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Glycosynthase Principle Transformed into Biocatalytic Process Technology: Lacto-N-Triose II Production with Engineered Exo-Hexosaminidase Katharina Schmölzer, Melanie Weingarten, Kai Baldenius, and Bernd Nidetzky ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b01288 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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ACS Catalysis

Glycosynthase Principle Transformed into Biocatalytic Process Technology: Lacto-N-Triose II Production with Engineered Exo-Hexosaminidase Katharina Schmölzer,† Melanie Weingarten,‡ Kai Baldenius,‡ Bernd Nidetzky*,†,§

†Austrian

Centre of Industrial Biotechnology, Petersgasse 14,

8010 Graz, Austria. ‡BASF

SE, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, Germany.

§Institute

of Biotechnology and Biochemical Engineering, Graz

University of Technology, NAWI Graz, Petersgasse 12/I, 8010 Graz, Austria.

ABSTRACT.

Glycosynthases

glycoside

synthesis.

mechanistic

are

Derived

repurposing

of

promising from

their

enzyme

glycoside

active

site,

catalysts

for

hydrolases

by

glycosynthases

ACS Paragon Plus Environment

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ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

utilize suitably

activated

Page 2 of 46

glycosyl donors for glycosylation,

yet, are unable to hydrolyze the products thus formed. Although primed for synthetic application by their design, glycosynthases have

yet

to

see

actual

uses

in

carbohydrate

production.

To

challenge limitations on glycosynthase applicability perceived from the process chemistry point of view, here, we developed a glycosynthase (D746E variant) from Bifidobacterium bifidum -Nacetyl-hexosaminidase highly active

synthetically (≥ 100 µmol

min-1 mg-1) and fully chemo- and regioselective in using N-acetylD-glucosamine

We

thus

1,2-oxazoline for -1,3-glycosylation of lactose.

established

chemo-enzymatic

process

technology

for

production of lacto-N-triose II, a core structural unit of human milk

oligosaccharides.

Using

equivalent

amounts

of

oxazoline

(prepared chemically in 40% yield from N-acetyl-D-glucosamine) and lactose, we obtain lacto-N-triose II (515 mM; 281 mg mL-1; 90% yield; ≤1 h reaction time) immediately recoverable from the reaction

in

85%

purity.

These

metrics

of

process

efficiency

reveal the prodigious potential of the glycosynthase for the trisaccharide production.

KEYWORDS:

biocatalytic

process,

glycosylation,

glycosynthase,

hexosaminidase, human milk oligosaccharides, lacto-N-triose II, oxazoline donor substrate


2

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ACS Catalysis

INTRODUCTION Complex oligosaccharides, like those contained in human milk, are gaining increased attention for applications in healthrelated nutrition.1-3 To further these applications, the oligosaccharides must be produced efficiently at large scale.4-5 Despite the advances in catalytic glycosylation by chemical or biological approaches,6-11 robust platforms for facile synthesis of defined oligosaccharides are not available. Limitations on the applicability of existing methodologies arise from their restricted transformability into efficient process technologies. A microorganism engineered for oligosaccharide synthesis from nutrients is therefore considered as the most promising system for process development.4,

6

However, microbial “fermentation” is

limited to space time yields ( 0.6 g L-1 h-1)4,

12-13

typically much

lower than space time yields (≥ 100 g L-1 h-1) often required in process chemistry.14-16 The maximum product concentration may be limited to a few g L-1 due to complex bottlenecks on metabolism, cellular transport, or both.4,

12-13

The substrate is predominantly

used for growth, with only a small portion of it remaining for synthesis. This renders the fermentation often an inherently atom-inefficient synthetic strategy. A biocatalytic technology which like fermentation exploits the substrate specificity and


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the selectivity control of enzymes but obviates the problems of the living cell to attain process efficiencies required in chemical production is, therefore, highly desirable. Glycosynthases6 embody a distinct enzymatic principle of oligosaccharide synthesis (for reviews, see refs

6,7, 17-19).

They

are engineered glycoside hydrolases in which the catalytic residue responsible for promoting nucleophilic attack on the substituted anomeric carbon (Scheme 1a) is replaced by a nonfunctional residue.20-21 Glycosynthases are therefore virtually inactive against their normal glycoside donor substrates. However, when offered a suitably activated glycoside donor of opposite anomeric configuration to that of the native substrate, glycosynthases catalyze glycosylation reactions, yet are unable to hydrolyze the glycoside products formed.20-21 Glycosynthases thus have enormous potential for use in oligosaccharide synthesis.7,

17-19

In theory, they are able to combine the specific

advantages of glycosyltransferases and glycoside hydrolases, while obviating the main drawbacks that these natural types of glycosylating enzymes involve.22-23 Whereas the absence of product hydrolysis removes the inherent shortcoming of the glycoside hydrolases,20-21 the glycosynthases can also overcome limitations arising from the high degree of substrate specificity exhibited by many glycosyltransferases.23 However, although several


4

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ACS Catalysis

glycoside hydrolases have been converted into glycosynthases,2021, 24-26

these engineered enzymes have yet to see application in

industrial oligosaccharide production. This lack of application, and the resulting underuse of their potential, stems from limitations on glycosynthase applicability perceived from the process chemistry point of view. a

Base O H O R1

O

HO

R'

O

FHO

R1 = sugar

F

O

O H OR1

O

R'

Mutated nucleophile

b Acid/base O

HO HN O

O H OR

O

O

O

Oxazolinium ion ROH

Oxazolinium ion formation

O

HO

O

O

HN

O H O R1 O H

O

Trans-glycosylation R1 = sugar

O

HO HN

O

Stabilizing residue Stabilizing residue

P hy rima dr ry ol ys is R1 = H

t uc od ysis r P rol d hy

O

O H OR1

O

O

O

Trans-glycosylation product

R1 = sugar

Hydrolysis product

Scheme 1. Trans-glycosylation by -glycosynthases and -N-acetylhexosaminidases. (a) Oligosaccharide synthesis by an inverting glycosynthase that uses glycosyl fluoride as the donor substrate. This “classic” glycosynthase is derived from a retaining -glycoside hydrolase by substitution of the catalytic nucleophile (e.g., Asp, Glu) by a non-nucleophilic residue (e.g., Gly, Ala, Ser). (b) Substrate-assisted reaction of a -N-


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acetyl-hexosaminidase leading to formation of oligosaccharide and hydrolysis products.

Any viable process technology for biocatalytic glycosylation will involve a matching pair of enzyme and donor substrate which combine to give the reaction efficiency demanded by the processing objective.7,

19, 22-23, 27

The intrinsic interdependence of

enzyme and donor in controlling reactivity and selectivity of the glycosylation makes their selection a fundamental problem of decisive importance for practical realization. In the case of glycosynthases, therefore, this selection implies a convergent strategy of enzyme engineering and synthetic design of the donor substrate.7,

17-19

The high degree of chemical activation

necessitated in typical glycosynthase donors renders them labile substrates for biocatalytic transformations.28-30 An enzymatic conversion rate substantially faster than the substrate decomposition rate is therefore required, but this may be difficult to obtain with glycosynthases possessing low specific activity. Directed evolution is useful to enhance the enzyme activity, yet such programs necessitate substantial effort to establish.31-32 The synthetic utility of the donor substrate hinges critically on a facile procedure for its preparation, ideally from an unprotected sugar.


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ACS Catalysis

The -glycosylation from 1,2-oxazoline-activated N-acetyl-Dglucosaminyl donors (Scheme 1b) represents an important synthetic strategy towards oligosaccharides.30,

33-34

Glycosynthases are promising catalysts for this reaction, as shown in seminal studies on the synthetic N-glycosylation of proteins and peptides by glycosynthases derived from protein endo--N-glucosaminidases.35-39 However, the protein glycoengineering thus performed is usually not targeted at the actual production. Studies of oligosaccharide synthesis with these enzymes40-43 suggest potential of the general synthetic principle for biocatalytic process development. Here, therefore, we designed a glycosynthase from the Bifidobacterium bifidum exo--N-acetyl-hexosaminidase44-45 that is synthetically highly active and fully regioselective in using N-acetyl-D-glucosamine 1,2-oxazoline (NAG-oxa) for -1,3-glycosylation of lactose (Scheme 2). The glycosynthase is hydrolytically deficient not only toward the trisaccharide product but also toward the NAGoxa donor. We show this salient characteristic of enzyme chemoselectivity to be decisive for a highly atom-efficient glycosylation (90%) of lactose from NAG-oxa. The wildtype enzyme, by contrast, hydrolyzes both the NAG-oxa donor and the trisaccharide product with significantly higher activity than the glycosynthase. This renders the wildtype enzyme rather


7

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unsuitable for synthetic application. Therefore, based on the glycosynthase, we thus develop chemo-enzymatic process technology for the production of lacto-N-triose II (LNT II), a core structural unit of the human milk oligosaccharides (HMOs).1 This involves NAG-oxa prepared chemically in 40% yield from Nacetyl-D-glucosamine (GlcNAc). Using equivalent amounts of NAGoxa and lactose at solubility limit (600 mM), we obtain LNT II (515 mM; 281 mg mL-1; 90% yield; ≤ 1 h reaction time) immediately recoverable from the biocatalytic reaction in 80% purity. These metrics of process efficiency reveal the prodigious potential of the glycosynthase for process chemistry application and identify it as far superior to alternative synthetic options for the trisaccharide production.

Scheme 2. Synthesis of lacto-N-triose II (LNT II) from lactose by enzymatic trans-glycosylation with NAG-oxa as the donor substrate. Wild-type and variant enzymes of family GH-20 -Nacetyl-hexosaminidase BbhI from B. bifidum JCM1254 were used.

RESULTS AND DISCUSSION


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ACS Catalysis

Glycosynthase design. A glycoside hydrolase that catalyzes hydrolysis of the synthetic target product with high activity and selectivity generally represents a promising starting point for glycosynthase development. We therefore selected the -N-acetyl-hexosaminidase BbhI from B. bifidum JCM1254 for the current study.45 Previously, BbhI was shown to catalyze -1,3-glycosylation of lactose using para-nitrophenyl-N-acetyl--D-glucosaminide (GlcNAc--pNP) as the donor substrate.44 However, despite extensive reaction optimization, the enzymatic synthesis of LNT II was inefficient due to fast hydrolysis of the product under all the conditions used. BbhI is an exo-acting -N-acetyl-hexosaminidase and belongs to family GH-20 of the glycoside hydrolase families. Enzymes of family GH-20 utilize neighboring group participation from the substrate’s 2-acetamido group in catalysis. The enzymatic reaction is promoted by a highly conserved triad of residues (Glu, Asp, Tyr; Figure 1) and proceeds through an 1,2oxazolinium ion intermediate (Scheme 1b). An active-site close up from a modeled structure of BbhI is shown in Figure S1. Based on evidence for endo--N-acetyl-glycosaminidases of families GH18 and GH-85,24-26,

36-38, 46

a promising design for BbhI


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glycosynthase was to substitute the residues (Asp746, Tyr827) involved in stabilization of the oxazolinium intermediate. The structure model (Figure S1) corroborates the evidence from sequence alignment (Figure 1) in suggesting that Asp746 and Tyr827 are positionally conserved in the BbhI active site. Within family GH-20, there is limited precedence on glycosynthase development. A D313A variant of the -hexosaminidase from Streptomyces plicatus40,41 (GH-20) and Tyr470 variants (Phe, His, Asn) of the -hexosaminidase from Talaromyces flavus42 (GH-20) reflect enzyme design strategies conceptually similar to the one applied here to BbhI. We prepared four site-directed variants (D746E, D746A, D746Q, Y827F) of BbhI. The Asp746 variants entail loss of electrostatic stabilization (D746A) of the intermediate, a probable steric conflict in substrate/intermediate positioning (D746E), or both (D746Q). The Y827F variant involves the removal of a hydrogen bond for substrate binding and catalysis.47 Anticipated to have low activity for LNT II hydrolysis, these BbhI variants might however utilize the 1,2-oxazoline of Nacetyl-D-glucosamine (NAG-oxa) as donor for -1,3-glycosylation of lactose. Purified preparations of wild‐type and variant enzymes were obtained from Escherichia coli over-expression cultures, with a C‐terminal His6‐tag for purification by metal


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ACS Catalysis

chelate chromatography. All enzymes were conveniently stable during storage (4°C, -20°C) for weeks.

Figure 1. Partial sequence alignment of pro- and eukaryotic GH20 -N-acetyl-hexosaminidases displaying residues critical for substrate-assisted catalysis. Sequence alignment was performed with Clustal Omega using default settings. Critical amino acids are highlighted in green. The mutation sites are indicated by arrows. Glycosynthase characterization. To assess the different enzymes for LNT II synthesis, we performed reactions using NAG-oxa (60 mM) as the donor and lactose (600 mM) as the acceptor. Wild-type BbhI is optimally active at 55°C and pH 5.8.44 We used a lower temperature of 37°C and a higher pH of 7.5 to minimize the extent of non-enzymatic


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hydrolysis of the NAG-oxa donor substrate in the reaction. NAGoxa has a reported half-life of 15 h (30°C, pH 7.8)30 and BbhI retains 80% of its maximum activity under the reaction conditions.44 Note: in the time span of the conversion experiments, no evidence of enzyme inactivation was noted. Table 1. Activity and selectivity parameters of wild-type and site-directed variants of BbhI.

BbhI

NAG-oxaa TransRT/Hd Product glycosyla hydrolysi tion s (µmol -1 (µmol mg mg min1 min-1)c 1)c

GlcNAc--pNPb TransRT/He Product glycosyla hydrolys tion is (µmol (µmol mg mg-1 min1 min-1)c 1)c

WT

38

1

1.9

12

3

1.1

D746E

11

7

4.410-1

40

D746A

2.210-1

4

6.010-2 n.d.

1.010-2

4

2.710-2 n.d.

D746Q

2.610-2 2.4

210 -1 4

n.d.

1.310-3

2

n.d.

Y827F

5 6.310-2 2.610-1 1.910-2 n.d., not detectable. a60 mM NAG-oxa, 600 mM lactose, 37°C, pH 7.5. b20 mM GlcNAc--pNP, 400 mM lactose, 20% DMSO, 55°C, pH 5.8. cActivities for trans-glycosylation are given as µmol LNT II formed per min and mg purified enzyme used. Activities for product hydrolysis are given as µmol LNT II formed per min and mg purified enzyme used. d,e RTH is the ratio of the enzyme activities for trans-glycosylation and donor substrate hydrolysis. The RTH value was determined as the ratio of the LNT II concentration and the GlcNAc concentration at the time of maximum LNT II yield. dFor the reactions with NAG-oxa, the points marked with arrows in Figure 2, panels (a) – (e), indicate the times of RTH determination. eThe GlcNAc concentration was determined from the difference between the total concentration of GlcNAc--pNP converted (measured as pNP released) and the LNT II concentration formed.


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ACS Catalysis

All enzymatic reactions yielded a single trans-glycosylation product, identified as LNT II by reference to an authentic standard in HPLC and TLC (Figures S2 – S3 of the Supporting Information) and later directly by NMR. This result importantly shows that all enzymes were specific for -1,3-glycosylation of lactose under the conditions used; and they did not perform iterative glycosylation to also form higher oligosaccharides, a potential problem of selectivity found in chitinases.42-43,

48-49

There was no glycosylation of the GlcNAc released due to enzymatic or spontaneous hydrolysis of the NAG-oxa donor substrate. Our choice of BbhI for glycosynthase development was thus supported. We summarize in Figure 2 the results of full time-course analysis of LNT II formation by the different enzymes. The enzymes can be classified according to whether the LNT II released initially was degraded later in the reaction. The reaction time course of wild-type BbhI was characterized by rapid hydrolysis of the LNT II (Figure 2a). The D746A and D746Q variants (Figure 2b – 2c) showed no product hydrolysis. The D746E and Y827F variants (Figure 2d – 2e) retained activity for hydrolysis of LNT II. They reflect partial conversion of wild-type BbhI into glycosynthase. Interestingly, the maximum yield of LNT II was much higher for the D746A as compared to the D746Q variant (Figure 2f), despite the fact that


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both enzymes are hydrolytically deficient towards the product. This result emphasizes the need to evaluate for each enzyme how the individual activities for trans-glycosylation of lactose, donor substrate hydrolysis and product hydrolysis (Scheme 1b) combine to the overall course of LNT II synthesis. A comparative assessment of the synthetic utility of each enzyme will thus be possible and the differences in yield thus become clear.


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ACS Catalysis

Figure 2. LNT II synthesis by wildtype BbhI and variants thereof. Time courses show synthesis from 60 mM NAG-oxa using a 10-fold molar excess of lactose. (a) Wildtype, 0.23 µM; (b) D746A, 18 µM; (c) D746Q, 9 µM; (d) D746E, 8.4 µM; (e) Y827F, 4


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µM; (f) comparison of enzymes regarding the maximum yield of LNT II (black bars) and the selectivity parameter RTH (white bars). The LNT II product in panels (a) – (e) is shown as filled circles. The arrows in panels (a) – (e) indicate the times of determination of RTH, the ratio of the LNT II formed by transglycosylation of lactose from NAG-oxa and the GlcNAc released due to hydrolysis of NAG-oxa (see also Figure S4 of the Supporting Information). RTH is determined at the maximum yield of LNT II. RTH is calculated as [LNT II]/[GlcNAc] at the respective time indicated. The relevant GlcNAc concentration is shown in panels (a) – (e) as a filled green triangle. For easier viewing, this green triangle is also circled.

The specific activities for trans-glycosylation and product hydrolysis were obtained conveniently from the LNT II time course, as shown in Figure S4. However, the NAG-oxa was hydrolyzed spontaneously during the HPLC analysis. The assessment of donor substrate hydrolysis by the enzymes therefore required special consideration based on measurement of the GlcNAc time course. Figure S4 illustrates for the reaction of the wildtype enzyme how the analysis was performed. In each enzymatic reaction, as shown in Figure 2 and in more detail in Figure S4, there was a time period in which the concentration of


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ACS Catalysis

LNT II was almost constant at its maximum value. In that period, the corresponding GlcNAc concentration was also constant (Figure S4b). The time period serves to separate the time domain of trans-glycosylation/donor hydrolysis from that of LNT II hydrolysis. It also marks the point of full utilization of the NAG-oxa donor substrate. The ratio of the LNT II and GlcNAc concentrations at the maximum LNT II yield thus provided an estimate of the ratio of the NAG-oxa utilization rates for trans-glycosylation and hydrolysis (RTH). Arrows in Figure 2a – 2e indicate the times of RTH determination. Non-enzymatic hydrolysis of NAG-oxa is expected to be low (≤ 15%) at reaction times shorter than 2 h. To counteract non-enzymatic NAG-oxa hydrolysis, enzyme loadings giving maximum yields within 10 min – 120 min were used. We additionally showed for the wildtype enzyme and the D746E variant that use of NAG-oxa in excess (3.2fold) over lactose did not improve LNT II yields (Figure S5 of the Supporting Information). A summary of the time course analysis is shown in Figure 2f and the corresponding rate parameters are given in Table 1. A synthetically useful glycosynthase will hydrolyze neither the product nor the donor substrate. For practical use, good activity for trans-glycosylation is also essential. Among the available options here (Table 1), the D746E variant represented


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the best choice by a three-pronged criterion thus implied. The variant was only 3.4-fold less active in trans-glycosylation than wild-type BbhI, yet it showed 7-fold improved NAG-oxa utilization (RTH) and 32-fold lower activity for LNT II hydrolysis. It was 4.6-fold more active and showed 1.8-fold higher RTH than the Y827F variant. The D746A and D746Q variants had low specific activities. While unable to hydrolyze LNT II, they did hydrolyze the NAG-oxa donor (Figure 2b,c). The RTH of the D746A variant was 20-times that of the D746Q variant. In a comparison of just these two BbhI glycosynthases, therefore, the D746A variant would be more useful synthetically. The D746Q variant acted primarily as a NAG-oxa hydrolase under the conditions used (Figure 2c). However, even for the D746A variant, the RTH was lower than the RTH of the D746E variant. The LNT II yield based on the NAG-oxa used in the reaction was thus highest for the D746E variant (85%; Figure 2d and 2f). Nitro-phenyl glycosides are important glycosylation donor substrates for the study of enzymatic reactions, including those of glycosynthases.17,

41-42

It was of interest, therefore, to

compare GlcNAc--pNP with NAG-oxa for LNT II synthesis by the different BbhI enzymes. Using GlcNAc--pNP (20 mM) and lactose (400 mM) at conditions optimal for activity of wild-type BbhI (55 °C; pH 5.8), we performed time-course analysis of each


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ACS Catalysis

enzymatic reaction (Figure S6 of the Supporting Information). The corresponding rate parameters are summarized in Table 1. GlcNAc--pNP was stable during the analytical procedure used. Therefore, the RTH

could be determined from measurements of LNT

II and pNP release in the initial phase of the reaction, as shown in Figure S5. In terms of specific activity for transglycosylation at the respective conditions used, NAG-oxa was the better donor substrate than GlcNAc--pNP. The preference for NAGoxa was pronounced (≥ 20-fold) in the Asp746 variants. It was relatively smaller (3.2-fold) in wild-type BbhI. The RTH

values

varied somewhat depending on the donor substrate used. However, the overall trend, that the D746E variant was the best enzyme according to the RTH

criterion, was the same when NAG-oxa or

GlcNAc--pNP was used. Using the D746E variant, LNT II was obtained in 80% yield based on GlcNAc--pNP used in the reaction. Therefore, these results support the idea of using NAG-oxa as the donor substrate for LNT II synthesis. They also suggest the D746E variant as the catalyst to be used for the reaction. NAG-oxa synthesis. Synthetic utility of the glycosynthase route toward LNT II hinges on efficient production of the NAG-oxa donor substrate. A


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process modified after Shoda and co-workers was developed (Scheme 3). The synthesis involved 2-chloro-1,3-dimethyl-1Hbenzimidazol-3-ium chloride (CDMBI) as a dehydrative agent in 2fold molar excess over GlcNAc (80% yield).34 CDMBI was prepared in 2 steps from 2-hydroxybenzimidazole via 1,3dimethylbenzimidazolone (DMBI). The DMBI yield was increased from 70% to 92% by using KOH instead of NaOH.34 The NaOH reference experiment yielded 75% of DMBI. The DMBI was converted to CDMBI in 54% yield (49% in literature)34 by using an additional 1.1 equiv. of total oxalyl chloride, decreasing the temperature from 80°C to 70°C, and longer reaction times (5.5 h). The CDMBI yield was increased from 34%34 to 50% by these modifications. OH OH HN

N

Toluene Bu 4NBr KOH MeI

Cl

O N

N

60°C

2-Hydroxybenzimidazole

Toluene (COCl) 2

N

Cl N

70°C 92%

54 %

DMBI

CDMBI

O

HO HO O

NH

Na 3PO 4*12H 2O H 2O 0 - 3°C

OH

OH O

HO HO

O

N 40 %

NAG-oxa

Scheme 3. Chemical synthesis of NAG-oxa. The final product yield is after desalting the NAG-oxa by extraction into acetonitrile. Initial downstream processing of NAG-oxa involved flashfiltration and freeze-drying to a lyophilized crude product. Product identity and NAG-oxa content of the product was shown by


20

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ACS Catalysis

quantitative

1H

NMR spectroscopy (Figure S7 of the Supporting

Information). NAG-oxa was thus synthesized on 30 mmol scale in 60% yield from GlcNAc. No DMBI was detectable, but a large excess (6.5-fold) of Na3PO4

remained in the product. Shoda

demonstrated applicability of a non-desalted disialooligosaccharide oxazoline (8.5 mM) in reaction of Endo-M N175Q.34 However, solubility of our lyophilized crude product was limited to about 130 mM NAG-oxa, so desalting, to enhance the donor substrate solubility to about 600 mM, was crucial for the intensification of LNT II synthesis. This was achieved by extraction with acetonitrile (9.5 g per g lyophilized crude product), giving NAG-oxa in 60% yield on a 3.5 mmol scale. Thus, the desalted NAG-oxa was obtained through easily scalable procedures in 40% overall yield from unprotected GlcNAc. Importantly, no chromatography was required in the isolation of NAG-oxa.21,

38

Glycosynthase process technology for LNT II production. The initial experiments of LNT II synthesis (Figure 2, Table 1) had used lactose (600 mM) in 10-fold molar excess over NAG-oxa (60 mM). Ideally, equivalent amounts of donor and acceptor would however be used to obtain a high product concentration.14 We therefore analyzed effect of varied NAG-oxa concentration (130 – 600 mM) on LNT II synthesis from lactose (600 mM) using the


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Page 22 of 46

D746E variant (Figure S8 of the Supporting Information). Increase in the NAG-oxa concentration benefited the LNT II production not only in terms of the final product concentration (Figure 3a), but also in terms of the enzymatic synthesis rate (Figure 3b). The synthesis rate increased linearly with the NAGoxa concentration (Figure 3b), giving rise to an outstanding specific enzyme activity of 120 µmol mg-1 min-1 at the highest donor concentration used (600 mM). The LNT II released approached theoretical yield (90%) based on the NAG-oxa used in the reaction (Figure 3a). Note, the 600 mM of each NAG-oxa and lactose represents the solubility limit for the two substrates used in combination.


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ACS Catalysis

Figure 3. Production of LNT II with the BbhI D746E glycosynthase. (a) Time-course of LNT II synthesis by the D746E variant (4 µM) using equimolar amounts of NAG-oxa and lactose (600 mM). (b) LNT II synthesis rate for varied NAG-oxa concentrations. The straight line shows the linear regression fit (R2 = 0.90). Overlay of HPLC-UV traces (c) and HPLC-RI traces (d) used to evaluate the purity of LNT II produced on gramscale. The concentrations of authentic standards were: (c) 0.7 mM LNT II, 1 mM GlcNAc; (d) 40 mM LNT II, 100 mM Lac, 50 mM


23

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GlcNAc.

Page 24 of 46

The final product was dissolved in water to a

concentration of 642 mM (350 mg mL-1). The 200-fold diluted (c) or undiluted (d) samples were applied. Note, the first peak in the HPLC-RI traces is the injection peak. A time course of bulk production of LNT II is shown in Figure 3a. The initial production rate was

2.2  102 mg mL-1 h-1. LNT II

was obtained in good yield (86%) at 281 mg mL-1 (515 mM) within only 0.5 h of reaction. This corresponds to a RTH value of 6.1. The space-time yield (STY) of the overall synthesis was 562 mg mL-1 h-1. The mass‐based turnover number (mg product formed/mg enzyme added; TON) was 388, useful for a biotransformation conducted in batch mode. Only marginal hydrolysis of LNT II occurred under the conditions used (Figure 3a). The ratio of synthesis and hydrolysis of LNT II was 2200. Note: in Table 1, the ratio of synthesis and hydrolysis for the D746E variant was lower (183 = 11/0.06). This obtains because the synthesis was enhanced (10-fold) in response to the increase in NAG-oxa concentration from 60 mM (Table 1; Figure 2) to 600 mM (Figure 3). The LNT II hydrolysis was similar under the different reaction conditions (cf. Figure 2 and 3). Thus, as shown in Figure 3, production of ∼1 g of LNT II was enabled in only 3.6 mL of batch volume.


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These useful characteristics of the biocatalytic synthesis made it easy to recover the LNT II in at least technical-grade purity (85%, based on HPLC analyses, Figures 3c – 3d) in high yield (≥ 85%) directly from the reaction mixture. Note that the commercial reagent quality of LNT II is about 90% (Carboynth, Berkshire, Compton, UK). Enzyme was removed by ultrafiltration and solid product (∼1 g) was obtained as a white powder after freeze drying. The LNT II was thus prepared from NAG-oxa in ∼73% overall yield. The solid product additionally contained lactose (10%) and GlcNAc (5%). If nonetheless a higher product purity was required, from a process chemistry point of view, nanofiltration of the product solution could be an efficient means of removing the lactose (10%) and the GlcNAc (5%) from the main LNT II.50-51

Due to high solvent consumption, one may want

to avoid well-established methods of chromatography. However, we would like to point out that industrially produced oligosaccharides usually have a purity similar to the LNT II obtained here (e.g., 2’-fucosyllactose, 90%; lacto-Nneotetraose, 90%; Jennewein Biotechnologie GmbH, Rheinbreitbach, DE). The LNT II preparation from this study, we suggest, could be considered a possible final product, for blending applications in nutritional additives for example. Product identity was confirmed by

1H

and

13C

NMR spectroscopic analysis

(Figures S9 – S11 of the Supporting Information) and reference


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Page 26 of 46

to published NMR data.44 LNT II was the only product regio-isomer detected (Figure S10 of the Supporting Information). Overlay of NMR spectra (13C, HSQC) of isolated LNT II (Figure S10 of the Supporting Information) and commercial standard (Figure S11 of the Supporting Information) showed exact match of the signal at 82 ppm, characteristic for -GlcNAc linked to the C-3 position of -galactosyl in lactose. To demonstrate the synthetic advance due to the D746E variant as compared to wild-type BbhI, we performed reactions at one-tenth of the enzyme concentration used in bulk synthesis. The NAG-oxa concentration was also lowered (255 mM). From the results (Figure S12 of the Supporting Information), one immediately sees the benefit of the glycosynthase, namely a doubling of the maximum LNT II yield and almost no product hydrolysis upon extended reaction. The initial LNT II production rate was comparable for the two enzymes, but conversion with wild-type BbhI slowed down already after 5 min while it remained constant over 0.5 h with the D746E variant (Figure S12 of the Supporting Information). We would like to emphasize that in terms of solubility in the presence of lactose (600 mM), NAG-oxa (600 mM; 37°C) has clear advantage over GlcNAc--pNP (100 mM; 55°C, 20% DMSO). However, even if GlcNAc--pNP was used as donor substrate,


26

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ACS Catalysis

it would be the D746E variant that enabled higher LNT II yields (Figure S13 of the Supporting Information). Implications for enzymatic production of NAG-capped oligosaccharides. Efficient glycosynthase technology. Robust methods for regiocontrolled synthesis of NAG-capped oligosaccharides via enzymatic trans-glycosylation are of high interest. Considering the options of donor substrate for the reaction (e.g., GlcNAc-pNP, chitobiose), NAG-oxa presented a promising choice, but enzymes able to use it efficiently are not broadly available. The glycosynthase technology developed here builds on two important discoveries from the current study. First, the family GH-20 -N-acetyl-hexosaminidase BbhI utilizes NAG-oxa with high activity (40 µmol mg-1 min-1; 111 s-1) for regioselective glycosylation of lactose. Second, “glycosynthase engineering” yields enzyme variants, most notably the D746E variant, which compared to wild-type BbhI show substantially enhanced aptitude for trisaccharide synthesis. Optimized preparation of the NAGoxa donor substrate, ready for immediate use in the biocatalytic reaction, completes an integrated development for process chemistry application.


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Page 28 of 46

In targeting the residues stabilizing the oxazolinium ion intermediate of the enzymatic reaction (Scheme 1b), engineering of BbhI followed a “classic” mechanism-based approach for glycosaminidases.24-26,

35-38, 46, 49, 52

Detailed evaluation of the

BbhI variants provided new insight important for glycosynthase development. The high efficiency (530 M-1 s-1) and turnover frequency (340 s-1; 600 mM NAG-oxa) of the best improved BbhI variant (D746E) in the trans-glycosylation of lactose are worth emphasizing. The useful synthetic activity in combination with favorable characteristics of selectivity renders the D746E variant a good candidate biocatalyst for LNT II production. Besides having low activity (0.15 s-1) for hydrolyzing the trisaccharide product, the D746E variant also minimizes donor substrate hydrolysis in the synthetic reaction (RTH = 7). Although some control of reaction time is still required for high-yield synthesis, as shown in Figure 3a, the LNT II production is conveniently performed using the D746E variant. In terms of RTH the D746E variant surpasses the wild-type enzyme and other glycosynthases (e.g., D746A, D746Q) that would be preferred if absence of product hydrolysis was the only criterion of enzyme selection. A comprehensive, three-pronged criterion for assessing the synthetic efficiency of the glycosynthase is thus suggested.


28

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ACS Catalysis

Glycoside hydrolase family GH-20 comprises various -N-acetylhexosaminidases of potential interest for oligosaccharide synthesis.40-42,

53-56.

These enzymes share a conserved active site.

NAG-oxa was accepted as a glycosyl donor by mutant enzymes of Streptomyces plicatus -N-acetyl-hexosaminidase.40-41 It was also used by variant of the enzyme HEX1.57 However, utilization of NAG-oxa as donor substrate for trans-glycosylation is not a general property of family GH-20 enzymes, as demonstrated by the -N-acetyl-hexosaminidase from Talaromyces flavus.42 Since conversion into glycosynthase cannot be expected to elicit donor activity completely absent from the enzyme originally, choice of the wild-type GH-20 “template” seems to be important. Process intensification for LNT II production. LNT II synthesis was previously shown with Leloir glycosyltransferases utilizing uridine 5’-diphospho--N-acetyl-glucosamine as donor substrate for the glycosylation of lactose.4,

6, 12

Multi-enzyme one-pot

reactions combine LNT II synthesis with in situ supply of the sugar nucleotide donor.6 Whole-cell bio-transformations are preferred for large-scale application.6,

12

Compared to both these

glycosyltransferase systems, the glycosynthase process technology enables the LNT II production to be intensified in terms of the final product concentration, the STY or both.58-65 The process intensification is typically by two magnitude orders


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Page 30 of 46

or even higher. The glycosynthase reaction between NAG-oxa and lactose is completely specific for the LNT II product. The glycosynthase process for LNT II production brings oligosaccharide synthesis from sugar oxazoline donors to an unprecedented level of efficiency. Using a GH-18 endo-type glycosaminidase (chitinase), N,N’-diacetylchitobiose (172 mM) was previously synthesized from GlcNAc (3-fold excess) and NAGoxa, in 43% yield.33 Tri- and tetrasaccharides were prepared from disaccharide oxazolines,66 however in yields (50 – 70%) and product concentrations (50 mM) considerably lower than in LNT II synthesis. Selectivity of chitinases was a problem for synthesis of defined oligosaccharides. Polyaddition of sugar oxazoline derivatives gave chitooligomers with varying degree of polymerization.43,

48-49

Therefore, the conversion of a GH-1 β-

glycosidase into an exo-β-N-acetyl-glucosaminidase able to use NAG-oxa for trans-glycosylation (10 mM product; 80% yield) represents an interesting development.30 However, chemical derivatization of the acceptor substrate was required for proper regiocontrol of the glycosylation.67 CONCLUSIONS The family GH-20 β-N-acetyl-hexosaminidase BbhI utilizes NAG-oxa as donor substrate for selective -1,3-glycosylation of lactose.


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ACS Catalysis

High activity for product hydrolysis however limits the applicability of wildtype BbhI for production of LNT II. Glycosynthase design applied to BbhI resulted in enzyme variants that retain wildtype regioselectivity in trans-glycosylation, but lack (D746A, D746Q), or have strongly decreased (D746E, Y827F), activity for LNT II hydrolysis. When lactose acceptor is present, the D746E variant strongly favors trans-glycosylation over donor substrate hydrolysis (RTH). This feature, together with a specific activity of 100 U mg-1 under the synthesis conditions, renders the D746E variant a promising candidate catalyst for enzymatic trans-glycosylation of lactose from NAGoxa. Thus, we have developed a glycosynthase-catalyzed LNT II synthesis with high potential for process chemistry application. A glycosynthase process suitable for bulk trisaccharide production has broad significance in the field. It demonstrates the successful transformation of the glycosynthase mechanistic principle into an efficient process technology. It thus expands the scope of application for these important enzymes of glycoside synthesis. The current LNT II synthesis is relevant in the context of preparation of HMO core-structures by enzymatic glycosylation. Significant intensification of bio-catalysis compared to -Nacetyl-hexosaminidase- and glycosyltransferase-catalyzed


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reactions was achieved.44,

58-65

Page 32 of 46

The simple recovery of LNT II in a

purity of about 80% also benefits the scalability of the glycosynthase process. LNT II is one of the major building blocks of human milk oligosaccharides.1 Our LNT II synthesis offers now an efficient entry into these oligosaccharide structures. For further decoration of LNT II with other sugars (e.g., -galactosyl), -galactosidases61, glycosyltransferases4,

6, 12

68

or Leloir

could be used, giving lacto-N-

neotetraose (LNnT) or lacto-N-tetraose (LNT) as important human milk oligosaccharide products. EXPERIMENTAL SECTION Materials. Media components and chemicals were of reagent grade from Sigma Aldrich/Fluka (Vienna, Austria), Roth (Karlsruhe, Germany) or Merck (Vienna, Austria). GlcNAc was from Sigma Aldrich (Vienna, Austria). GlcNAc--pNP and LNT II were from Carbosynth (Compton, Berkshire, UK). Enzyme preparation. Production of the enzymes (without signal peptide and transmembrane region) and their purification were done according to literature.44 Briefly, synthetic BbhI genes (wild-type -N-acetyl-hexosaminidase from B. bifidum JCM1254 (GenBank: AB504521.1, aa 33-1599)44 and D746E, D746A, D746Q, Y827F variants) codon-optimized for E. coli expression were


32

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ACS Catalysis

ligated into HindIII-XhoI-cut pET21b(+) plasmid (BioCat GmbH, Heidelberg, Germany). Residue numbering of the full-length enzyme is used. All inserts were confirmed by DNA sequencing. BbhI enzymes were expressed in E. coli BL21(DE3) at 25°C for 20 h by induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside, using LB-medium supplemented with 115 mg L-1 ampicillin. Each enzyme was produced as C-terminal His6-tag fusion protein. Enzyme purification was done by single-step His6-tag affinity chromatography. The enzyme preparations used were pure by the criterion of migration as single protein band in SDS PAGE. Preparation of CDMBI. Synthesis modified after Shoda and coworkers34 was used. Full experimental details of the synthetic procedure, including relevant

1H

and

13C

NMR data, are provided

in the Supporting Information. Preparation of NAG-oxa. NAG-oxa was synthesized as described previously.34 Then the reaction mixture was flash-filtered over C18 resin (Chromabond Flash FM 70/10 C18 ac; Macherey Nagel, Düren, Germany) and lyophilized overnight (AdVantage Pro BenchTop; SP Scientific, New York, USA). Extraction with acetonitrile (9.5 g per g lyophilized crude product) under magnetic stirring at room temperature for 1 h was used for desalting. After concentration under reduced pressure at 25°C,


33

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Page 34 of 46

the sample was dried under oil-pump vacuum and stored at -20°C. See the Supporting Information for full details. Synthetic evaluation of enzymes. Reactions were performed in 600 µL total volume using 50 mM sodium phosphate buffer, pH 7.5 (with NAG-oxa) or pH 5.8 (with GlcNAc--pNP). Reactions contained 60 mM NAG-oxa, 600 mM lactose and 0.23 – 18 µM enzyme or 20 mM GlcNAc--pNP, 20% DMSO, 400 mM lactose and 0.12 – 24 µM enzyme. Temperature was 37°C (NAG-oxa) or 55°C (GlcNAc--pNP). An agitation rate of 650 rpm was used (Thermomixer comfort; Eppendorf, Hamburg, Germany). Samples taken at certain times were heat-treated (10 min, 99°C) and precipitated protein was removed by centrifugation (13,200 rpm, 10 min). The supernatant was analyzed by HPLC and TLC. LNT II and GlcNAc were measured from reactions with NAG-oxa. LNT II and pNP were measured from reactions with GlcNAc--pNP. Specific enzyme activities for formation and hydrolysis of LNT II were obtained from the reaction time course (see Figure 2 and Figure S4 of the Supporting Information). Specific activities (µmol min-1 mg-1) were calculated from the volumetric reaction rates (µmol mL-1 min-1) and the concentration of purified protein used (mg mL-1). The parameter RTH was obtained from the ratio of the LNT II and GlcNAc concentrations measured at the maximum LNT


34

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ACS Catalysis

II yield in the reaction (see Figure S4b of the Supporting Information). For reactions with GlcNAc--pNP, the difference of total GlcNAc--pNP consumption (measured as pNP release) and trans-glycosylation (measured as LNT II formation) was used to calculate the GlcNAc released by donor substrate hydrolysis. The total GlcNAc--pNP consumption (based on pNP release) was obtained from the reaction time courses (Figure S6 of the Supporting Information). Note: in spite of slightly different analytical procedures applied to the reactions with NAG-oxa and GlcNAc--pNP donor substrate, the definition of the parameter RTH is not changed. The RTH values obtained from the two experimental series can be compared to each other in this sense, however with the proviso that the incubation conditions with the two donor substrate were not the same. LNT II production. Enzymatic conversions, using NAG-oxa (130 – 600 mM) and lactose (600 mM), were carried out at pH 7.5, 37°C and 650 rpm (Thermomixer comfort; Eppendorf, Hamburg, Germany) in a total volume of 600 µL. The reaction was started by adding 0.73 mg mL-1 (4 µM) of the D746E variant. Samples were taken at certain times and analyzed by HPLC. Bulk production of LNT II was carried out as described above in a total volume of 3.6 mL, using 600 mM of each NAG-oxa and


35

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Page 36 of 46

lactose. It was performed in a 50 mL Sarstedt tube (diameter 2.8 cm, height 11.5 cm) under magnetic stirring (stir bar: 18  5 mm; 250 rpm) and in a water bath. Samples were taken at certain times and analyzed by HPLC. For DSP of LNT II, the reaction (600 mM of each NAG-oxa and lactose) was stopped after 45 min by heat-treatment (15 min, 99°C) and centrifuged (13,200 rpm, 15 min). The supernatant was freeze-dried overnight (Christ Alpha 1-4, B. Braun Biotech International, Melsungen, Germany). The product was analyzed by HPLC and its chemical identity confirmed by NMR. Analytics. LNT II, GlcNAc, pNP and lactose were analyzed by HILIC-HPLC using a Luna® NH2 column (3 µm, 100 Å, 250  4.6 mm; Phenomenex, Aschaffenburg, Germany). HPLC analysis was performed at 30°C with a 75% acetonitrile and 25% water at an isocratic flow rate of 1 mL min-1. UV-detection at 195 nm was used for quantification of LNT II, GlcNAc and pNP. Lactose was monitored by refractive index (RI) detection. TLC was performed on silica gel 60 F254 aluminum sheet (Merck, Darmstadt, Germany) using 1butanol/acetic acid/water (2/1/1 by volume). TLC plates were first analyzed under UV light (254 nm). Carbohydrates were then visualized with thymol – sulfuric acid reagent. LNT II, GlcNAc, pNP and lactose were used as authentic standards.


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ACS Catalysis

A Varian (Agilent) INOVA 500-MHz NMR spectrometer (Agilent Technologies, Santa Clara, California, USA) and the VNMRJ 2.2D software were used for all NMR measurements. 200 mg of isolated LNT II were dissolved in 600 µL D2O. LNT II standard (65 mM) was dissolved in D2O – H2O (11.5:1 v/v).

1H

NMR spectra (499.98 MHz)

were measured on a 5 mm indirect detection PFG-probe, while a 5 mm dual direct detection probe with z-gradients was used for

13C

NMR spectra (125.71 MHz). The HSQC spectrum was measured with 128 scans per increment and adiabatic carbon 180° pulses. Mnova 9.0 was used for evaluation of spectra. ASSOCIATED CONTENT Supporting Information. The Supporting Information is free of charge on the ACS Publications website. Detailed methods and materials, a computational model of the BbhI structure (Figure S1), HPLC analysis of LNT II synthesis from NAG-oxa (Figure S2), TLC analysis of LNT II synthesis from NAG-oxa (Figure S3), synthetic evaluation of BbhI enzymes (Figure S4), LNT II synthesis using NAG-oxa in excess (Figure S5), LNT II synthesis from GlcNAc--pNP (Figure S6), quantitative

1H

NMR spectrum of

crude NAG-oxa (Figure S7), effect of increasing NAG-oxa concentration on LNT II synthesis (Figure S8),

1H

and

13C

NMR

spectra of isolated LNT II (Figures S9 – S10), HSQC NMR spectrum of authentic LNT II (Figure S11), comparison of wild-type BbhI


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Page 38 of 46

and D746E mutant for LNT II synthesis (Figure S12), LNT II synthesis at maximum concentration of GlcNAc--pNP (100 mM; Figure S13) (PDF).

Corresponding Author *E-mail for B.N.: [email protected] Author Contributions The manuscript was written through contributions of all authors. K.B. and B.N. designed the research. K.S. and M.W. performed experiments. K.S. and M.W. analyzed data. K.S. and B.N. wrote the paper. All authors have given approval to the final version of the manuscript. Conflict of Interest M.W. and K.B. are employees of BASF SE with an interest in the commercial production of chemicals. There are no conflicts to declare for K.S. and B.N. ABBREVIATIONS CDMBI, 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride; DMBI, 1,3-dimethylbenzimidazolone; GlcNAc, N-acetyl-Dglucosamine; GlcNAc--pNP, para-nitrophenyl-N-acetyl--Dglucosaminide; LNT II, lacto-N-triose II; NAG-oxa, N-acetyl-Dglucosamine 1,2-oxazoline; TLC, thin layer chromatography.


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ACKNOWLEDGMENT This work was supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (BMVIT), the Styrian Business Promotion Agency, SFG, the Standortagentur Tirol and ZITTechnology Agency of the City of Vienna through the COMETFunding Programme managed by the Austrian Research Promotion Agency FFG. The authors thank Prof. Hansjörg Weber from the Graz University of Technology for NMR analysis and Margaretha Schiller for her excellent technical support. REFERENCES (1) Bode, L. Human Milk Oligosaccharides: Every Baby Needs a Sugar Mama. Glycobiology 2012, 22, 1147-1162. (2) Kunz, C.; Rudloff, S. Health Promoting Aspects of Milk Oligosaccharides. Int. Dairy J. 2006, 16, 1341-1346. (3) Smilowitz, J. T.; Lebrilla, C. B.; Mills, D. A.; German, J. B.; Freeman, S. L. Breast Milk Oligosaccharides: StructureFunction Relationships in the Neonate. Annu. Rev. Nutr. 2014, 34, 143-169. (4) Han, N. S.; Kim, T.-J.; Park, Y.-C.; Kim, J.; Seo, J.-H. Biotechnological Production of Human Milk Oligosaccharides. Biotechnol. Adv. 2012, 30, 1268-1278. (5) Bode, L.; Contractor, N.; Barile, D.; Pohl, N.; Prudden, A. R.; Boons, G. J.; Jin, Y. S.; Jennewein, S. Overcoming the Limited Availability of Human Milk Oligosaccharides: Challenges and Opportunities for Research and Application. Nutr. Rev. 2016, 74, 635-644. (6) Chen, X., Human Milk Oligosaccharides (HMOS): Structure, Function, and Enzyme-Catalyzed Synthesis. In Adv. Carbohydr. Chem. Biochem., Baker, D. C.; Horton, D., Eds. Academic Press: 2015; Vol. 72, pp 113-190. (7) Danby, P. M.; Withers, S. G. Advances in Enzymatic Glycoside Synthesis. ACS Chem. Biol. 2016, 11, 1784-1794. (8) Kulkarni, S. S.; Wang, C.-C.; Sabbavarapu, N. M.; Podilapu, A. R.; Liao, P.-H.; Hung, S.-C. “One-Pot” Protection,


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