Cyclodextrin Glycosyltransferase Research Articles

Structures of some novel α-glucosyl diterpene glycosides from the glycosylation of steviol glycosides.

Four new minor diterpene glycosides with a rare α-glucosyl linkage were isolated from a cyclodextrin glycosyltransferase glucosylated stevia extract containing more than 98% steviol glycosides. The new compounds were identified as 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-α-d-glucopyranosyl-β-d-glucopyranosyl) ester] (1), 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl ester] (2), 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (3), and 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-α-d-glucopyranosyl-β-d-glucopyranosyl) ester] (4) on the basis of extensive NMR and mass spectral (MS) data as well as hydrolysis studies.

Acceptor-induced modification of regioselectivity in CGTase-catalyzed glycosylations of p-nitrophenyl-glucopyranosides

Cyclodextrin glycosyltransferases (CGTase) are reported to selectively catalyze α(1→4)-glycosyl transfer reactions besides showing low hydrolytic activity. Here, the effect of the anomeric configuration of the glycosyl acceptor on the regioselectivity of CGTase catalyzed glycosylations was investigated. For this purpose, the α and β anomers of p-nitrophenyl-d-glucopyranoside were used as glycosyl acceptors, Bacillus macerans and Thermoanaerobacter sp. CGTases were used as biocatalysts and β-cyclodextrin as the glycosyl donor. As expected, p-nitrophenyl-α-d-glucopyranosyl-(1→4)-O-α-d-glucopyranoside was produced when p-nitrophenyl-α-d-glucopyranoside was used as acceptor with B. macerans CGTase. Surprisingly, when p-nitrophenyl-β-d-glucopyranoside was used as glycosyl acceptor, besides the expected α(1→4)-glycosylation products both α(1→3)- and α(1→6)-transfer products were also obtained. This unexpected change in B. macerans CGTase regioselectivity leading to α(1→4)-, α(1→3)- and α(1→6)-glycosylation products was also observed for Thermoanaerobacter sp. CGTase with the β anomer. It is shown, applying time course analyses, that all isomers can be synthesized efficiently by adequate selection of enzyme and reaction conditions. In fact, when using Thermoanaerobacter sp. CGTase the yield of p-nitrophenyl-β-d-isomaltoside (the α(1→6)-transfer product) was the highest at long reaction time (19% yield). The previously unknown capacity of α(1→6)-glycosidic linkages formation by CGTases demonstrates an unexpected broader regioselectivity of CGTases in glycosyl-transfer reactions as well as an acceptor dependent transfer selectivity.

Mutations in cyclodextrin glycosyltransferase from Bacillus circulans enhance β-cyclization activity and β-cyclodextrin production.

Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) is used to produce cyclodextrins, which are cyclic glucans with many industrial applications. In the present study, the effects of the amino acid residue at position 577, which is located in calcium-binding site III (CaIII), on cyclization activity and cyclodextrin production were investigated by replacing Asp577 in CGTase from Bacillus circulans STB01 with glutamate, arginine, lysine, and histidine. The results showed that mutations D577E and D577R significantly increased the β-cyclization activity. The D577R mutant, in particular, displayed a 30.7% increase in the β-cyclization activity when compared to the wild-type CGTase. Furthermore, under conditions resembling industrial production processes, the D577R and D577E mutants displayed 9.1 and 2.0% enhancement in β-cyclodextrin production, respectively. More importantly, the higher β-cyclization activities resulted in a significant reduction in the amount of mutant protein required during the process. Thus, the two mutants were much more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme.

Recovery of Bacillus cereus cyclodextrin glycosyltransferase using ionic liquid-based aqueous two-phase system

Ionic liquids-based aqueous two-phase system (ILATPS) offers immediate phase separation and thus reduces the overall processing time, is significantly advantageous as compared to conventional ATPS such as polymer/polymer ATPS and polymer/salt ATPS. In this study, ILATPSs composed of imidazolium-based ionic liquid (IL) and salt were experimentally evaluated for their efficiencies in recovering Bacillus cereus cyclodextrin glycosyltransferase (CGTase) from fermentation broth. The phase-forming behavior of 1-ethyl-3-methylimidazolium tetrafluroborate, (Emim)BF4/sodium citrate ILATPS and (Emim)BF4/sodium carbonate ILATPS were first studied by constructing the binodal curves. Effects of the ILs concentration, pH value, feedstock loading, and addition of sodium chloride (NaCl) on the recovery of CGTase in ILATPS were investigated. The optimum conditions for the recovery of CGTase were obtained in an ILATPS consisting of 35% (w/w) (Emim)BF4, 18% (w/w) sodium carbonate and 3% (w/w) NaCl. Experimental results showed that 78% of CGTase could be recovered in the IL-rich phase in single-step purification with a purification fold (PF) of 15.4. The high PF indicates that this ILATPS is feasible to be applied in the recovery and separation of CGTase from the fermentation broth.

Optimization of β-Cyclodextrin Production from Sago Starch using Recombinant Cyclodextrin Glycocyltransferase from Bacillus sp. A2-5a

Cyclodextrin (CD) is a cyclic oligosaccharide molecule which is classified into three types: α-CD, β-CD, and γ-CD, each consists of 6, 7, and 8 glucose units. CDs are produced enzymatically using starch as the substrate catalyzed by CD glycosyltransferase (CGTase). This research was aimed to determine optimum condition of β-CD production from sago starch using recombinant CGTase (rCGTase) from Bacillus sp. A2-5a, and using the optimum condition, β-CD production was done at 100 mL scale using complexing agents. In this study, rCGTase was overproduced in Escherichia coli BL21(DE3) and purified applying Ni-NTA affinity chromatography with gradual elution of imidazole and concentrated using Nanosep column. The purified rCGTase was 76.39 kDa in size and displayed β-cyclization and hydrolysis activities using zymography. The results showed that optimum conditions for β-CD production was achieved when preheated sago starch of 0.5% (w/v), enzyme of 2.6x10-2 U, and N,N-dimethylformamide (DMF) as complexing agent of 1% (v/v) were used and the reaction time was 8 h. When DMF of 1% (v/v) was added repeatedly at 100 mL scale production, the highest concentration of β-CD was obtained at the reaction time of 8 h. This research reported for the first time the optimum condition of b-CD production at 100 mL scale using rCGTase from Bacillus sp. A2-5a with DMF as complexing agent.

COMPARATIVE STUDY OF Β-CYCLODEXTRIN PRODUCTION FROM THE CGTASE PRODUCING NOVEL STRAINS- ITS CHARACTERIZATION AND HOST-GUEST COMPLEX FORMATION

The enzymatic synthesis of β-CD by CGTase produced from different strains of alkaliphilic bacterial culture which was isolated from the cultivated sugarcane fields and standard MTCC cultures using starch as substrate. An alkalophilic bacteria were grown for six days at static conditions at pH 10.5. The time course of CGTase activity was studied with the maximum activity observed on 6th day. The activity check and its confirmation were done by Dextrinising and Phenolphthalein assay. The soluble starch was best substrate to produce the cyclodextrin. Extraction of β-CD was done using complexing agents, these agents binds only to β-CD and forms complex, the complex thus formed was recovered. The obtained β-CD was made inclusion complex with guest molecule and was further characterized using UV absorption spectrophotometer, FT-IR and melting point.  Keywords: Cyclodextrin glycosyltransferases, Cyclodextrins, Bacillus licheniformis.

Hydrophobic Complexation Promotes Enzymatic Surfactant Synthesis from Alkyl Glucoside/Cyclodextrin Mixtures

The unique ability of cyclodextrin glycosyltransferase to form and utilize the cyclic maltooligosaccharide cyclodextrin (CD) makes this enzyme an attractive catalyst for the synthesis of alkyl glycosides. Here, we characterize the sugar headgroup elongation of alkyl glucosides (acceptor) via two transglycosylation reactions from either a linear (maltohexose) or a cyclic (CD) glycosyl donor. Inclusion complex formation overcomes both poor substrate solubility and aggregation. We have used pure alkyl glucosides and αCD as model compounds. The complex between CD and alkyl glucoside was efficiently used as a substrate. Kinetic and thermodynamic measurements allow the prediction of the optimal synthesis conditions. This optimum corresponds to the transition between a donor-limiting and an acceptor-limiting regime. The resulting rational design should lead to the practical development of a cost-efficient industrial synthesis. Our findings with respect to the importance of complexation should also readily apply to other enzymatic systems.

BIOPROSPECTING FOR CYCLODEXTRIN GLYCOSYLTRANSFERASE PRODUCING BACTERIA

Cyclodextrin glycosyltransferase- (CGTase), (EC.2.4.1.19) producing micro-organisms were isolated from different sources of soil (paddy plantation, potato plantation, garden and Malpe beach) from places in and around Udupi, India. A potential isolate MBS 24 was identified as Virgibacillus pantothenticus. This organism was cultivated on modified Horikoshi medium at 130 rpm for 24 h at 32 °C. The maximum production of CGTase enzyme was observed (0.308μmol/ml β-CD units) after 36 h of incubation at 32°C and pH 10.0. CGTase was precipitated using ammonium sulphate at 60% saturation. Cyclodextrin (CD) produced by incubation of CGTase with starch was extracted using toluene and analyzed by thin layer chromatography. Rf values of the solvent extracted CD were similar to the standard α, β and γ CD confirming that all three types of CD’s were formed during enzymatic conversion. These CDs could be used as drug carrier for delivering BCS class II drugs.

Fusion of self-assembling amphipathic oligopeptides with cyclodextrin glycosyltransferase improves 2-O-D-glucopyranosyl-L-ascorbic acid synthesis with soluble starch as the glycosyl donor.

In this study, we fused six self-assembling amphipathic peptides (SAPs) with cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans to catalyze 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) production with cheap substrates, including maltose, maltodextrin, and soluble starch as glycosyl donors. The results showed that two fusion enzymes, SAP5-CGTase and SAP6-CGTase, increased AA-2G yields to 2.33- and 3.36-fold that of wild-type CGTase when soluble starch was used as a substrate. The cyclization activities of these enzymes decreased, while disproportionation activities increased. Enzymatic characterization of the two fusion enzymes was performed, and kinetics analysis of AA-2G synthesis confirmed the enhanced soluble starch specificity of SAP5-CGTase and SAP6-CGTase compared to that in the wild-type CGTase. As revealed by structure modeling of the fusion and wild-type CGTases, enhanced substrate-binding capacity may result from the increased number of hydrogen bonds present after fusion. This study demonstrates an effective protein fusion approach to improving the substrate specificity of CGTase for AA-2G synthesis. Fusion enzymes, especially SAP6-CGTase, are promising starting points for further development through protein engineering.

Nanosilica Sol Leads to Further Increase in Polyethylene Glycol (PEG) 1000-Enhanced Thermostability of β-Cyclodextrin Glycosyltransferase from Bacillus circulans

A major disadvantage of cyclodextrin production is the limited thermostability of cyclodextrin glycosyltransferase. The ability of combinations of nanosilica sol with polyethylene glycol (PEG) 1000 to enhance the thermostability of the β-cyclodextrin glycosyltransferase from Bacillus circulans was investigated. It was found that 10% PEG 1000 combined with 0.05% nanosilica sol could activate the β-cyclodextrin glycosyltransferase by 17.2%. Furthermore, 0.05% nanosilica sol leads to further increase in PEG 1000-enhanced thermostability of β-cyclodextrin glycosyltransferase. With the simultaneous addition of 10% PEG 1000 and 0.05% nanosilica into the enzyme solution, which was allowed to incubate for 60 min at 60 °C, 61.3% of β-cyclodextrin-forming activity could be retained, which was much higher than that with only 10% PEG 1000 added. Atomic force microscopy, fluorescence spectroscopy, and circular dichroism analysis indicated that silica nanoparticles helped PEG 1000 further protect the tertiary and secondary structures of β-cyclodextrin glycosyltransferase. This study provides an effective approach for improving the thermostability of cyclodextrin glycosyltransferase and related enzymes.

Structural basis of a mutant Y195I α-cyclodextrin glycosyltransferase with switched product specificity from α-cyclodextrin to β-/γ-cyclodextrin

Cyclodextrin glycosyltransferase (EC 2.4.1.19) (CGTase) is an extracellular bacterial enzyme which has the unique capability of forming cyclodextrins from starch. Our previous investigation revealed that a mutant Y195I α-CGTase drastically altered the cyclodextrin specificity by switching toward the synthesis of both β- and γ-CDs (Xie et al., 2013a,b). In this study, we determined one X-ray structure of the mutant Y195I α-CGTase at 2.3Å. The overall structure was similar to that of the typical β-CGTase from Bacillus circulans 251, with minor difference in flexible domains since they showed about 70% homogeneity of amino acid sequences. The central site with isoleucine tended to be more flexible than tyrosine thus made the sugar chain, during the cyclization process, form a larger cyclodextrin like β- and γ-CDs surrounding the central site instead of α-CD. Superposition of the structure of Y195I α-CGTase with those of β-CGTase and γ-CGTase showed that residues Lys232, Lys89 and Arg177 at subsites +2, −3 and −7 could form smaller substrate binding cavity. In summary, the crystal structure revealed that moderate increase of mobility of the central site resulted in the switched product specificity from α-CD to β- and γ-CDs of the mutant Y195I α-CGTase. The space differences alongside the active domain may be another factor that impacts the product specificity of the CGTase.

Mutations enhance β-cyclodextrin specificity of cyclodextrin glycosyltransferase from Bacillus circulans

The effects of amino acid residue at position 31 in the neighborhood of calcium binding site I (CaI) on product specificity of cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) were investigated by replacing Ala31 in the CGTase from Bacillus circulans STB01 with arginine, proline, threonine, serine and glycine. The results showed that the mutations A31R, A31P, and A31T resulted in the increases in β-cyclodextrin-forming activity and β-cyclodextrin production, indicating that these mutations enhanced β-cyclodextrin specificity of the CGTase. Especially the mutant A31R displayed approximately 26% increase in β-cyclodextrin production with a concomitant 41% decrease in α-cyclodextrin production when compared to the wild-type CGTase. Thus, it was much more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhanced β-cyclodextrin specificity of the mutants might be a result of stabilizing CaI, which also suggested that CaI might play an important role in cyclodextrin product specificity of CGTase.

Two-step process for preparation of oligosaccharide propionates and acrylates using lipase and Cyclodextrin Glycosyl Transferase (CGTase)

Abstract Background Oligosaccharide esters are attractive candidates for applications as surfactants, hydrogels and other materials, but direct enzymatic acylation is difficult with carbohydrates longer than disaccharides. Results A combination of one lipase-catalyzed step and one transglycosylation step catalyzed by a cyclodextrin glycosyl transferase (CGTase) was used to synthesize oligosaccharide esters. The conversion of glucose and maltose with vinyl propionate catalyzed by Candida antarctica lipase B (Novozym 435) in dioxane proceeded to full conversion to mixtures of mono and diesters. When ethyl acrylate was used as acyl donor, mono and diesters were formed, but full conversion was not reached. The CGTase catalyzed reactions between the glucose and maltose esters and α-cyclodextrin were carried out in water. In the initial phase, addition of the glucose residues of the cyclodextrin to the ester substrate occurred (coupling reaction), followed by disproportionation reactions yielding a range of oligosaccharide esters with varying chain length. The monoesters were efficient acceptors in the CGTase-catalyzed reactions, while the diesters were not converted to a significant extent. As a consequence, the glucose propionate which contained large amounts of diesters was converted to 40% conversion while the maltose propionate which contained mainly monoesters was converted to 86% conversion. Conclusions A two-step enzymatic process for preparation of oligosaccharide esters has been developed. Oligosaccharide propionates were produced in high yield with a total reaction time of 5 h. The double bond of the acrylate moiety reduced the reaction rate of the lipase catalyzed transesterification, but in both cases, the CGTase efficiently converted the monoesters to oligosaccharide esters.

Cyclodextrin production by Bacillus lehensis isolated from cassava starch: Characterisation of a novel enzyme

The properties of a previously unknown enzyme, denominated cyclodextrin glycosyltransferase, produced from Bacillus lehensis, were evaluated using affinity chromatography for protein purification. Enzyme characteristics (optimum pH and temperature; pH and temperature stability), the influence of substances on the enzyme activity, enzyme kinetics, and cyclodextrin production were analysed. Cyclodextrin glycosyltransferase was purified up to 320.74-fold by affinity chromatography using β-cyclodextrin as the binder and it exhibited 8.71% activity recovery. This enzyme is a monomer with a molecular weight of 81.27 kDa, as estimated by SDS-PAGE. Optimum temperature and pH for cyclodextrin glycosyltransferase were 55°C and 8.0, respectively. The Michaelis-Menten constant was 8.62 g/l during maximum velocity of 0.858 g/l.h.  

Novel insight into the secretory expression of recombinant enzymes in Escherichia coli

The secretory expression of recombinant enzymes in Escherichia coli has generally been a challenging task. In the present study, we investigated the expression of the extracellular enzyme cyclodextrin glycosyltransferase in E. coli. Our results indicated that when the overexpressed pre-proteins were not translocated across the inner membrane in a timely manner, they aggregated near the inner side of the E. coli inner membrane, resulting in the formation of insoluble inclusion bodies, which eventually blocked the pre-protein translocation channels and subsequently impeded further protein secretion. This mechanism suggests that for the efficient production of extracellular enzymes in E. coli, it is very important to maintain a balance between the rate of pre-protein synthesis and translocation, which can be achieved by altering the cultivation process. Our findings provide novel insight into the secretory expression of extracellular enzymes and may shed light on the further development of new strategies for extracellular protein production in E. coli.

Effect of substrate and enzyme concentration on cyclodextrin production in a hollow fibre membrane reactor system

The batch and continuous production of cyclodextrins (CDs) was assessed by employing an enzymatic membrane reactor (EMR) system. The effects of tapioca starch substrate and cyclodextrin glycosyltransferase (CGTase) concentrations on the yield of CDs were studied. A similar effect on the behaviour of the ultrafiltration membrane in the EMR system (integration system) was also evaluated. The results for the batch process showed that incremental doses of CGTase caused gradual increments in CD yield; however, further addition of CGTase (above 1.0%) showed a 16% reduction in the total CD production. Further incremental in the tapioca starch concentration increased CD concentration (23g/L). However, addition above 8% w/v resulted in an insignificant yield of CDs. In the case of integration system, tapioca starch feeding rate that is higher than 4.41g/h caused adverse effects (lower CD yield and membrane flux). In particular at higher tapioca starch feeding rate (5.0g/h), the hydraulic resistance would reach as high as 1.31×1013m−1. Presumably this phenomenon was due to the unreacted substrates that were adsorbing onto the membrane surface and pores that subsequently led to greater fouling conditions and a severe flux decline. In addition, the weak adsorption (ra1) has been found to be the major fouling mechanism attributed to starch and its by products. Therefore, hydraulic cleaning is highly suggested as the procedure to be used for this EMR system.

Production and characterization of cyclodextrin glycosyltransferase from Bacillus sp. isolated from Cuban soil

Abstract A cyclodextrin glycosyltransferase (CGTase) from an alkaliphilic Bacillus sp. strain, isolated from Cuban soil, was purified with Sephadex G-50 with a yield of 66.5%. The CGTase was stable over a very wide pH range, 6.0–10, at 25°C and was most active at pH 7.5. The enzyme exhibited an optimum temperature of 60°C and was stable to 50°C for at least 8 h. The T 50 value – defined as the temperature at which 50% of the initial activity was retained–was 63°C in this enzyme. The influence of substrate or product concentration on the initial rate of CD production was studied, and the kinetic parameters were determined. The analysis of kinetic parameters K m and V max was obtained by the action of CGTase on the starch of corn with respect to β-CD, and the values were 4.1 g/L and 5.2 μM β-CD/min ml, respectively. The purified CGTase from Bacillus sp. could be used for an efficient cyclodextrin (CD) production which is the significant yield of γ- CDs.

Biosynthesis of CGTase by immobilized alkalophilic bacilli and crystallization of beta-cyclodextrin: Effective techniques to investigate cell immobilization and the production of cyclodextrins

Cyclodextrin glycosyltransferase (CGTase) catalyses the degradation of starch, producing α-, β-, and γ-cyclodextrins (CDs). Immobilized cells offer several advantages, such as their prolonged and repeated use, ease separation from the fermentation medium, and reduced risk of contamination. The biosynthesis of CGTase and β-CD was optimized by immobilization of Bacillus firmus strain 37 and Bacillus sphaericus strain 41 cells on a loofa sponge. Both microorganisms produced significant levels of CGTase for three consecutive cycles (94.2U/mL on average), increased the relative formation of the β-CD, and inhibited the formation of other CDs. The crystallization of the CD produced by both bacterial strains, when corn starch was used as the substrate, resulted in a 64% recovery of β-CD. The purity of the β-CD was 89.5% when maltodextrin was used as the substrate for B. firmus strain 37. The Tilden–Hudson technique was used as a simple, inexpensive, and efficient method for monitoring the continuous production of CDs using immobilized cells. The innovative use of the Fourier Transform Infrared Spectroscopy technique by means of the Attenuated Total Reflectance method suggested that the interaction between the B. sphaericus strain 41 cells and the loofa sponge occurred by natural adsorption.

Immobilization of cyclodextrin glucanotransferase on aminopropyl-functionalized silica-coated superparamagnetic nanoparticles

Background: Cyclodextrin glycosyltransferase (CGTase) from Amphibacillus sp. NPST-10 was successfully covalently immobilized on aminopropyl-functionalized silica coated superparamagnetic nanoparticles; and the properties of immobilized enzyme were investigated. The synthesis process included preparing of core magnetic magnetite (Fe3O4) nanoparticles using solvothermal synthesis; followed by coating of Fe3O4 nanoparticles with dense amino-functionalized silica (NH2-SiO2) layer using in situ functionalization method. The structure of synthesized Fe3O4@NH2-SiO2 nanoparticles was characterized using TEM, XRD, and FT-IR analysis. Fe3O4@NH2-SiO2 nanoparticles were further activated by gluteraaldehyde as bifunctional cross linker, and the activated nanoparticles were used for CGTase immobilization by covalent attachment. Results: Magnetite nanoparticles was successfully synthesized and coated with and amino functionalized silica layer (Fe3O4/NH2-SiO2), with particle size of 50-70 nm. The silica coated magnetite nanoparticles showed with saturation magnetization of 65 emug -1 , and can be quickly recovered from the bulk solution using an external magnet within 10 sec. The activated support was effective for CGTase immobilization, which was confirmed by comparison of FT-IR spectra of free and immobilized enzyme. The applied approach for support preparation, activation, and optimization of immobilization conditions, led to high yields of CGTase immobilization (92.3%), activity recovery (73%), and loading efficiency (95.2%); which is one of the highest so far reported for CGTase. Immobilized enzyme showed shift in the optimal temperature from 50 to 55oC, and significant enhancement in the thermal stability compared with free enzyme. The optimum pH for enzyme activity was pH 8 and pH 7.5 for free and immobilized CGTase, respectively, with slight improvement of pH stability of immobilized enzyme. Furthermore, kinetic studies revealed that immobilized CGTase had higher affinity toward substrate; with km values of 1.18 ± 0.05 mg/ml and 1.75 ± 0.07 mg/ml for immobilized and free CGTase, respectively. Immobilized CGTase retained 87% and 67 of its initial activity after 5 and 10 repeated batches reaction, indicating that immobilized CGTase on Fe3O4/NH2-SiO2 had good durability and magnetic recovery. Conclusion: The improvement in kinetic and stability parameters of immobilized CGTase makes the proposed method a suitable candidate for industrial applications of CGTase. To best of our knowledge, this is the first report about CGTase immobilization on silica coated magnetite nanoparticles.

Site-saturation mutagenesis of central tyrosine 195 leading to diverse product specificities of an α-cyclodextrin glycosyltransferase from Paenibacillus sp. 602-1

Central tyrosine 195 plays an important role in the active site of cyclodextrin glycosyltransferase (CGTase) that is highly conservative among various CGTases. However, a detailed functional understanding of this subsite is lacking. In this study, we applied site-directed saturation mutagenesis to investigate the effect of tyrosine 195 on the hydrolytic activity and cyclization specificity of an α-CGTase. A total of 17 mutant CGTases were obtained and heterologously expressed in E. coli. The mutant Y195F α-CGTase showed similar characteristics with wild-type α-CGTase. The other mutant α-CGTases showed considerably lower activity for starch-degradation and cyclodextrin (CD) formation. Interestingly, we found that the main product of mutant Y195R α-CGTase was γ-CDs (50%), not α-CDs (35%). The mutant Y195I α-CGTase drastically altered the CD specificity of α-CGTase, which showed a switch toward the synthesis of both β- and γ-CDs with percentages of 34% and 38%, respectively. Other mutant CGTases retained the α-CD as the main product but with lower percentages than wild-type α-CGTase. Mutant Y195F, Y195I, and Y195R CGTases showed an optimal temperature of 50°C and pH 6.5. The mutants Y195I and Y195R also showed better thermostability. These findings suggested that aromatic amino acids Tyr or Phe at the 195 position were important for the amylolytic activity and cyclization specificity of α-CGTase. The mutants Y195I CGTase and Y195R CGTase have potential applications for γ-CD production in the future.