522 Carbohydrate Biotechnology

Enzymes in Carbohydrate Production Two main classes (IUB Classification) • Glycosyl Hydrolase (GH, E.C. 3.2) • Glycosyl Transferase (GT, E.C. 2.4)

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Terms Define 

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Enzymes for in vivo production of CHOs • enzymes used by living cells to produce CHOs Enzymes for industrial production of CHOs • enzymes produced in large scale to hydrolyze / synthesize / modify CHOs for industrial benefits Bulk enzymes • industrial enzymes produced in bulk quantities (>102 tons annually), most from bacterial fermentation, produced as technical (e.g.food) grade • 80% of industrial enzymes are “Hydrolase”, used in depolymerization of proteins, CHOs, and lipids ( Protease, Carbohydrase, and Lipase )

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Carbohydrate Production In vivo GH-Amylases

-Cellulase,Hemicellulase -Pectinase -Lactase -Pullulanases -Invertase - etc.

In Industry GH-Amylases*

-Cellulase*Hemicellulase -Pectinase* -Lactase

(ß-galactosidase)*

-Pullulanases -Invertase * Bulk enzymes

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Carbohydrate production

In vivo GT-Specific GT e.g.

In Industry GT- CGTase*

glucosyl/galactosyl/ fucosyl/sialyl,etc.

-starch synthase -CGTase I -Glucose isomerase* The only one isomerase (EC 5.3) important as industrial enzyme

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O-Glycosyl Hydrolase, E.C. 3.2.1.x (Carbohydrase, Glycosidase)  

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Hydrolyze glycosidic bonds Most important in hydrolysis of CHO polymers in industry (food,textile,detergent,paper,pharmaceutics) Amylase family (  -, ß -, and glucoamylase) has greatest use in starch processing to produce dextrin, glucose, maltose Cellulase, lactase, and pectinase – used to hydrolyze cellulose,lactose, and pectin Grouped into ~ 70 GH families based on amino acid similarities Two categories : retaining vs inverting enzyme 6

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General Catalytic Reaction by GH 

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Hydrolysis of glycosidic bond between monomers, with water molecules as glycosyl acceptor (Glycosyl)n-OR + H2O (Glycosyl)n-OH + H-O-R R = oligosaccharide at the reducing end of a glycosyl chain (see frame 13)

Depending on specificity of each enzyme, hydrolysis may be • endo- or exo•  - or ß • 1,4- or 1,6- linkage specific 8

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For synthetic side Reverse hydrolysis results in oligosaccharide synthesis, but in a very low yield. Equilibrium shift could be performed by using high [s], low water content, high temperature, or long incubation time Many glycosidases, in addition to hydrolysis, also have transglycosylation activity, which can be activated by attaching good leaving groups on substrate 9

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Action modes of amylases and their reaction products from starch and related enzymes

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Enzymes in -amylase family according to E.C. classification

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Bulk industrial enzymes in Amylase family 

 - , ß - , and glucoamylase - GH

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Cyclodextrin glycosyltansferase ( CGTase ) - GT

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Catalytic Mechanisms of GH 

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GHs have 2 -3 acidic amino acids as active site residues Hydrolysis takes place via general acid catalysis that requires 2 residues, a proton donor and a nucleophile or base Two mechanisms have been proposed, giving rise to retention or inversion of anomeric configuration

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Nucleophilic displacement mechanism (SN2 reaction) 

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Main characteristics : covalent-glycosyl enzyme intermediate Double displacement yields retaining configuration COO – makes nucleophilic attack at C-1, covalent intermediate is formed (anomer inversion obtained), then OH – of water nucleophilically attack C-1 from the reverse side of the covalent bond of the enzyme intermediate, glycosyl with reducing end releases, and product with retaining configuration is obtained.

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Single displacement yields inverting configuration COO – promotes nucleophilic attack by OH – of water at C-1 from ß (front) anomeric side, then covalent intermediate formed, another COO – helps stabilized, release of reducing end and inverting anomer is obtained.

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Oxocarbonium ion intermediate mechanism (SN1 reaction)  

Main characteristics : oxocarbonium ion intermediate For anomer retaining and inverting products protonation of glycosidic O2 by COOH of enzyme, release of reducing end, oxocarbonium ion intermediate formed, then COO – promotes nucleophilic attack of OH – of water from the  - side (by the retaining enzyme) or ß - side (by the inverting enzyme)

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For retaining enzyme, distance between 2 catalytic residues is ~ 5.5 Å For inverting enzyme, distance between 2 catalytic residues is ~ 10 Å

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Catalytic mechanism of industrial GH GH Enzyme  - amylase ß – amylase

E.C.number

Catalytic mech.

3.2.1.1

retention(SN1,SN2)

3.2.1.2

inversion(SN1,SN2)

glucoamylase

3.2.1.3

inversion(SN1,SN2)

cellulase

3.2.1.4 / 91

(EG,endoglucanase CBH,cellobiohydrolase)

pectinase (PG,polygalacturonase lactase CGTase (GT)

retention(SN2 for EG,CBH I);inversion (SN2 for CBH II)

3.2.1.15 /67/82

inversion

3.2.1.23

retention(SN2)

2.4.1.19

retention(SN2)

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Topology of Active site of GH  

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Three types of topology are found Pocket (Crater) - optimal for recognition of a saccharide with non-reducing extremity, not efficient for fibrous substrate (with no free chain ends), found mostly in exo-enzyme e.g. glucoamylase, ß amylase, ß - galactosidase, ß – glucosidase Cleft (Groove) – open structure, allows random binding of several sugar units in polymeric substrate, commonly found in endo-enzyme e.g.  - amylase, endocellulase, chitinase, xylanase, glucanase 22

Pocket

Cleft

Tunnel

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Tunnel – formed from the Cleft topology in which enzyme evoles to have long loops cover the cleft part, found only in Cellobiohydrolase II (CBH II), enzyme can release product while still firmly bound to polysaccharide chain, creating processitivity (which may be important in efficient degradation of insoluble cellulose)

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Active site residues and topology of industrial GH GH Enzyme  - amylase

Topology

Residues

cleft

Asp231,Glu261,Asp 328 B.licheniformis

ß - amylase

pocket

Glu186,Glu380 soybean

glucoamylase

pocket

Glu179,Glu400

cellulase (PG)

cleft (for EG), tunnel (for CBH II)

pectinase

A.awamori

Glu63,Asp76 (EG I) Glu244,Asp263(CBH II) Asp180,201,202

A.niger

lactase

pocket

Glu537,461

CGTase (GT)

cleft

Glu257,Asp229,Asp328

B.circulans

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3D-Structure and Classification in GH Family of Industrial GH GH Enzyme  - amylase some thermostable - amylase

GH Family GH 13 57

3D-structure GH 3 domains, (ß/)8 barrel catalytic domain, ß-fold raw starch binding domain

ß - amylase

GH 14

3 domains, (ß/)8 catalytic domain,many loops

glucoamylase

GH 15

3 domains, (/)6 catalytic domain, ß strands of raw starch binding domain

lactase

GH 2 (lac Z)

2-6 subunits,each has 5 domains, /ß barrel

(ß-galactosidase)

GH 42 (lac G)

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GH Enzyme cellulase

pectinase (PG,PME,PAL,PL) CGTase (GT)

GH Family

3D-structure

14 Families (GH 5,6,7,8,9,10,12, 26,44,45,48,51, 61,74) GH 28

3 domains: catalytic core (some CBH has (ß/8), peptide linker, and cellulose binding domain (except plant)

GH 13

5 domains, 3 similar to -amylase with ß/8 , 2 extra domains D&E

All have one topology,right handed parallel ß-helix (ß sheets twisted into helix structure)

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(/)8 model structure

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Ribbon representation of the main fold of catalytic domain in various GH families

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A new class of Glycosynthase 

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In food industry, glycosidases are often used for industrial OS synthesis by a reverse hydrolysis reaction, however, a high [E] and [S] is required and a usually low product yield is obtained since hydrolysis direction is more favored for glycosidases. “Glycosynthase”, a novel class of enzyme with the inactive/low hydrolytic reaction has been produced by engineering of the glycosidases

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Examples of glycosynthase Mutation at Glu358, the nucleophile residue of βglucosidase (GH family1) of Agrobacterium sp., the G358A mutant led to OS products with yields of >60% (Withers, 1998) The G358S mutant showed a 24-fold improvement of enzyme synthetic rate compared to G358A. This mutant synthesized 4-nitrophenyl-β-Nacetyllactosamine, a precursor of several cell-surface antigens with 65% yield. 31

Glycosyltransferase (E.C.2.4) 

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Specific glycosyltransferase of vertebrates – endomembrane proteins (endoplasmic reticulus and golgi apparatus), involve in N- and O- glycan and glycoprotein biosynthesis Activated Donor(X-sugar) + Acceptor (R-OH or R-NH)

R-O (or N) – sugar + X 32

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sugar = mono / oligosaccharide R = a free saccharide or a saccharide linked to an aglycone, a protein or a lipid X = nucleotide / dolichol phosphate

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e.g. ß 1,4- galactosyltransferase EC. 2.4.1.38 (X=UDP)  2,6-sialyltransferase EC.2.4.99.3 (X=CMP)

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Cyclodextrin Glycosyltransferase (CGTase) EC. 2.4.1.19 

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for transferase class, only Transglutaminase and CGTase are industrial enzymes. But for Glycosyltransferase, CGTase is the only one. has high transglycosylation but low hydrolysis activity a member of - amylase family, endo, -1,4 cleavage CGTase

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Starch

cyclodextrins + Gn + limit dextrin

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different from other bulk industrial enzymes, that is ; CDs, not CGTase, is used in industry 34

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Reaction Mechanisms of CGTase

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Model of active site of CGTase and how the enzyme cleaves the substrate

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Domain Arrangement in Starch-hydrolysis Enzymes of -Amylase Family -amylase CGTase Maltogenic -amylase G4-forming -amylase

Cyclodextrinase Isoamylase

Pullulanase Glucoamylase 38

Domain Comparison between -amylase and CGTase

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Isomerase : Glucose isomerase, EC.5.3.1.15 

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The only one isomerase used as bulk industrial enzyme Isomerize glucose to fructose, an important sweetener An intracellular enzyme in microorganisms e.g.

Actinoplanes, Streptomyces 

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Classified into 2 families by amino acid sequence similarities, family I (390 ± 4 aa) and II (442 ± 7 aa) Most are homotetramers

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Reaction catalyzed

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Mechanism of Isomerization by Glucose Isomerase 

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Enzyme binds glucose in pyranose form (forward reaction) or fructose in furanose form (reverse reaction) ring open (stimulated by Asp57,His54) Then isomerization occurs, via 1,2 hydride shift, stimulated by metal ions ( H2O at M2 withdraw proton from C-2 OH resulting in a transition intermediate stabilized by M1, M2, and Lys183, polarization of C-O bond and hydride ion formation, which is then shifted to C-1, formed a ketose which then closed ring as furanose) 42

Hydride shift mechanism of glucose isomerase

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3D-Structure of Glucose Isomerase 

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has 2 domains, (ß/) 8 catalytic domain at N-terminal side, and C-terminal domain (helix & loop) two metal ion binding sites per subunit around active site, bind Mn+2 / Mg+2 (consist of several Glu & Asp) pocket active site, consists of His54, Asp57, Lys183 (A.missouriensis)

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References 

Essentials of Carbohydrate Chemistry and Biochemistry (2003) T.K.Lindhorst, Wiley VCH, Weinheim

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Glycoenzymes (2000) M.Ohnishi, ed., Japan Scientific Societies Press, Japan, p.5-26, 84

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เอนไซม์ ดดั แปรคาร์ โบไฮเดรตในอุตสาหกรรม (2551) , เปี่ ยมสุ ข พงษ์ สวัสดิ์ , โรงพิมพ์จุฬาฯ กรุงเทพฯ 392 หน้ า Davies G., and Henrissat B. (1995) Structures and Mechanisms of Glycosyl Hydrolases. Structure 3, 853-859.

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