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Designing active site pKa values to shift optimum pH of Bacillus circulans xylanase
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
2.3.044 Designing active site pKa values to shift optimum pH of Bacillus circulans xylanase S. Pokhrel ∗ , Y.J. Yoo School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea
pH-dependence of enzyme catalysis is considered to be modulated by change in electrostatic interactions. Introduction or disruption of long-range electrostatic interactions can be an effective tool to change pKa of active sites and pH optima of enzymes. Our strategy is to alter titration behavior of strongly interacting catalytic glutamates in Bacillus circulans xylanase to shift pH activity profile by introducing basic amino acid residues in juxtaposition of the acidic residues surrounding the catalytic sites. Here, we study the possibility of designing long-range electrostatic interactions to modulate the pKa s of catalytic residues to shift optimum pH of the enzyme towards alkaline or acidic side. Mutation ´˚ from the catalytic sites. sites were kept at farther distance (>8 A) pKa was predicted in mutant models and verified by site directed mutagenesis experiment. The results show that pH optima can significantly be changed by up to +1.5 pH unit even when the ´˚ from the catalytic sites. From the result, mutation sites are >15 A it is concluded that the titration behaviour of strongly interacting catalytic residues in Bacillus circulans xylanase that differ in their intrinsic pKa by ∼1 unit can effectively be changed by interfering titration behaviour of titratable residues in their immediate surrounding.
847 amino acids. The multiple sequence alignment of the deduced protein was done using Clustal V program of DNASTAR which showed high similarity with the sequences of glycoside hydrolase family 3 members. Highest sequence identity was observed with members of genus Kluyveromyces; 98.6% with unnamed protein product of 765 aa of Kluyveromyces lactis NRRL Y-1140 and 73.4% with -glucosidase protein of Kluyveromyces fragilis. The complete gene encoding BGL I was cloned in-frame with the native Saccharomyces cerevisiae ␣-factor secretion signal in yeast shuttle expression vector, pPIC9 and active protein was secreted in culture supernatant of Pichia pastoris GS115 strain. A maximum of 560 U/L activity was obtained on eighth day in case of clone pPIC9 BGL1-5 and 415 U/L for clone pPIC9 BGL1-41. A model was built of BGL I using EXO I of Hordeum vulgare as a template which predicted the protein to be of multi-domain nature with at least one (␣/)8 barrel domain. Asp227 and E590 were predicted to be catalytically important. The role of Asp227 was confirmed by site-directed mutagenesis. The utility of these mutant enzymes is being investigated in increased oligosaccharide synthesis. doi:10.1016/j.nbt.2009.06.428
2.3.046 Immobilization of invertase from Saccharomyces cerevisiae on core/shell silica supports M. Antov ∗ , M. Nikolic, V. Srdic Faculty of Technology, Novi Sad, Serbia
doi:10.1016/j.nbt.2009.06.427
2.3.045 Elucidation of catalytically important residues in a large family 3 -glucosidase from Pichia etchellsii R. Baranwal ∗ , S. Jain, M.A. Shah, S. Mishra Dept. of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New-Delhi, India
Oligosaccharides and other glycoconjugates have considerable potential as therapeutics and therefore their synthesis is being looked at using enzymatic routes. -Glucosidases (BGL) are important enzymes for this purpose as stereo- and regio-specific molecules can be synthesized. No studies have been conducted on family 3 enzymes due to lack of information on the structure and catalytically important residues of these proteins. The thermotolerant yeast Pichia etchellsii produces multiple -glucosidases that include two well characterized wall-bound enzymes BGL I and BGL II. Both are large molecular weight enzymes and have been assigned to family 3 of the glycosylhydrolase families. Three different PCR strategies were used, based on the known internal peptide sequences of BGL I, to fish out the gene and determine the complete nucleotide sequence encoding this protein. The gene, bgl1 (GenBank Accession No. EU914813), was found to be intron-less and the ORF consisted of 2544 nucleotides encoding a protein of
S126
www.elsevier.com/locate/nbt
Immobilization of biomolecules on different solid surfaces is an area of great interest for both science and industry — obtained biomaterials have important applications in biotechnology, immunosensing, biomedicine, etc. Among the numerous methods of enzyme immobilization, physical adsorption is easy to perform but at the same time enzyme is easily desorbed. Physical adsorption within mesoporous matrixes considerably overcame the desorption problem but the entrapped enzyme usually has a slow enzymatic reaction rate because the substrate has to diffuse from the exterior environment to the interior part of the matrix to interact to the entrapped enzyme. To overcome this limitation, the objective of the present study was to investigate the synthesis of the core/shell silica materials and their application for the immobilization of industrially important enzyme. Supports of core/shell silica structure were synthesized and used for the immobilization of invertase from Sacharomyces cerevisiae. The synthesis involved deposition of silica nanoparticles prepared from sodium silicate on silica cores synthesized via the hydrolysis and condensation of silicon alkoxide. External surface of the silica cores was in addition modified to enable attractive electrostatic interaction between oppositely charged surfaces of silica cores and shell silica particles. The pore structure and thickness of the mesoporous silica shell were tuned by changing the processing parameters. Transmission electron microscopy revealed that silica supports had well defined core which was evenly coated with shell. It
ABSTRACTS
2.3.044 Designing active site pKa values to shift optimum pH of Bacillus circulans xylanase S. Pokhrel ∗ , Y.J. Yoo School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea
pH-dependence of enzyme catalysis is considered to be modulated by change in electrostatic interactions. Introduction or disruption of long-range electrostatic interactions can be an effective tool to change pKa of active sites and pH optima of enzymes. Our strategy is to alter titration behavior of strongly interacting catalytic glutamates in Bacillus circulans xylanase to shift pH activity profile by introducing basic amino acid residues in juxtaposition of the acidic residues surrounding the catalytic sites. Here, we study the possibility of designing long-range electrostatic interactions to modulate the pKa s of catalytic residues to shift optimum pH of the enzyme towards alkaline or acidic side. Mutation ´˚ from the catalytic sites. sites were kept at farther distance (>8 A) pKa was predicted in mutant models and verified by site directed mutagenesis experiment. The results show that pH optima can significantly be changed by up to +1.5 pH unit even when the ´˚ from the catalytic sites. From the result, mutation sites are >15 A it is concluded that the titration behaviour of strongly interacting catalytic residues in Bacillus circulans xylanase that differ in their intrinsic pKa by ∼1 unit can effectively be changed by interfering titration behaviour of titratable residues in their immediate surrounding.
847 amino acids. The multiple sequence alignment of the deduced protein was done using Clustal V program of DNASTAR which showed high similarity with the sequences of glycoside hydrolase family 3 members. Highest sequence identity was observed with members of genus Kluyveromyces; 98.6% with unnamed protein product of 765 aa of Kluyveromyces lactis NRRL Y-1140 and 73.4% with -glucosidase protein of Kluyveromyces fragilis. The complete gene encoding BGL I was cloned in-frame with the native Saccharomyces cerevisiae ␣-factor secretion signal in yeast shuttle expression vector, pPIC9 and active protein was secreted in culture supernatant of Pichia pastoris GS115 strain. A maximum of 560 U/L activity was obtained on eighth day in case of clone pPIC9 BGL1-5 and 415 U/L for clone pPIC9 BGL1-41. A model was built of BGL I using EXO I of Hordeum vulgare as a template which predicted the protein to be of multi-domain nature with at least one (␣/)8 barrel domain. Asp227 and E590 were predicted to be catalytically important. The role of Asp227 was confirmed by site-directed mutagenesis. The utility of these mutant enzymes is being investigated in increased oligosaccharide synthesis. doi:10.1016/j.nbt.2009.06.428
2.3.046 Immobilization of invertase from Saccharomyces cerevisiae on core/shell silica supports M. Antov ∗ , M. Nikolic, V. Srdic Faculty of Technology, Novi Sad, Serbia
doi:10.1016/j.nbt.2009.06.427
2.3.045 Elucidation of catalytically important residues in a large family 3 -glucosidase from Pichia etchellsii R. Baranwal ∗ , S. Jain, M.A. Shah, S. Mishra Dept. of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New-Delhi, India
Oligosaccharides and other glycoconjugates have considerable potential as therapeutics and therefore their synthesis is being looked at using enzymatic routes. -Glucosidases (BGL) are important enzymes for this purpose as stereo- and regio-specific molecules can be synthesized. No studies have been conducted on family 3 enzymes due to lack of information on the structure and catalytically important residues of these proteins. The thermotolerant yeast Pichia etchellsii produces multiple -glucosidases that include two well characterized wall-bound enzymes BGL I and BGL II. Both are large molecular weight enzymes and have been assigned to family 3 of the glycosylhydrolase families. Three different PCR strategies were used, based on the known internal peptide sequences of BGL I, to fish out the gene and determine the complete nucleotide sequence encoding this protein. The gene, bgl1 (GenBank Accession No. EU914813), was found to be intron-less and the ORF consisted of 2544 nucleotides encoding a protein of
S126
www.elsevier.com/locate/nbt
Immobilization of biomolecules on different solid surfaces is an area of great interest for both science and industry — obtained biomaterials have important applications in biotechnology, immunosensing, biomedicine, etc. Among the numerous methods of enzyme immobilization, physical adsorption is easy to perform but at the same time enzyme is easily desorbed. Physical adsorption within mesoporous matrixes considerably overcame the desorption problem but the entrapped enzyme usually has a slow enzymatic reaction rate because the substrate has to diffuse from the exterior environment to the interior part of the matrix to interact to the entrapped enzyme. To overcome this limitation, the objective of the present study was to investigate the synthesis of the core/shell silica materials and their application for the immobilization of industrially important enzyme. Supports of core/shell silica structure were synthesized and used for the immobilization of invertase from Sacharomyces cerevisiae. The synthesis involved deposition of silica nanoparticles prepared from sodium silicate on silica cores synthesized via the hydrolysis and condensation of silicon alkoxide. External surface of the silica cores was in addition modified to enable attractive electrostatic interaction between oppositely charged surfaces of silica cores and shell silica particles. The pore structure and thickness of the mesoporous silica shell were tuned by changing the processing parameters. Transmission electron microscopy revealed that silica supports had well defined core which was evenly coated with shell. It