<p><i>Lactococcus lactis,</i> prolidase (<i>Lla</i>prol) hydrolyzes Xaa-Pro dipeptides. Since Xaa-Pro is known as bitter peptides, <i>Lla</i>prol is potentially applicable to reduce bitterness of fermented foods. <i>Lla</i>prol shows allosteric behaviour and substrate inhibition, which are not reported in other prolidases. Computer models of <i>Lla</i>prol based on an X-ray structure of non-allosteric <i>Pyrococcus furiosus</i> prolidase showed that a loop structure (Loop^32-43) is located at the interface of the protomers of this homodimeric metallodipeptidase. This study investigated roles of four charged residues (Asp³⁶, His³⁸, Glu³⁹, and Arg⁴⁰) of Loop^32-43 in <i>Lla</i>prol using a combination of kinetic examinations of ten mutant enzymes and their molecular models. Deletion of the loop structure by Î36-40 mutant resulted in a loss of activity, indicating Loop^32-43 is crucial for the activity of <i>Lla</i>prol. D36S and H38S exhibited 96.2 % and 10.3 % activity of WT, whereas little activities (less than 1.0 % of WT activity) were observed for mutants E39S, D36S/E39S, R40S, R40E, R40K and H38S/R40S. These results implied that Glu³⁹ and/or Arg⁴⁰ play critical role(s) in maintaining the catalytic activity of <i>Lla</i>prol. These observations suggested that the loop structure is flexible and this attribute, relying on charge-charge interactions contributed by Arg⁴⁰, Glu³⁹ and Lys¹⁰⁸, is important in maintaining the activity of <i>Lla</i>prol. When the loop takes a conformation close to the active site (closed state), Asp³⁶ and His³⁸ at the tip of the loop can be involved in the catalytic reaction of <i>Lla</i>prol. The two active mutant prolidases (D36S and H38S) resulted in modifications of the unique characteristics; the allosteric behaviour was not observed for D36S, and H38S <i>Lla</i>prol showed no substrate inhibition. D36E/R293K, maintaining the negative charge of position 36 and positive charge of position 293, still possessed the allosteric behaviour, whereas the loss of the charges at these positions (D36S of this study and R293S of a previous study (Zhang et al., 2009 BBA-Proteins Proteom 1794, 968-975) eliminated the allosteric behaviour. These results indicated the charge-charge attraction between Asp³⁶ and Arg²⁹³ is important for the allostery of <i>Lla</i>prol. In the presence of either zinc or manganese divalent cations as the metal catalytic centre, D36S and H38S enzymes also showed different substrate preferences from WT <i>Lla</i>prol, implying the influence of Asp³⁶ and His³⁸ on the substrate binding. D36S and H38S also showed higher activities at pH 5.0 to 6.0, in which range WT <i>Lla</i>prol steeply decreased its activity, indicating Asp³⁶ and His³⁸ are involved in the active centre and influence the microenvironment of catalytic His²⁹⁶. The above observations are attributed to modifications of their local structure in the active centre since the temperature dependency and thermal denaturing temperature indicated little effects on the overall structure of the <i>Lla</i>prol mutants.</p> <p>From these results, we concluded that the unique behaviours of <i>Lla</i>prol are correlated to Loop^32-43 and Asp³⁶ and His³⁸ on it. When Loop^32-43 takes a closed conformation, Asp³⁶ interacts with Arg²⁹³ via charge-charge attraction to form an allosteric subsite. The saturation of the allosteric site with substrates further allowed the communications of His³⁸ with S₁ site residues to complete the active site. When the substrate concentration becomes higher than it is required to saturated productive S₁' site, His³⁸, Phe¹⁹⁰ and Arg²⁹³ would resemble the residue arrangement of S₁' site residues (His²⁹², Tyr³²⁹, and Arg³³⁷) and bind to the proline residue of substrates. This non-productive binding would prevent the conformational change of Loop^32-43, which further results in the substrate inhibition. For further confirmation of this mechanism, crystallographic studies will be conducted. In this thesis, we have indentified the conditions to produce crystals of <i>Lla</i>prol proteins.</p>

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. ACC deaminases break down ACC, the immediate precursor of ethylene in plants, into ammonia and α-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. It was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather that it utilizes D-cysteine as a substrate, and in fact encodes a D-cysteine desulfhydrase. Kinetic characterization of the enzyme has shown that it is similar to other previously characterized D-cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering two amino acid residues within the predicted active site changed the enzyme from D-cysteine desulfhydrase to ACC deaminase. Concomitantly, it was shown that by altering two amino acids residues at the same position within the active site of ACC deaminase from Pseudomonas putida UW4 changed this enzyme into D-cysteine desulfhydrase.

<p><i>Lactococcus lactis,</i> prolidase (<i>Lla</i>prol) hydrolyzes Xaa-Pro dipeptides. Since Xaa-Pro is known as bitter peptides, <i>Lla</i>prol is potentially applicable to reduce bitterness of fermented foods. <i>Lla</i>prol shows allosteric behaviour and substrate inhibition, which are not reported in other prolidases. Computer models of <i>Lla</i>prol based on an X-ray structure of non-allosteric <i>Pyrococcus furiosus</i> prolidase showed that a loop structure (Loop^32-43) is located at the interface of the protomers of this homodimeric metallodipeptidase. This study investigated roles of four charged residues (Asp³⁶, His³⁸, Glu³⁹, and Arg⁴⁰) of Loop^32-43 in <i>Lla</i>prol using a combination of kinetic examinations of ten mutant enzymes and their molecular models. Deletion of the loop structure by Î36-40 mutant resulted in a loss of activity, indicating Loop^32-43 is crucial for the activity of <i>Lla</i>prol. D36S and H38S exhibited 96.2 % and 10.3 % activity of WT, whereas little activities (less than 1.0 % of WT activity) were observed for mutants E39S, D36S/E39S, R40S, R40E, R40K and H38S/R40S. These results implied that Glu³⁹ and/or Arg⁴⁰ play critical role(s) in maintaining the catalytic activity of <i>Lla</i>prol. These observations suggested that the loop structure is flexible and this attribute, relying on charge-charge interactions contributed by Arg⁴⁰, Glu³⁹ and Lys¹⁰⁸, is important in maintaining the activity of <i>Lla</i>prol. When the loop takes a conformation close to the active site (closed state), Asp³⁶ and His³⁸ at the tip of the loop can be involved in the catalytic reaction of <i>Lla</i>prol. The two active mutant prolidases (D36S and H38S) resulted in modifications of the unique characteristics; the allosteric behaviour was not observed for D36S, and H38S <i>Lla</i>prol showed no substrate inhibition. D36E/R293K, maintaining the negative charge of position 36 and positive charge of position 293, still possessed the allosteric behaviour, whereas the loss of the charges at these positions (D36S of this study and R293S of a previous study (Zhang et al., 2009 BBA-Proteins Proteom 1794, 968-975) eliminated the allosteric behaviour. These results indicated the charge-charge attraction between Asp³⁶ and Arg²⁹³ is important for the allostery of <i>Lla</i>prol. In the presence of either zinc or manganese divalent cations as the metal catalytic centre, D36S and H38S enzymes also showed different substrate preferences from WT <i>Lla</i>prol, implying the influence of Asp³⁶ and His³⁸ on the substrate binding. D36S and H38S also showed higher activities at pH 5.0 to 6.0, in which range WT <i>Lla</i>prol steeply decreased its activity, indicating Asp³⁶ and His³⁸ are involved in the active centre and influence the microenvironment of catalytic His²⁹⁶. The above observations are attributed to modifications of their local structure in the active centre since the temperature dependency and thermal denaturing temperature indicated little effects on the overall structure of the <i>Lla</i>prol mutants.</p> <p>From these results, we concluded that the unique behaviours of <i>Lla</i>prol are correlated to Loop^32-43 and Asp³⁶ and His³⁸ on it. When Loop^32-43 takes a closed conformation, Asp³⁶ interacts with Arg²⁹³ via charge-charge attraction to form an allosteric subsite. The saturation of the allosteric site with substrates further allowed the communications of His³⁸ with S₁ site residues to complete the active site. When the substrate concentration becomes higher than it is required to saturated productive S₁' site, His³⁸, Phe¹⁹⁰ and Arg²⁹³ would resemble the residue arrangement of S₁' site residues (His²⁹², Tyr³²⁹, and Arg³³⁷) and bind to the proline residue of substrates. This non-productive binding would prevent the conformational change of Loop^32-43, which further results in the substrate inhibition. For further confirmation of this mechanism, crystallographic studies will be conducted. In this thesis, we have indentified the conditions to produce crystals of <i>Lla</i>prol proteins.</p>

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. ACC deaminases break down ACC, the immediate precursor of ethylene in plants, into ammonia and α-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. It was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather that it utilizes D-cysteine as a substrate, and in fact encodes a D-cysteine desulfhydrase. Kinetic characterization of the enzyme has shown that it is similar to other previously characterized D-cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering two amino acid residues within the predicted active site changed the enzyme from D-cysteine desulfhydrase to ACC deaminase. Concomitantly, it was shown that by altering two amino acids residues at the same position within the active site of ACC deaminase from Pseudomonas putida UW4 changed this enzyme into D-cysteine desulfhydrase.

Chlorate and perchlorate pollutions have become a problem in the environment in the last decades. Studies have shown that some bacteria can degrade these substances into unharmful substances such as chloride and molecular oxygen. One of these chlorate degrading bacteria is Ideonella dechloratans that uses chlorate reductase and chlorite dismutase to process chlorate. In the promoter gene sequence of chlorite dismutase there might be regulator sequences such as fumarate and nitrate reductase regulator (FNR) and aerobic respiration control protein (ArcA) that might control the transcription of this enzyme. This promoter sequence was placed in a pBBR1MCS-4-LacZ reporter vector and the possible regulatory sequences were changed through site-directed mutagenesis and tested on activity through beta-galactosidase assays. The changes in the FNR binding sequence gave beta-galactosidase activity that was close to a negative control which might give conclusions that either FNR has an important role or an important part of the promoter was hit. The changes in the ArcA regulator binding sequence did not give such big differences and no certainty can be given if this made important changes to the promoter.

Altering the substrate specificity of proteases is a powerful process with possible applications in many areas of therapeutics as well as proteomics. Although the field is still developing, several proteases have been successfully engineered to recognize novel substrates. Previously in our laboratory, eight highly active OmpT variants were engineered with novel catalytic sites. The present study examined the roles of several residues surrounding the active site of OmpT while attempting to use rational design to modulate fine specificity enough to create a novel protease that prefers phosphotyrosine containing substrates relative to sulfotyrosine or unmodified tyrosine residues. In particular, a previously engineered sulfotyrosine-specific OmpT variant (Varadarajan et al., 2008) was the starting point for rationally designing fifteen new OmpT variants in an attempt to create a highly active protease that would selectively cleave phosphotyrosine substrates. Our design approach was to mimic the most selective phosphoryl-specific enzymes and binding proteins by increasing positive charge around the active site. Sulfonyl esters have a net overall charge of -1 near neutral pH, while phosphate monoesters have a net overall charge of -2. Selected active site residues were mutated by site-directed mutagenesis to lysine, arginine, and histidine. The catalytic activities and substrate specificities of each variant were characterized. Although several variants displayed altered substrate specificity, none preferred phosphotyrosine over sulfotyrosine containing peptides. Taken together, our results have underscored the subtle nature of protease substrate specificity and how elusive it can be to engineer fine specificity. Apparently, phosphotyrosine specific variants were not possible within the context of our starting sulfotyrosine specific OmpT derivative mutated to have single amino acid changes chosen on the basis of differential charge interactions. / text

Of the organisms with sequenced genomes, plants appear to possess the most rhomboid protease-encoding genes. However, our knowledge of processes in plants that involve Regulated Intramembrane Proteolysis (RIP) and rhomboid proteases remains low. As expressed recently by other researchers, finding a natural substrate for a rhomboid protease represents the biggest experimental challenge. Using yeast mitochondria-based assays, a potential link between the plastid translocon component Tic40 and organellar rhomboid proteases was recently uncovered. In this particular link, rhomboid proteases appear capable of influencing the pattern of imported Tic40 in yeast mitochondria. Tic40 may thus represent a natural plant target of organellar rhomboid proteases. Here, we obtained further motif-oriented evidence supporting Tic40 as a natural plant rhomboid substrate. A comparative analysis of sequences revealed that Tic40 may also possess similar TMD motifs found in the model substrate, Spitz. Rhomboid proteases often require these motifs to cleave substrates within intramembrane environments. Using site-directed mutagenesis and yeast mitochondria assays, the impact of mutations occurring in the motifs ASISS, GV, QP, and GVGVG of Tic40 was assessed. In terms of cleavage and changing the pattern of imported Tic40, some of the mutations showed decreased activities and a few showed enhancements. More importantly, the overall observed pattern associated with select Tic40 mutations resembled the characteristics reported for the model substrates. In particular, mutations in the Tic40 GV motif produced similar results as that observed with Spitz, by drastically decreasing or increasing cleavage as a function of amino acid sequence. / Thesis (Master, Biology) -- Queen's University, 2010-05-30 10:22:07.72

Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. / Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.

Retinoblastoma Binding Protein 6 (RBBP6) is a 200 KDa protein shown to play a role in 3'- polyadenylation of mRNA transcripts, as well as to function as an E3 ligase catalysing ubiquitination of cancer-associated proteins. RBBP6 has been previously reported to localise to nuclear speckles, which are thought to play a role in mRNA splicing, presumably as a result of its RS domain, which is known to target mRNA splicing factors to nuclear speckles. However recent studies in our laboratory have shown that isoform 3 of RBBP6, consisting mainly of the DWNN domain, also localises to speckles in resting cells, but more strongly in cells subjected to various stresses, suggesting that the DWNN domain may be the speckle-targeting domain. / Magister Scientiae - MSc (Biotechnology)

Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. / Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.