound activity. This is the activity bound to the solid carrier. It

was calculated as difference in the soluble enzyme activity (U)

before (A₀) and after the immobilization (A). The activity A is

the total activity present in the supernatant of the immobiliza-

tion and the washing solutions. The bound activity is calculated

as ΔA (=A₀À A) divided by the carrier mass (g). The bound

activity is expressed as U/g.

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Unless mentioned, a 1.2 activity ratio of Z-OsCGT and Z-

GmSuSy loaded on the carrier was used. Especially note, the

term of solid catalyst refers to the enzymes co-immobilized on

Relisorb SP400 throughout the manuscript. The volumetric

activities varied as indicated in the text. The temperature was

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30 C and agitation was at 1000 rpm in an Eppendorf

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enzymatic reaction, the solid carrier was centrifuged off

°

(4000 rpm, 20 min, 4 C; Centrifuge 5424R, Eppendorf) and the

supernatant withdrawn for analysis. The carrier washed twice

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Acknowledgements

Martin Pfeiffer, Peter Wied and Chao Zhong (Institute of

Biotechnology and Biochemical Engineering, Graz University

of Technology) are acknowledged for their support. H.L. is

recipient of fellowship from the China Scholarship Council

(grant number 201906380025).

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Article Doi

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

DOI: 10.1002/adsc.202001549

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