Biotechnology for Improved Foods and Flavors - American Chemical

Chapter 13

Chimeric β-Glucosidases with Increased Heat Stability

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 5, 2016 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch013

Kiyoshi Hayashi, Ajay Singh, Chika Aoyagi, Atsushi Nakatani, Ken Tokuyasu, and Yutaka Kashiwagi Enzyme Applications Laboratory, National Food Research Institute, 1-2-1 Kannondai, Tsukuba, Ibaraki 305, Japan In order to improve thermal stability of the β-glucosidases from Cellvibrio gilvus, chimeric enzymes were constructed. Two homologous domains were found between C. gilvus and Agrobacterium tumefaciens β -glucosidase. Since the active center of the enzyme locates at the N-terminal domain, the C-terminal domain, showing 40 % homology, was selected to construct chimeric enzymes. Four chimeric enzymes of CHSTY, CHBSA, CHAGE and CHBSM possessing 8%, 22%, 30% and 39% of the amino acid sequence of A. tumefaciensβ-glucosidaseat the C-terminal were prepared by shuffling the gene. The properties of four chimeric enzymes to pH and temperature were an admixture of the two parental enzymes. It is especially interesting that the thermal stability of the chimeric enzymes was increased 9-16 °C when compared to that of the C. gilvus enzyme. Regarding the substrate specificity of the four chimeric enzymes, they are more similar to A. tumefaciens than the C. gilvus enzyme. This result suggests that enzyme character can be improved by constructing chimeric enzymes.

Several kinds of enzymes have been used in many industries including food industries because enzymes catalyze the reaction at very moderate conditions and the reaction is very specific. Since enzymes are so useful and important, searching for a new enzyme with required characteristics such as higher heat stability, broader substrate specificity, etc. is inevitable. Altering some character of the currently used enzyme is extremely difficult. Immobilization of enzymes can sometimes stabilize enzymes but this is not applicable for all cases (7). Chemical modification of the enzyme also does not help so much to improve the enzyme character (2). There have been no appropriate methods to improve a character of the enzyme. The ordinal way to find a new enzyme is to carry out screening for the enzymes, since enormous varieties of enzymes have been created during the long time span of evolution (5). During the long process of evolution of 3 to 4 billion years, point mutation and gene shuffling occurred in the genes of enzymes. By point mutation in the gene, only one amino acid will be altered, sometime resulting suitable character for living things. By gene shuffling, a wider region of the gene has been exchanged, resulting in drastic alteration of the enzyme character. Of course most of 0097-6156/%/0637-0134$15.00/0 © 1996 American Chemical Society

In Biotechnology for Improved Foods and Flavors; Takeoka, Gary R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


13. HAYASHI ET AL.

Chimeric (3-Glucosidases with Increased Heat Stability 135

these changes occur very slowly during evolution. As a result, varieties of enzymes with different amino acid sequences, that is different in character, have been created. Screening for a new enzyme is to find a suitable enzyme which is created naturally during this long process. Because of the advance in biotechnology, enzyme genes can be manipulated in laboratories. Enzymes possessing one or two different amino acids can easily be produced by point mutation of the gene. However, changing only one or few amino acids is not sufficient to improve the enzyme character, though it is easy to inactivate the enzyme by changing one amino acid in the active site (4). Gene shuffling will be a good answer to create a new enzyme of desired character (5-7). More than 40,000 thousands of available sequences of the protein can be used for designing chimeric enzymes. In this paper, we examined the improvement of heat stability of a enzyme by preparation of chimeric enzymes by employing heat sensitive B-glucosidase produced by Cellvibrio gilvus (8) and a heat resistant one from Agrobacterium tumefaciens (9). Preparation of Chimeric B-glucosidase ft-glucosidase of C. gilvus. Cellobiose rather than glucose accumulated when C. gilvus ATCC 13127 was cultivated in a medium containing acid swollen cellulose (70). Then, accumulated cellobiose has been considered to be utilized in the cell by hydrolysing it to glucose and glucose-1-phosphate by cellobiose phosphorylase (EC 2.4.1.20) (11-13). It was found that this unique accumulation of the disaccharide has been caused by a specificity of B-glucosidase (EC 3.2.1.21) of this strain; substantially lower activity toward cellobiose than cellotriose, cellotetraose, cellopentaose and cellohexaose (8). This B-glucosidase can be regarded as a suitable enzyme for production of cellobiose which is used as non-dietary sugar. However, heat stability of this enzyme is not high enough for the production of disaccharide. Homology Analysis. C. gilvus' B-glucosidase which belongs to the BGB group of B-glucosidases (14-17) share conserved regions with B-glucosidases from different organisms including bacteria, yeast and fungi, as shown in Table I. It was also found that the homologous region can be separated into two domains. Among eleven homologous proteins two enzymes produced by Butyrivibrio fibrisovens and Ruminococcus albus showed that the two homologous domains are inverted as shown in Fig. 1, indicating that these domains may have independent functions. Highest homology scores in the amino acid sequence were obtained between C. gilvus B-glucosidase and A. tumefaciens B-glucosidase (9). In particular, the region from Ala-541 to Pro-811 of B-glucosidase from A. tumefaciens is quite similar (about 40%) to the region from Ala-472 to Pro-741 of B-glucosidase from C. gilvus as shown in Fig. 2. Importance of C-domain. Since deletion of more than 70 bp fragment from the Cterminal part of C. gilvus B-glucosidase gene resulted in the loss of enzyme activity (T.T. Hoa and K. Hayashi, unpublished), the C-terminal region seems to be important for B-glucosidase activity. Although, the deletion of about 100 amino acid residues near the C-terminal region of oc-amylase gene did not affect enzyme activity (18), cyclomaltodextrin glucanotransferases lacking 30 amino acids (79) and an endoglucanase lacking 75 amino acids (20) from the C-terminal end showed no enzyme activity. Keeping in view the importance of the C-terminal region and the location of estimated catalytic center at Asp-291 in the N-terminal region of C. gilvus (16), the C-terminal region was selected for the construction of chimeric enzymes.


136

BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS


Table I. Homology with C.gilvus B-glucosidases Microorganisms

Homology Score

Agrobacterium tumefaciens (Bacterium) Clostridium thermocellum (Bacterium) Kluyveromyces fragilis (Yeast) Saccharomycopsisfibligera B (Yeast) SaccharomycopsisfibligeraA (Yeast) Butyrivibriofibrisovens(Bacterium) Hansenula anomala (Yeast) Ruminococcus albus (Bacterium) Schizophyllum commune (Fungi) Aspergillus wentii (Fungi)

1013 839 784 710 706 610 596 582 208 101

Ruminococcus albus

Agrobacterium tumefaciens Figure 1. Homologous regions in amino acid sequences of B-gluco sidases from R. albus, C.gilvus and A.tumefaciens. Two homologous regions were found in N- terminal (Shaded) and C-terminal (Darkly shaded) regions of C Gilvus B-glucosidase genes. The two homologous domains were inverted in case of Ruminococcus albus B-glucosidase genes.


13.

HAYASHI ET AL.

Chimeric 0-Glucosidases with Increased Heat Stability 137

CHBSM-^i CG YPVGGIAVKGLLPATWPGPWYYPSSPLRAIQAQAPNAKVVFDDGRDPARAARVAAGADV • • •

AT

mm

•

AVTLGAARRYRVVVEYEAPKASLDGINICALRFGVEKPLGDAGIAEAVETARKSDIVLL

1

549

CHBSM-^ CHAGE—•)

CG ALVFANQWIGEANDAQTLALPDGQEELITSVAGANGRTVVVLQTGGPVTMPWLARVPAVL Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 5, 2016 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch013

480

m m

540

AT LVGREGEWDTEGLDLPDMRLPGRQEELIEAVAETNPNVVVVLQTGGPIEMPWLGKVRAVL 608

CHBSA—'

CHAGE—•

CG EAWYPGTSGGEAIANVLFGAVNPSGHLPATFPQSEQQLPRPKLDGuPKNPELQFAVDYHE 600 .

W

W .

W . # W

.

.

*

*

*

AT QMWYPGQELGNALADVLFGDVEPAGRLPQTFPKALTD-NSAITDDPSIYPGQDGHVRYAE 667 -

'——-CHBSA CG GAAVGYKWFOLKGHKPLFPFGHGLSYTTFAYSG--LSG-QLKDGRLHVRFKVTNTGNVAG

657

AT GIFVGYRHHDTREIEPLFPFGFGLGYTRFTWGAPQLSGTEMGADGLTVTVDVTNIGDRAG

727

* ***. *

. ****** **. ** *

*** .. .. * * .***.*. **

CIICTY •! CG KDVPQVYAAPMSTKWEAP-KRLAAWSKVALLPGETKfeVEVAVEPRVLAMFDEKSRTWRRP 717 .***.* * * * * * . . * . * **. * *****•.. .* AT SDVVQLYVHSPNARVERPFKELRAFAKLKLAPGATGTAVLKIAPRDLAYFDVEAGRFRAD 787

I

CHSTY—•

CG KGKIRLTLAEDASAANATSVTVELPASTLDARGRAR

752

AT AGKYELIVAASAIDIRA-SVSIHLPVDHVMEP

818

Figure 2. Homology of the amino acid sequences in C-terminal region of C. Gilvus and A. tumefaciens 13-glucosidases. Identical and similar amino acid residues are designated by (*) and (.), respectively. Four chimeric enzymes were constructed by shuffling the regions marked by arrow heads. Construction of Four Chimeric Enzymes. Considering the translation frame and similar regions of both genes, four regions in C. gilvus B-glucosidase gene were selected for substitution with A. tumefaciens B-glucosidase gene. The structures of the four chimeric B-glucosidase genes are shown in Figure 3. In order to obtain the chimeric enzyme gene of CHSTY where 8% of amino acid sequence are originating from A. tumefaciens B-glucosidase, the region between two Aval site starting from Pro-759 over stop codon in the plasmid of pcbgl coding A. tumefaciens B-glucosidase (9) were substituted at Styl site in the plasmid of pCG5 coding C. gilvus B-glucosidase (21). Three other chimeric enzymes were similarly prepared by substituting the region between Sfil and Hinfl sites starting from Asp-660 in pcbgl at BsaBI site of pCG5 (CHBSA, 22% amino acid sequence originating from A. tumefaciens B-glucosidase), the region between two Avail sites starting from Ile-594 in pcbgl at Agel site of pCG5 (CHAGE, 30% amino acid sequence originating from A. tumefaciens B-gluco sidase), and the region between Ndel and Hinfl sites starting from Cys-517 in pcbgl at



138


Figure 3. Construction of four chimeric B-glucosidase genes. Light and dark shadow represent regions derived from C. gilvus and A. tumefaciens, respectively. Restriction enzymes used for the construction of chimeric enzymes are shown with arrowheads. BsmI site of pCG5 (CHBSM, 39% amino acid sequence originating from A. tumefaciens B-glucosidase) (22-23). The clones expressing chimeric enzymes were isolated on agar plates containing ampicillin (50 |Xg/ml) and fluorescent substrates of 4-methylumbelliferyl-B-glucoside (1 mM). Four chimeric plasmids were characterized and confirmed by restriction enzyme digestion. Characterization of Chimeric B-glucosidases Protein Behavior in Column Chromatography. Distinctive differences in the behavior of B-glucosidases from C. gilvus and A. tumefaciens have been observed during the purification step using ion-exchange chromatography. Cation-exchange column (SP Sepharose) was used at pH 5.0 to elute B-glucosidases from C. gilvus, whereas anion-exchange column (Q Sepharose) was used to elute A. tumefaciens' Bglucosidases at pH 6.5. All four chimeric B-glucosidases were found to show similar behavior to that of C. gilvus B-glucosidase in ion exchange chromatography, indicating that outer surface of the four chimeric enzymes is close to C. gilvus enzyme. 1

Effect of pH on the Chimeric Enzyme Activity. The pH optima for C. gilvus and A. tumefaciens enzymes were found to be 6.2-6.4 and 7.2-1 A, respectively. Chimeric enzymes showed intermediate profiles of their parents as shown in Figure 4A. The optimum pH of CHSTY, CHBSA, CHAGE and CHBSM enzyme was 6.0, 6.6, 6.8-7.0 and 6.6-7.0, respectively. All the chimeras were stable between pH 4


HAYASHI ET AL.

Chimeric 0-Glucosidases with Increased Heat Stability 139


13.

P

H

Figure 4. pH-activity (A) and pH stability (B) profiles of four chimeric and two parental B-glucosidases. The pH was adjusted with buffers: citrate (pH 3.0-4.0); 2-(N-morpholino) ethane sulphonic acid (pH 5.0-6.8); 3-(N-morpholino) propane sulphonic acid (pH 7.0-8.0); 2-(N-cyclohexylamino) ethane sulphonic acid (pH 9.0-10.0). For pH stability experiments, enzyme was incubated at different pHs for 1 h at 25°C. The residual activities were measured under standard assay condition. C. gilvus, A ; CHSTY, A; CHBSA, • ; CHAGE, O; CHBSM, O; A tumefaciens, •.


140



and 9, whereas the B-glucosidases from C.gilvus and A. tumefaciens were stable at pH 4-8 and pH 5-10, respectively, as shown in Fig. 4B. Substitution of segments in homologous C-terminal region seems to have an influence on pH-activity and stability. In Bacillus cyclomaltodextrin glucanotransferase (19) and cellulase (24), pH-activity profile were found to be influenced by the N- and the C-terminal parts. Improved Thermal Stability in Chimeric Enzymes. B-Glucosidase from C. gilvus is optimally active at 35°C, whereas that of A. tumefaciens exhibit maximum activity at 60°C. With regards to heat stability, B-glucosidase from C. gilvus shows complete activity up to 30°C, retains about 80% of its maximum activity at 35°C, and inactivates completely at 55°C. On the other hand, A. tumefaciens' enzyme is stable up to 55°C and even at 65°C, it retains 60% of its maximum activity. Four chimeric enzymes showed marked differences in their temperature optima (Fig. 5A). The chimeric enzymes were optimally active at 45-50°C, showing an intermediate temperature optimum between C. gilvus' and A. tumefaciens' enzymes. CHSTY showed the temperature optima of 45 °C with complete loss of activity at 65 °C. CHBSA showed the temperature optima of 45 °C with no activity at 70°C. On the other hand, CHAGE was optimally active at 50°C and exhibited 52% of its maximum activity at 60°C. CHBSM exhibited maximum activity at 50°C and 61% of its maximum activity at 60°C. The temperatures at which 50% loss of the enzyme activities occurred were 41, 47, 50, 55 and 57 and 67°C for C. gilvus, CHSTY, CHBSA, CHAGE, CHBSM and A. Tumefaciens enzymes, respectively (Fig. 5B). Thus heat stability of chimeric enzymes was increased by 6-16°C as compared to C. gilvus enzyme though none of them exceeded that of A. Tumefaciens enzyme. Heat stability may be influenced by only a few amino acid substitutions (22, 25). In the case of chimeric isopropylmalate dehydrogenases produced by shuffling genes of Thermus thermophilus and Bacillus subtilis, the heat stability of chimeric enzymes was approximately proportional to the content of the amino acid sequence from the T thermophilus enzyme (26). In general, protein stability increases with the insertion into an oc-helix of helix-forming amino acids (alanine, glutamic acid, etc.) and decreases with the insertion of helix-breaking amino acids (proline, glycine, etc.). The secondary structures of the parental and chimeric enzymes were predicted by Robson's method (27). There were similar numbers of helix-breaking but more helixforming amino acid residues in the a-helix regions of chimeric enzymes than C. gilvus enzyme, suggesting that it could be one of the factors influencing heat stability of chimeras. Hydrophobic interaction inside the protein molecule is another important factor in stabilizing protein structure. Hydrophobic cluster analysis (28,29) of native and chimeric enzymes revealed that the amino acid substitution from C. gilvus to A. tumefaciens significantly increased the hydrophobic properties of the chimeric enzymes. These substitutions might be important for heat stability of B-glucosidase. Substrate Specificity of Chimeric Enzymes Difference in Parental Enzymes. B-Glucosidase from C. gilvus has rather strict specificity toward glucose residues in aryl-glycosides (Table II). This enzyme hydrolysed p-nitrophenyl-B-D-xyloside (pNPXyl) only at 0.81% of the level of pnitrophenyl-B-D-glucoside (pNPGlu) and p-nitrophenyl-B-D-galactoside (pNPGal) at 0.15%. It showed no activity to p-nitrophenyl-B-D-fucoside (pNPFuc), p-nitrophenyl -B-D-m anno side (pNPMan), p-nitrophenyl-N-acetyl-B-D-glucosaminide (pNPGlunAc), p-nitrophenyl-N-acetyl-B-D-galactosaminide (pNPGalnAc), p-nitrophenyl-a-D-glucoside (pNP-a-Glu) (Table III). On the other hand, A. tumefaciens


HAYASHI ET AL.

Chimericfi-Glucosidaseswith Increased Heat Stability 141


13.

Temperature (°C) Figure 5. Temperature optima (A) and heat stability (B) profiles of four chimeric B-glucosidases. For heat stability experiments, each enzyme at its optimum pH was treated at different temperatures for 1 h. The residual activities were measured under standard assay condition. C gilvus, A ; CHSTY, A; CHBSA, • ; CHAGE, 0\ CHBSM, 0;A. tumefaciens, #.


142


Table II. Kinetic Parameters of Three Chimeric Enzymes and Two Parental Enzymes


Substrate

CG

CHBSA

CHAGE

CHBSM

AT

p-nitrophenyl-6-D-glucoside Km(mM) Vmax (%)a

1.806 100

0.273 100

0.291 100

0.270 100

0.032 100

p-nitrophenyl-6-D-xyloside Km(mM) Vmax (%)

6.261 0.81

0.005 7.7

0.004 7.2

0.004 9.1

0.005 14

p-nitrophenyl-B-D-galactoside Km(mM) Vmax (%)

10.8 0.15

34.4 49

15.1 32

16.6 43

13.1 63

p-nitrophenyl-B-D-fucoside Km(mM) Vmax (%)

Biotechnology for Improved Foods and Flavors - American Chemical

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