Study of chitinase and chitin binding protein from molecular
cloning, structure and mechanistic action to the applications
(Yue-Jin Wu)
iii
(ChiNCTU2) (Serratia
marcescens) Chi_NCTU
(chitin binding protein,
CBP) CBP
ChiNCTU2 18
27 (signal peptide) 333
E145 Y227 ChiNCTU2
( )
E145G 2800
E145Q+(NAG)2 -1 (chair form) E145Q/Y227F+(NAG)2
E145G/Y227F+(NAG)4 -1 (boat form)
E145Q/Y227F+(NAG)2 E145G/Y227F+(NAG)4
D143 E145 E190 Y193
ChiNCTU2 ChiNCTU2
Serratia marcescens DNA
(20mM NaOAc, pH 5.5)
(K367D368 367G368P T416A417Y418T419 416G417G418G419G) ChiA_NCTU_m1
ChiA
ChiA_NCTU chitin
N-Serratia marcescens DNA (chitin binding protein,
CBP) CBP chitin CBP21
2 CBP pH 8.0
pH<7
CBP
linker protease cut site MCS DNA
pRSET A MCS 90 % 40 % CBP CBP (EAAAK)5 CBP EAAAK pH 6-7 (protease)
v
Study of chitinase and chitin binding protein from
molecular cloning, structure and mechanistic action to the
applications
Student Yue-Jin Wu Advisor Dr. Yaw-Kuen Li
Department of Applied Chemistry
National Chiao Tung University
ABSTRACT
This thesis discussed the function, structure, catalysis and potential applications of proteins that involved in chitin degradation. Two bacterial chitinases were cloned and overexpressed for these subjects. Chitin-binding protein (CBP), recognized as an enhancer for the catalysis of chitinase, was further introduced in the study. An unexpected outcome was derived from the study of CBP that, consequently, allowed us to develop a simple and cost-effective system for protein purification. The results for all subjects were summarized as follows:
A chitinase gene from Bacillus cereus NCTU2 (ChiNCTU2) was previously cloned and identified as a member of family 18 glycoside hydrolases. ChiNCTU2 is a hydrolytic enzyme, which cleaves -1,4-glycosidic bonds of the natural biopolymer chitin to generate di-N-acetyl-chitobiose. The matured protein containing 333-aa was over-expressed in E. coli. Amino acid multi-alignment revealed that E145 and Y227 of ChiNCTU2 are conserved. Mutagenic study showed that the catalytic activity of E145G mutant was reduced by 2900 fold, indicating the essential role of E145 for ChiNCTU2 catalysis. However, the function of Y227 is different from that of conserved Y390 (ChiA) and Y214 (ChiB) in Serratia marcescens. Besides of the mutagenic examination, we present the crystal structure of ChiNCTU2. The structure composes of only a catalytic domain without the commonly observed chitin-binding domain in other chitinases. The structures of the cocrystallized mutants, E145Q+(NAG)2, E145Q/Y227F+(NAG)2 and
E145G/Y227F(NAG)4, have been refined at high resolution and the interactions with the
and rotates the substrate in the vicinity of the scissile bond. Furthermore, the enzyme imposes a critical “chair” to “boat” conformational change on the sugar residue bound to the -1 subsite. The presence of substrate induces a significant conformational change (about 5.7Å) on a dynamic loop (from residues I106 to V112). The distance is equal to the size of a mono-saccharide imply the possibility that the saccharide can slide along the cleft. The residues, D143, E145, E190 and Y193, were also found in double conformations. Through the kinetic analysese of various mutants, the extensive inspection of sugar-complex protein structure, and a sequence comparison with homologous chitinases, the mechanistic action of ChiNCTU2 can be extensively elucidated.
Another chitinase (chiA) from Serratia Marcescens was cloned by PCR. The recombinant enzyme was characterized and tested for the preparation of chitobiose and other oligosaccharides. In general, the recombinant chtinase A exhibited an exo-type catalytic activity toward colloidal chitin and released both N-acetylglucosamine and N,N-diacetyl chitobiose as products. Although massive efforts were spent for developing mutants with anticipated power to produce uniform oligosaccharide was unsuccessful, we discovered when the enzymatic reaction was performed in sodium acetate buffer at pH 5.5 N,N-diacetyl chitobiose were produced as the predominant product; under such conditions, an enzymatic process is established for the production of the disaccharide on a 100-g scale.
In order to enhance the catalytic hydrolysis of chitin by chitinase, CBP from Serratia marcescens was cloned and employed to furnish the goal. In the presence of CBP, the catalytic activity chitinase was enhanced by 15-20%. In addition to the application of CBP as an activity enhancer of chitinase, we attempted to use CBP as the protein carrier for protein purification by -chitin affinity column. We reported herein an unexpected discovery that the presence of the repeated EAAAK peptide linker in a CBP-fusion protein possessed a pH-dependent auto-cleavage feature. An expression vector derived from pREST was constructed to compose the gene of the CBP and the nucleotide sequence of the (EAAAK)5 peptide linker following restriction sites for target gene insertion. Fusion
proteins were expressed with E. coli and purified with a chitin column. In the range pH 6-7, the target protein becomes automatically released from the fusion protein without proteolytic treatment. Although the mechanism of this auto-cleavage property of an (EAAAK) 5 linker remains unclear, this feature has been successfully employed for many
cases of protein purification without the tag of a fusion protein. Proteins with high purity (> 90% homogeneity for all cases) were easily obtained. The recovery yield was more than 40 %.
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..………...…………...2 1-1 (chitin) ...2 1-2 ………...………..3 1-3 N- ……….5 1-4 (chitinase) ………6 1-4-1 ……...…6 1-4-2 ………6 1-4-3 ………...………...7 1-4-4 ...9 1-5 Family 18 ………9 1-6 Family 18 ...12 1-7 ………..…….…16………...………....………..…..17
2-0 ..………..………..…...17
2-1 …………..………..……...18
2-2 ………..………...18
2-2-1 Phenyl Sepharose High Performance (Hydrophobic interaction column,HIC) ………..………...18
2-2-2 Q Sepharose High Performance ………..…...19
2-3 ………...………...….19 2-4 ………...………....19 2-5 ………...………....20 2-6 ...………...….20 ………...………....…………....21 3-1 ChiNCTU2 ………...21 3-2 ………..………....…...22 3-3 ChiNCTU2 ……...…..….23 3-4 ………...………...….25 3-4-1 ………...………..….25
3-4-2 E145Q E145Q/Y227F E145G/Y227F cocrystal ...………....26
3-4-3 E145G/Y227F cocrystal ………..27
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3-4-5 ChiNCTU2 binding dynamic loop………..…..…....30
3-5 ChiNCTU2 ………33
DNA shuffling
..………...…………...364-1 Serratia marcescens ………...36
4-2 Chitin binding protein (CBP21)…………...37
4-3 DNA shuffling ………..……..37 4-4 ………....…...…...…..41 ………...………....…………....42 5-0 ………..………..……….….…….42 5-1 ( colloidal chitin ) …………..………...………...43 5-2 0.5 Chitin LBA ………..………..………...43 5-3 ………..…….…43 5-4 ChiA pH ………..……….44 5-5 100 N- ………..…....44 5-6 DNA shuffling……….…….….44 5-6-1 ………..……….44 5-6-2 ………...47
5-7 chitinase A exo type endo type………..…...…...48
5-9 KD/GP TAYT/GGGG TAYT/GGGG chitin ………...51 5-10 50 N- ……….52 5-11 CBP21 chitinase ………...52 ……….…...……….….53 6-1 Chi A ……..………....…...53 6-2 100 N- ………...54 6-3 DNA shuffling………..…………...……….…….55
6-3-1 DNA shuffling ChiA ………..…………55
6-3-2 ………...56 6-3-3 ChiA ………..57 6-4 ChiA ……….…….…58 6-5 50 N- ……….………...62 6-6 CBP21 chitinase ……….……..64 ..………...…………...66
7-1 Chitin binding protein CBP21 ...66
7-2 CBP21 ...68
7-3 ...70
xi 7-4 …...………....75 7-5 ………....…...…...…..77 ………...………....…………....78 8-1 …………..………..………...78 8-2 CBP21 ………..………..………...79 8-3 CBP21/pRSET_A ……….80 8-4 CBP21 ……….81 8-5 CBP21 ……….…82 8-6 30-50 mesh ………..……..…83
8-7 CBP21 CBP21 Fusion Protein Vector ……...83
8-7-1 CBP21 ………..………86
8-8 CBP21 (A. fumigatus Y2K chitosanase)………....92
8-9 CBP21 Streptomyces matensis LPHase and Bacillius cereus NCTU2 chitinase ……..……….….93
8-10 Genenase protease cutting site ……….….94
8-11 pH Linker 5 (EAAAK)5 ……….……….94
8-12 linker (EAAAK=2~4) V5-AF de-Genenase protease cutting site V5-AF de-CBP21 V5-AF…….………...….95
8-13 Mass protein ………...96
…
………...………....…………....979-2 CBP21 ..………..………..98
9-3 CBP21 ……...……….99
9-4 30-50 mesh ………...………...100
9-5 CBP21 ………...101
9-6 CBP21 ………..………...102
9-7 CBP21 S. matensis LPHase B. cereus NCTU2 chitinase A. fumigatus Y2K chitosanase……….103
9-8 genenase protease ………..109
9-9 Linker 5 (EAAAK)5 ……….……....110
9-10 linker (EAAAK)2-5 de-Genenase protease cutting site V5-AF de-CBP21 V5-AF ….………..……111
9-11 CBP21 .………....…....119
..………
……….………..………121…
..……….………124.………..………132
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1-1 family 18 19 ……….9
3-1 Wild Type Mutants ChiNCTU2 …………..…...25
3-2 ChiNCTU2 E145G/Y227F (NAG)4 …………...28
7-1 ………...73 8-1 CBP21 7 (Linker)……….86 9-1 LPHase ……..……….….104 9-2 V5-LPH ( )………105 9-3 V5-LPH ( )……….…...106 9-4 V5-NCTU2 ( )……….…107 9-5 V5-NCTU2 ( )………...107 9-6 V5-AF ( )…...108 9-7 V5-AF ( )……….…109 9-8 ………..……...112 1-1 -1,4-….………....3 1-2 ……….4 1-3 ………...………..…7 1-4 ChiNCTU2 ...8
1-5 (GlcNAc)6 S.marcescens ChiA -1 boat
………...10
1-6 S.marcescens ChiA (E315L) 3D , (binding cleft) ………..…...11 1-7 ……….…..…12 1-8 substrate-assisted catalysis………...…13 1-9 S.marcescens ChiA ………..………..……..….15 3-1 ChiNCTU2 3D ...22 3-2 ……...23 3-3 ChiNCTU2 ………..…23
3-4 ChiNCTU2 family 18 chitinase chitinase ……….………..……....24
3-5 chitin cocrystal………..…..….26
3-6 E145G/Y227F chitin cocrystal.……….…..…….27
3-7 ChiNCTU2 ...28
3-8 Allosamidin chitinase inhibitor cyclo-(L-His-L-Pro) allosamidin ………....…29
3-9 E145G/Y227F cyclo-(L-His-L-Pro) cocrystal………….…30
3-10 ChiNCTU2 chitin binding dynamic loops...31
3-11 Family 18 3D ………..32
xv 3-13 ChiNCTU2 ………..…34 4-1 CBP21 ………..37 4-2 DNA shuffling …...40 6-1 TLC ………...54 6-2 Chi A ………...….54 6-3 MASS ……….….55 6-4 MASS ……….….55 6-5 DNA shuffling ……….56 6-6 chitin LBA ………...……….57
6-7 S.m. Chi A (NAG)8 co-crystal ...58
6-8 SWISS-model ………..….59
6-9 ChiA …………...….60
6-10 Mass …………...60
6-11 Mass N- ……...61
6-12 wild type mutant KD/GP mutant KD/GP+TAYT/GGGG ………...61
6-13 N- ……….…….62
6-14 ………...63
6-16 S. marcescens chitinase A B. cereus NCTU2 chitinase CBP21 CBP21 ……….….64 7-1 CBP21 chitinase A …………..….68 7-2 pH CBP21 A ………..….69 8-1 CBP21 ………..…83 8-2 CBP21 ………...84 8-3 ChiC 3D ………85 8-4 Vector 5 ………...93 9-1 CBP21 SDS-PAGE……….…….98 9-2 CBP21 ………..99 9-3 pH CBP21 SDS PAGE………...100 9-4 1M NH4 2SO4 pH CBP21 SDS PAGE………..100 9-5 30-50 mesh 300 mg CBP21 ...101 9-6 7 linker……….………...102
9-7 V5-AF V6-AF V7-AF SDS-PAGE…………103
9-8 V5-LPH SDS-PAGE ………..105
9-9 V5-NCTU2 SDS-PAGE ……….…106
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9-11 genenase protease V5-AF ………..………...109
9-12 V5-AF ……….………..….110
9-13 V5-AF pH overnight …….111
9-14 V5-AF 100 10 pH overnight ………...111
9-15 linker de-genenase cut site de-CBP21 gene ………..…...112
9-16 proteins 100 mM Pi buffer 300mM NaCl pH 7.0 20 overnight ………113
9-17 Linker (EAAAK)5 mass ……….…….114
9-18 Linker (EAAAK)4 mass ………115
9-19 Linker (EAAAK)3 mass ………115
9-20 Linker (EAAAK)2 mass ………116
9-21 V5-AF(de-genenase cutting site) mass ………117
9-22 V5-AF(de-CBP21 gene) mass ……….………118
9-23 CBP21 ………...………..119
