All living organisms need energy from their environment for maintenance and growth. The carbohydrate starch is such an energy source. It is an abundant carbohydrate composed of amylose and amylopectin polymers. Before organisms can utilize the starch, it has to be degraded to smaller sugars suitable for uptake into the cells. Therefore, these organisms secrete several starch degrading enzymes of which cyclodextrin glycosyltransferase (CGTase) is an example.

Zie: Summary in "thesis"

Leemhuis, Reinder Johannes,

English

Leemhuis, Reinder Johannes

University of Groningen

   University of GroningenWhat makes cyclodextrin glycosyltransferase a transglycosylaseLeemhuis, Reinder JohannesIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2003Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Leemhuis, R. J. (2003). What makes cyclodextrin glycosyltransferase a transglycosylase. Groningen: s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019 89  Chapter 6   Mutations Converting Cyclodextrin Glycosyltransferase from a Transglycosylase into a Starch Hydrolase  Hans Leemhuis, Bauke W. Dijkstra and Lubbert Dijkhuizen  Published in FEBS Letters, volume 514, pages189-192, 2002.       SUMMARY Cyclodextrin glycosyltransferase (CGTase) efficiently catalyses trans-glycosylation of oligo-maltodextrins, although the enzyme has also a low hydrolytic activity. Its +2 substrate binding subsite, which contains the conserved Phe184 and Phe260 residues, has been shown to be important for this transglycosylation activity [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. Here we show that the amino acid side chain at position 260 also controls the hydrolytic activity of CGTase. Three Phe260 mutants of Thermoanaero-bacterium thermosulfurigenes CGTase were obtained with a higher hydrolytic activity than ever observed before for a CGTase. These Phe260 mutations even changed CGTase from a transglycosylase into a starch hydrolase.   INTRODUCTION The α-amylase family, or glycoside hydrolase family 13, is a large family of starch processing enzymes [23;24]. The catalytic site residues and the α-retaining bond cleavage mechanism are strictly conserved in all members of this family [7;11], but the product and reaction specificity vary widely. Cyclodextrin glycosyltransferase (CGTase) is also a member of this family. It forms circular α-(1,4)-linked oligosaccharides (cyclodextrins) from linear α-(1,4)-linked oligosaccharide substrates. This reaction proceeds via a covalent Chapter 6 90  intermediate [11;16]. The non-reducing end of this intermediate is subsequently used as the acceptor that cleaves the covalent enzyme-substrate bond, and a cyclodextrin is released. CGTase can also use water or the non-reducing end of a free oligosaccharide as acceptor, which results in a hydrolysis or a disproportionation reaction, respectively.    Figure 1. Schematic overview of the interactions between Tabium CGTase and a maltohexaose inhibitor bound from subsites –3 to +3. For clarity, not all interactions at subsites –1 and +1 are shown [58].  Whereas α-amylase is a strongly hydrolytic enzyme, CGTase is first of all a transglycosylase. The hydrolysis activity of CGTases is in general much lower than the disproportionation and cyclization activities. Only the CGTases from Thermoanaerobacter [147] and Thermoanaerobacterium thermosulfurigenes strain EM1 (Tabium) [120] have relatively high hydrolysis activities, although still very low compared to α-amylases. The ratio between hydrolysis and transglycosylation is determined by the nature of the acceptor used and by the properties of the CGTase acceptor subsites. While acceptor subsite +1 is conserved in CGTases and α-amylases, the +2 subsite is only conserved in CGTase [40;44;58;73;77;135;148]. At this subsite Phe184 and Phe260 interact with bound oligosaccharides (Fig. 1) [124;149]. Furthermore, it has been suggested that binding of sugars at the acceptor subsites, but not water, activates CGTases for catalysis [42]. This induced-fit model can explain the Acceptor subsite +2 in Tabium CGTase   91 transglycosylation specificity of CGTase with its associated low hydrolysis activity.  Here we describe mutations of three aromatic residues in Tabium CGTase. Phe184 and Phe260 are part of subsite +2, while Phe196 is located in the centre of the active site cleft (Fig. 1). All three residues interact with substrates and mutagenesis experiments have already demonstrated the importance of these residues in the transglycosylation activities of CGTase [77;123;124;149;150]. Mutation of Phe260 strongly enhanced the hydrolysis activity, converting CGTase into an enzyme preferring hydrolysis to transglycosylation.   MATERIALS AND METHODS Bacterial strains, plasmids and growth conditions – Escherichia coli strain DH5α [141] was used for DNA manipulations. Plasmid pCT2, with the Tabium cgt gene [115], was used for site-directed mutagenesis and sequencing. CGTase proteins were produced with plasmid pCScgt-tt [131] and Bacillus subtilis strain DB104A as host [116]. Plasmid carrying strains were grown on LB medium [132] at 37° C in the presence of ampicillin (100 µg/ml) or kanamycin (50 µg/ml for E. coli and 5 µg/ml for B. subtilis).  Production and purification of CGTase proteins – CGTase (mutant) proteins were produced and purified as described [33]. Purity and molecular weight were checked by SDS-polyacrylamide gel electrophoresis. Enzyme concen-trations were determined using the Bradford reagent from Bio-Rad (München, Germany) and bovine serum albumin as standard. DNA manipulations – Mutants were constructed in pCT2 as described [58] and verified by DNA sequencing. Mutations were subsequently introduced in pCScgt-tt by exchanging its 1300 bps ScaI/DraIII fragment for the corresponding fragment of pCT2 carrying the mutation. The following oligonucleotides were used:  F184S, 5’-CATTATGGAGGTACCGATTCTTCATCTTATG-‘3 (KpnI); F196G, 5’-TATCGTAACTTAGGTGGTGATTTAGCAGATCTAAATCAAC-‘3 (BglII); F260L, 5’-CCAGTATTTACATTTGGAGAGTGGTTACTTGGAA CG-‘3; F260N, 5’-CCAGTATTTACATTTGGAGAGTGGAATCTTGGAAC G-‘3; F260H, 5’-CCAGTATTTACATTTGGAGAGTGGCATCTTGGAACG-‘3; F260R, 5’-CCAGTATTTACATTTGGAGAGTGGCGTCTTGGAACG-‘3; F260E, 5’-CCAGTATTTACATTTGGAGAGTGGGAACTTGGAACG-‘3; F260I, 5’-CCAGTATTTACATTTGGAGAGTGGATTCTTGGAACG-‘3. Restriction sites are underlined. Mutations in Phe260 resulted in loss of a StuI Chapter 6 92  restriction site. The double mutants F184S/F260N and F196G/F260N were made using plasmid pCT2 with the F260N mutation as template in the PCR reactions. Enzyme assays – All enzyme assays were performed in 10 mM sodium citrate buffer (pH 6.0) at 60º C. β-Cyclodextrin forming activity was determined by incubating 0.1–5.0 µg enzyme/ml with a 2.5% (w/v) solution of partially hydrolysed potato starch with an average degree of polymerisation of 50 (Paselli SA2; AVEBE, Foxhol, The Netherlands). The amount of β-cyclodextrin produced was quantified with phenolphthalein [118]. Disproportionation activity was determined as described before [75;121], using 0.1–5.0 µg enzyme/ml, 1 mM 4-nitrophenyl-α-D-maltoheptaoside-4-6-O-ethylidene (EPS; Roche) and 10 mM maltose as donor and acceptor substrates, respectively. Hydrolysing activity was measured by following the increase in reducing power, using 1% (w/v) soluble starch (Lamers & Pleuger, Belgium) and 1 µg/ml of enzyme [33]. Formation of cyclodextrins and linear products from 10% (w/w) pregelatinised starch (Paselli WA4; AVEBE) was measured by incubating the starch solution for 24 h with 0.25 U/ml β-cyclodextrin forming activity (0.05 U/ml for F260R and F196G/F260N). Products formed were analysed by HPLC equipped with an Econosphere NH2 5U column (250 by 4.6 mm; Alltech Nederland bv, Breda, The Netherlands) eluted with acetonitrile/water (60/40, v/v; 1 ml/min).   RESULTS AND DISCUSSION Phenylalanine residues 184, 196 and 260 are important for trans-glycosylation activity – To investigate the importance of subsite +2 and residue 196 for the various activities of CGTase we mutated three conserved phenylalanines (Phe184, Phe196 and Phe260 in Tabium CGTase). The strongly reduced cyclization and disproportionation activities of the Phe184, Phe196 and Phe260 mutants (Table 1) are in agreement with results of mutations of the corresponding residues in other CGTases [33;77;123;124;149;150]. The double mutants F184S/F260N and F196G/F260N showed even a stronger reduction in cyclization and disproportionation activity (Table 1) than would be expected from a combination of the single mutants suggesting that Phe184/Phe260 and Phe196/Phe260 act synergistically in these reactions. These results are consistent with a proposed induced-fit mechanism for CGTase, in which sugar binding at the acceptor subsites induces structural rearrangements that activate Acceptor subsite +2 in Tabium CGTase   93 catalysis [42]. Thus, the decreased transglycosylation activities show that phenylalanine residues 184, 196 and 260 are important in the cyclization and disproportionation reactions.  Table 1. Hydrolysis, cyclization and disproportionation activities of Tabium wild-type and mutant CGTases at pH 6.0 and 60° C. Enzyme Hydrolysis of starch (µmol min-1 mg-1) β-Cyclization (µmol min-1 mg-1) Disproportionation (µmol min-1 mg-1) Wild-type 54 ±   4 240 ± 9 510 ± 23 F184S 60 ±   7 52 ± 6 230 ± 12 F196G 30 ±   3 27 ± 2 36 ±   2 F260N 117 ± 12 31 ± 3 95 ±   7 F260L 174 ± 10 33 ± 2 167 ± 10 F260I 174 ±   7 17 ± 1 218 ±   5 F260E 177 ± 13 19 ± 1 21 ±   4 F260H 25 ±   3 74 ± 3 177 ± 14 F260R 17 ±   2 0.7 ± 0.2 < 1 F184S/F260N 118 ± 11 5 ± 1 20 ±   2 F196G/F260N 15 ±   1 0.8 ± 0.2 < 1  Phe196 is important for transglycosylation and hydrolysis – The centrally located Phe196 residue (Fig. 1) has been suggested to play a role in limiting the hydrolysis activity of CGTase, since α-amylases (hydrolases) have a small residue (glycine, alanine, serine) at this position [86]. Indeed the Y195G mutation in BC251 CGTase, which is equivalent to position 196 in Tabium CGTase, shows an increased hydrolytic activity [68]. Unexpectedly, however, the corresponding F196G mutant of Tabium CGTase has a two-fold lower hydrolytic activity (Table 1). Thus, Phe196 is not only important for the transglycosylation activity, but also for the enzyme’s hydrolytic activity. Despite this, mutant F196G produced three times more linear products from starch than wild-type CGTase over a 24 h incubation period (Fig. 2). This indicates that residue Phe196 is a clear determinant for the linear versus circular product specificity of CGTase. Phe260 limits the hydrolytic activity – Hydrolysis is a minor activity of CGTases. Nevertheless, several mutations are known to increase the enzyme’s hydrolytic activity [33;75;76;124]. The most substantial enhancement of hydrolytic activity is, however, achieved by mutations of subsite +2 residues (Table 2). It has been suggested that the hydrophobicity of Phe183 and Phe259 Chapter 6 94  at subsite +2 limits the hydrolytic activity of BC251 CGTase [124]. Mutation of the corresponding residues in Tabium CGTase showed that the F184S mutation changed the hydrolysis rate only slightly (Table 1). In contrast, mutations in Phe260 either significantly decreased (F260H,R) or increased (F260N,L,I,E) the hydrolytic activity (Table 1). Mutation F260N doubled the hydrolytic activity, whereas the corresponding F259N mutation in BC251 CGTase increased the hydrolytic activity 20-fold (Tables 1 and 2). Both mutants, however, enhanced the hydrolytic activity with about 60 units, suggesting that the mutations have a similar effect in both CGTases and that the 20-fold enhancement is a consequence of the low hydrolytic activity of BC251 CGTase. The hydrolytic activity of CGTase thus is strongly modulated by the identity of residue 260.    Figure 2. Cyclodextrin and linear oligosaccharide (glucose to malto-octaose) products formed during incubation of the Tabium CGTase variants with 10% (w/v) starch for 24 h at 60° C. From bottom to top, α-CD (black), β-CD (gray), γ-CD (dark gray) and linear oligosaccharides (light gray).  Furthermore, our mutations reveal that it is not the hydrophobicity of the side chain at position 260 that is important for the enzyme’s hydrolytic activity, since similar sized hydrophobic (F260I,L) as well as hydrophilic (F260N,E) substitutions increase the hydrolytic activity to a similar extent (Table 1). Acceptor subsite +2 in Tabium CGTase   95 Therefore we conclude that the aromatic side chain of Phe260 is instrumental in limiting the hydrolytic activity of CGTase.  Table 2. Subsite +2 mutant CGTases described in literature and their hydrolysis activities. The Thermoanaerobacter CGTase is included, because the wild-type enzyme has a high hydrolytic activity. CGTase Hydrolysis activity  (µmol min-1 mg-1) Reference Tabium    Wild-type 54 this study B. circulans strain 251   Wild-type  3 [124] F183S 9 [124] F183N 11 [124] F259S 33 [124] F259N 60 [124] B. stearothermophilus   Wild-type 2 [149] F260I 4 [149] B. sp. 1011   Wild-type ND [123] F183L ND [123] F259L ND [123] Thermoanaerobacter   Wild-type 82 [152] Chemically modified 127 [152] ND, not determined.  Until now wild-type Thermoanaerobacter CGTase displayed the highest hydrolytic activity [152]. Three Tabium mutants (F260L,I,E), however, possess even higher hydrolytic activities, and significantly lower cyclization activities (Table 1). The data shows that single mutations can change CGTase reaction specificity from cyclization into hydrolysis. A product analysis after 24 h incubation of starch with (mutant) CGTases also showed that these Phe260 mutants produced substantially more linear than circular products (Fig. 2). Single mutations thus change CGTase product specificity from cyclodextrins into oligo-maltodextrins. Chapter 6 96  Conclusions – Our results show that Phe260 has an important role in limiting the hydrolytic activity of CGTase. Mutation of Phe260 into a Leu, Ile, or Glu resulted in mutants with the highest hydrolysis activity ever reported for a CGTase. Furthermore, CGTase can be changed from a transglycosylase into a hydrolase by mutations in Phe260, a residue that is strongly conserved in CGTase proteins. The data presented provide new insights into the hydrolysis reaction catalysed by CGTase and factors determining the transglycosylation versus hydrolysis specificity in the α-amylase family. Acknowledgements – H.L. acknowledges financial support from Danisco.       

What makes cyclodextrin glycosyltransferase a transglycosylase

All living organisms need energy from their environment for maintenance and growth. The carbohydrate starch is such an energy source. It is an abundant carbohydrate composed of amylose and amylopectin polymers. Before organisms can utilize the starch, it has to be degraded to smaller sugars suitable for uptake into the cells. Therefore, these organisms secrete several starch degrading enzymes of which cyclodextrin glycosyltransferase (CGTase) is an example. Zie: Summary in "thesis

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 University of Groningen
What makes cyclodextrin glycosyltransferase a transglycosylase
Leemhuis, Reinder Johannes
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2003
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Leemhuis, R. J. (2003). What makes cyclodextrin glycosyltransferase a transglycosylase Groningen: s.n.
Copyright
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Download date: 10-02-2018
What makes cyclodextrin 
glycosyltransferase a transglycosylase 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Hans Leemhuis
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The cover shows a maltononaose substrate (left), a maltoheptaose reaction 
intermediate (right) and a cyclodextrin product (lower) bound in the active site 
of the cyclodextrin glycosyltransferase from Bacillus circulans strain 251. The 
back cover shows the structure of the native enzyme. 
 
The author gratefully acknowledges support by Danisco and GBB for printing 
this thesis. 
 
 
RIJKSUNIVERSITEIT GRONINGEN 
 
 
 
 
What makes cyclodextrin 
glycosyltransferase a transglycosylase 
 
 
 
 
Proefschrift 
 
 
ter verkrijging van het doctoraat in de  
Wiskunde en Natuurwetenschappen 
 aan de Rijksuniversiteit Groningen 
 op gezag van de  
Rector Magnificus, dr. F. Zwarts, 
in het openbaar te verdedigen op 
 maandag 14 april 2003 
 om 16.00 uur 
 
 
door 
 
 
Reinder Johannes Leemhuis 
geboren op 9 november 1973 
te Groningen 
 
Promotores:  Prof. dr. L. Dijkhuizen 
Prof. dr. B.W. Dijkstra 
 
 
Beoordelingscommissie: Prof. dr. D. B. Janssen 
Prof. dr. A. J. M. Driessen 
Prof. dr. O. P. Kuipers 
 
VOORWOORD 
 
 
Na bijna vier jaar onderzoek in het lab van Prof. Lubbert Dijkhuizen 
(Microbial Physiology) is het tijd om dit promotieonderzoek af te ronden door 
het schrijven van het voorwoord van dit proefschrift. Voor het tot stand komen 
van dit boekje wil ik ten eerste Lubbert bedanken voor de mogelijkheid om in 
zijn groep onderzoek te verrichten. Hierdoor heb ik met veel plezier een 
bijdrage kunnen leveren aan het ontrafelen van de reactiespecificiteit van het 
enzym CGTase. De jaarlijkse meetings met Danisco, te Arhus en Kopenhagen, 
en de discussies over CGTase die wij tijdens het reizen hadden, hebben hieraan 
bijgedragen. Binnen Danisco liepen de contacten via Karsten. Karsten, I thank 
you for the nice collaboration, and for giving me the opportunity to do 
fundamental research on CGTase, the enzyme of common interest.  
Verder wil ik Prof. Bauke Dijkstra (Protein Crystallography) bedanken. 
Hoewel we niet op regelmatige basis samengewerkt hebben is jouw bijdrage 
aan dit onderzoek van grote waarde geweest. Vooral denk ik dan aan de 
discussies achter een monitor om de effecten van mutaties te verklaren aan de 
hand van driedimensionale structuren en natuurlijk aan de scherpe 
vragen/opmerkingen in de manuscripten.  
Ook Henriëtte heeft een belangrijke bijdrage aan dit boekje geleverd door de 
structuren van mutant CGTases te bepalen, hetgeen de kwaliteit van vier 
hoofdstukken heeft verhoogd. In het begin van mijn promotieonderzoek hebben 
de discussies met Joost en Bart gezorgd voor een snelle overdracht van de 
CGTase kennis, waarvoor dank. Verder wil ik Maarten (student) bedanken 
voor zijn screenings werk (hoofdstuk 7), Gea voor de hulp, uitleg en onderhoud 
van de DSC apparatuur (hoofdstuk 9), Gert-Jan voor het programmeren van 
Plato (de pipetteerrobot; hoofdstuk 7) en Robert, Michael, Edwin en Janine 
voor hun hulp en tips bij het layout werk.  
 Tenslotte is deze periode plezierig geweest door de vele microbiologie 
collega’s, met wie koffie gedronken en geborreld werd. 
 
Hans 
Groningen, januari, 2003 

CONTENTS 
 
 
Chapter 1          9 
General intorduction: The α-amylase family: transglycosylation  
and hydrolysis reaction specificity 
Chapter 2          29 
Thermoanaerobacterium thermosulfurigenes cyclodextrin  
glycosyltransferase: mechanism and kinetics of inhibition by acarbose  
and cyclodextrins 
Chapter 3          45 
The fully conserved Asp residue in conserved sequence region I of the  
α-amylase family is crucial for the catalytic site architecture and activity 
Chapter 4          55 
The remote substrate binding subsite -6 in cyclodextrin glycosyltransferase  
controls the enzyme’s transferase activity via an induced-fit mechanism 
Chapter 5          73 
Hydrophobic amino acid residues in the acceptor binding site are main  
determinants for reaction mechanism and specificity of cyclodextrin 
glycosyltransferase 
Chapter 6          89 
Mutations converting cyclodextrin glycosyltransferase from a  
transglycosylase into a starch hydrolase 
Chapter 7          99 
Conversion of cyclodextrin glycosyltransferase into a starch hydrolase  
by directed evolution: the role of Ala230 in acceptor subsite +1 
Chapter 8                  117 
A five-residue amino acid insertion converts cyclodextrin  
glycosyltransferase into a starch hydrolase with a high exo-specificity 
Chapter 9                  129 
Improved thermostability of Bacillus circulans cyclodextrin  
glycosyltransferase by the introduction of a salt-bridge 
 
Summary                  143 
Samenvatting                 151 
References                  157 
Overview of mutant CGTases               179 
Abbreviations                 182 
Publication list                           183 


University of Groningen Digital Archive

A fiveresidue amino acid insertion converts cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity.

Ala Essential for the conformation of

Ala General activity [135;148]

Ala General avtivity b

Asn Catalytic nucleophile [11;16;135;148]

Asn, Arg General activity [45;157]

Asn, Arg General activity [45;157] Subsite –1 Catalytic subsite

Asn, Arg General activity, less α-cyclodextrin a [58]

Asp His Reduced activity,

Characterization of the gene cluster involved in isoprene metabolism in Rhodococcus sp. strain AD45.

Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution: The role of Ala230 in acceptor subsite +1.

Cyclization, limitation of hydrolysis [53;123;124;149] Subsite +1 Acceptor subsite

Enzyme structure and mechanism, 2

Gln Acid-base catalyst

Gln Ala Cyclization, disproportionation [124] Subsite +2 Acceptor subsite

Glu Cyclodextrin specificity a

Gly Leu Selection of long substrates [74;chapter 4]

Gly Selection of long substrates [74;chapter 4]

Hemopexin and affection of vascular Ecto-ATP diphosphohydrolase.

His Asp General activity [157]

His Trp,

Hydrolysis and Transglycosylation Reaction Specificity of Cyclodextrin Glycosyltransferases.

Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin glycosyltransferase.

Improved thermostability of Bacillus circulans cyclodextrin glycosyltransferase by the introduction of a salt-bridge.

Lys Ala Reduced binding to raw starch [33]

Lys General activity [chapter 3]

Lys Leu General activity [124]

Mechanism of Thermoanaerobacterium thermosulfurigenes CGTase: Mechanism and kinetics of inhibition by acarbose and cyclodextrins.

Mutations converting cyclodextrin glycosyltransferase from a transglycosylase into a starch hydrolase.

Phe All possibilties Cyclization [53;68;77;123; 149;150]

Properties and applications of starch-converting enzymes of the α-amylase family.

Ser, Leu Cyclization,

The fully Asp residue in conserved sequence region I of the α-amylase family is crucial for the catalytic site architecture and activity.

The reaction cycle of cyclodextrin glycosyltransferase.

The remote substrate binding subsite -6 in cyclodextringlycosyltransferase controls the transferase activity of the enzyme via an induced-fit mechanism.

Thr Cyclization [77]

Transglycosylation specificity

Trp Ala Reduced binding to raw starch [33]

Trp Ala Reduced binding to raw starch a

Trp Cyclodextrin specificity a Replacement of loop 145-151 by an Asp residue Reduced activity and cyclodextrin specificity [77] Insertion of Ile residue between 145 and 146 Increased γ-cyclodextrin specificity a MBS 1 Maltose binding site [32]

What makes cyclodextrin glycosyltransferase a transglycosylase

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