Cgt Gene Research Articles

Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production.

In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20°C for 18 h with 0.4 mM of isopropyl β-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80°C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40°C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G.

Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110

Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-binding function of the Cgt protein in a binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant ∆cgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8–16% higher product titers when grown in maltose-containing medium.

Improved extracellular secretion of β-cyclodextrin glycosyltransferase from Escherichia coli by glycine supplementation without apparent cell lysis

The use of an effective inducer feeding strategy without causing cell lysis presents significant advantage to enhance the secretion of an enzyme to the culture medium of Escherichia coli. The cgt gene encoding β-cyclodextrin glycosyltransferase (β-CGTase) was cloned into pQE30xa as an N-terminal His-tagged protein and transformed into E. coli. The induction strategy was applied towards enhancing the extracellular secretion of the recombinant β-CGTase by increasing permeability of the outer membrane of E. coli. The supplementation of 1.2 mM glycine following 2 h of fermentation at 37°C enhanced the activity of β-CGTase to 38.295 U/mL, which was approximately 1.3-fold higher than the control (without induction). Further flow cytometry analysis was adopted as a rapid and highly reproducible approach to determine the effect of glycine supplementation on the viability of E. coli cells. The supplementation of glycine did not contribute to apparent cell lysis, with no adverse effects on cell viability, hence indicating the effectiveness of glycine in enhancing the extracellular secretion of β-CGTase. The recombinant β-CGTase was then purified through a combination of diafiltration and Ni-NTA affinity chromatography with 18.4-fold increase in purity. An effective glycine feeding strategy could enhance the extracellular secretion of β-CGTase without adverse effects on cell viability. This strategy could be applied potentially to enhance the secretion of a recombinant protein to the culture medium from E. coli cells without having cell lysis.

Heterologous expression of cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli and its application in 2-O-\u03b1-D-glucopyranosyl-L-ascorbic acid production

BackgroundCyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), which shows diverse applications in food, cosmetic and pharmaceutical industries.ResultsIn this study, the cgt gene encoding α-CGTase from Paenibacillus macerans was codon-optimized (opt-cgt) and cloned into pET-28a (+) for intracellular expression in E. coli BL21 (DE3). The Opt-CGT was purified by Ni2+-NTA resin with a 55% recovery, and specific activity was increased significantly from 1.17 to 190.75 U·mg− 1. In addition, the enzyme was adopted to transform L-AA into 9.1 g/L of AA-2G. Finally, more economic substrates, including β-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5 g/L of AA-2G were obtained, respectively.ConclusionsN-terminal amino acid is critical to the activity of CGTase suggested by its truncation study. Furthermore, when the Opt-CGT was flanked by His6-tags on the C- and N-terminal, the recovery of purification by Ni2+-NTA resin is appreciably enhanced. α-cyclodextrin was the ideal glycosyl donor for AA-2G production. In addition, the selection of low cost glycosyl donors would make the process of AA-2G production more economically competitive.

High-level secretion and characterization of cyclodextrin glycosyltransferase in recombinant Komagataella phaffii

Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch into cyclodextrin (CD), which is widely applied in food, pharmaceutical, cosmetic, and agricultural industries. For efficient production of CD, high yield of CGTase with good characteristics is necessary. In this study, the cgt gene from Bacillus pseudalcaliphilus was expressed in Komagataella phaffii after codon optimization and expression vector selection. The β-CGTase activity in the transformant reached 3885.1UmL−1, which is the highest value reported so far, at 28°C, 6% inoculum ratio, and 1.5% methanol addition following 24h of incubation. The recombinant CGTase showed high specific activity at 80°C without any γ-CGTase activity, and had good stability in a wide pH and temperature range. These results demonstrate that the recombinant CGTase could have potential industrial applications.

Mutations at calcium binding site III in cyclodextrin glycosyltransferase improve β-cyclodextrin specificity

Cyclodextrin glycosyltransferases (CGTases, EC 2.4.1.19) are industrially important enzymes that produce cyclodextrins from starch by intramolecular transglycosylation. In this study, the effects of amino acid residue at position 315 in calcium binding site III (CaIII) on product specificity of CGTase were investigated by replacing Ala315 in the CGTase from Bacillus circulans STB01 with arginine, aspartic acid, threonine, leucine and valine. The cgt gene, which encodes this enzyme, was expressed in B. subtilis WB600 alongside site-directed mutants A315R, A315D, A315T, A315L and A315V. The results showed that CaIII plays an important role in cyclodextrin product specificity. Replacement of Ala315 by charged amino acid residues enhanced β-cyclodextrin specificity, compared with the wild-type CGTase. Mutations A315R and A315D resulted in an approximately 10% increase in β-cyclodextrin activity. Furthermore, under conditions resembling the industrial production processes, the mutants A315R and A315D displayed obvious increases in the production of β-cyclodextrin, indicating they were much more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhancement of β-cyclodextrin specificity for the mutants might be due to the stability of CaIII by charged amino acid substitutions.

Glycine and Triton X-100 enhanced secretion of recombinant α-CGTase mediated by OmpA signal peptide in Escherichia coli

OmpA signal peptide mediated cgt gene from Paenibacillus macerans JFB05-01 was cloned and expressed in E. coli BL21 (DE3). The effects of glycine and Triton X-100 on extracellular production of α-cyclodextrin glycosyltransferase (α-CGTase) were investigated. When supplemented with Gly or Triton X-100 to the culture media individually, the secreted extracellular enzyme reached 32 or 33 U/mL at 48 h of cultivation, respectively. When supplemented with Gly and Triton X-100 together, the extracellular α-CGTase activity reached 48 U/mL after 48 h cultivation, which was 20-fold of the control group without any additives. Analysis of membrane permeability demonstrated that addition of glycine and Triton X-100 enhanced the permeability of both outer and inner membrane. The potential mechanism of the enhanced protein secretion was discussed.

Extracellular expression and biochemical characterization of α-cyclodextrin glycosyltransferase from Paenibacillus macerans

The cgt gene encoding α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90 h of induction, the activity of α-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant α-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45 °C, and half-lives are approximately 8 h at 40 °C, 1.25 h at 45 °C and 0.5 h at 50 °C. The recombinant α-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca 2+, Ba 2+, and Zn 2+ in a concentration-dependent manner, while it is dramatically inhibited by Hg 2+. The kinetics of the α-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation.

Gene cloning and enzymatic characteristics of a novel γ-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364

A new gene, cda, was found in the downstream region of the cgt gene encoding cyclodextrin (CD) glucanotransferase from Bacillus clarkii 7364. Cda encoded by the cda was a cyclodextrinase that has extremely high specificity for γ-CD. The rates of hydrolysis toward α- and β-CD, maltooctaose and polysaccharides were less than 4% of that toward γ-CD. Cda also has a transglycosylation activity, by which the maltotriose moiety was transferred from maltohexaose and maltopentaose. The comparison of the amino acid sequences between Cda and CD-degrading enzymes revealed the sequence of Cda has unique features. One of them is Gly247 next to the catalytic nucleophile Asp246. Most enzymes in GH family 13 have more bulky amino acids at this position. Other features in Cda are the lack of the N-domain in CD-degrading enzymes involving in the dimerization contributing to the preference of CDs and the existence of a long extra sequence in the C-terminus. Despite the lack of N-domain, Cda showed a dodecameric structure. The long extra sequence in the C-terminus might contribute to the oligomerization of Cda through a new mechanism. These unique features indicate that Cda is a novel type of CD-degrading enzyme.

Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution

Thermoanaerobacterium thermosulfurigenes cyclodextrin glucanotransferase primarily catalyses the formation of cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. This side reaction is undesirable, since it produces short saccharides that are responsible for the breakdown of the cyclodextrins formed, thus limiting the yield of cyclodextrins produced. To reduce the competing hydrolysis reaction, while maintaining the cyclization activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone PCR. Mutations in two residues, Ser-77 and Trp-239, on the outer region of the active site, lowered the hydrolytic activity up to 15-fold with retention of cyclization activity. In contrast, mutations within the active site could not lower hydrolytic rates, indicating an evolutionary optimized role for cyclodextrin formation by residues within this region. The crystal structure of the most effective mutant, S77P, showed no alterations to the peptide backbone. However, subtle conformational changes to the side chains of active-site residues had occurred, which may explain the increased cyclization/hydrolysis ratio. This indicates that secondary effects of mutations located on the outer regions of the catalytic site are required to lower the rates of competing side reactions, while maintaining the primary catalytic function. Subsequent functional analysis of various glucanotransferases from the superfamily of glycoside hydrolases also suggests a gradual evolutionary progression of these enzymes from a common 'intermediate-like' ancestor towards specific transglycosylation activity.

Regulation of cyclodextrin glucanotransferase synthesis in Bacillus ohbensis.

The production of cyclodextrin glucanotransferase (CGTase) by Bacillus ohbensis is dependent on the presence of starch and inhibited by glucose in the medium. Northern blot analysis revealed that the CGTase gene (cgt) was transcribed to almost the same level irrespective of the presence or absence of starch, but glucose completely repressed the transcription. Furthermore, a relatively high amount of CGTase protein was detected on Western blotting only in the medium with starch, showing the lack of posttranslational control of the CGTase activity. These findings suggest some starch induction mechanism for the cgt gene, possibly at the posttranscriptional level, besides negative transcriptional regulation by glucose.

Cloning and expression of a putative cyclodextrin glucosyltransferase from the symbiotically competent cyanobacterium Nostoc sp. PCC 9229

A polymerase chain reaction-based method was used to isolate a Nostoc sp. PCC 9229 cDNA from infected glands of Gunnera chilensis. The complete gene sequence was isolated from a genomic Nostoc sp. PCC 9229 library. Sequence analysis showed 84% amino acid similarity to a putative cyclodextrin glycosyltransferase from Nostoc sp. PCC 7120 and the gene was therefore termed cgt. Southern blot revealed that the cgt gene was present in symbiotically competent cyanobacteria. The cgt gene was expressed in free-living nitrogen-fixing cultures in light or in darkness when supplemented with fructose. This is the first expression analysis of a cgt gene from a cyanobacterium.

Transcriptional regulation of the human UDP-galactose:ceramide galactosyltransferase (hCGT) gene expression: functional role of GC-box and CRE.

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthetic pathway of galactocerebroside (GalC), the most abundant glycolipid in myelin. Using a GalC expressing cell line, human oligodendroglioma (HOG), one which does not express GalC, human neuroblastoma (LAN-5), we previously demonstrated that the human CGT (hCGT) gene promoter functions in a cell-specific manner. Because the proximal (-292/-256) and distal (-747/-688) positive domains were shown to be critically involved in regulating the expression of several myelin-specific genes, we further investigated the functional roles of these two motifs in hCGT expression. Mutation analysis confirmed that a GC-box (-267/-259) and a CRE (-697/-690) were critical for hCGT expression. Electrophoretic mobility shift assay (EMSA) demonstrated that these motifs specifically bound to nuclear extracts from both cell lines. Using antibodies to Sp1, Sp3, pCREB-1, and ATF-1, these proteins were shown to be components of the EMSA complexes. However, the only difference between the HOG and LAN-5 cells was found in the EMSA profile of the CRE complexes. This difference may account for the differential transcription of the hCGT gene in the two cell types. Furthermore, the expression levels of ATF-1 detected were much higher in HOG cells than in LAN-5 cells. Thus, our data suggest that the GC-box and CRE function cooperatively, and that the CRE regulates the cell-specific expression of the hCGT gene.

Characterization of the human UDP-galactose:ceramide galactosyltransferase gene promoter

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthesis of galactocerebroside, the most abundant glycosphingolipid in the myelin sheath. An 8 kb fragment upstream from the transcription initiation site of CGT gene was isolated from a human genomic DNA library. Primer extension analysis revealed a single transcription initiation site 329 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start site; however, this region contains a high GC content and multiple putative regulatory elements. To investigate the transcriptional regulation of CGT, a series of 5′ deletion constructs of the 5′-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in the human oligodendroglioma (HOG) and human neuroblastoma (LAN-5) cell lines, we found that the CGT promoter functions in a cell type-specific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at −292/−256 which contains the potential binding sites for known transcription factors (TFs) such as Ets and SP1 (GC box), a distal region at −747/−688 comprising a number of binding sites such as the ERE half-site, NF1-like, TGGCA-BP, and CRE, and a third positive cis-acting region distally localized at −1325/−1083 consisting of binding sites for TFs such as nitrogen regulatory, TCF-1, TGGCA-BP, NF-IL6, CF1, bHLH, NF1-like, GATA, and γ-IRE. A negative cis-acting domain localized in a far distal region at −1594/−1326 was also identified. Our results suggest the presence of both positive and negative cis-regulatory regions essential for the cell-specific expression in the TATA-less promoter of the human CGT gene.

Kidney lipids in galactosylceramide synthase-deficient mice: absence of galactosylsulfatide and compensatory increase in more polar sulfoglycolipids

UDP-galactose:ceramide galactosyltransferase (CGT) catalyzes the final step in the synthesis of galactosylceramide (GalCer). It has previously been shown that CGT-deficient mice do not synthesize GalCer and its sulfated derivative GalCer I(3)-sulfate (galactosylsulfatide, SM4s) but form myelin containing glucosylceramide (GlcCer) and sphingomyelin with 2-hydroxy fatty acids. Because relatively high concentrations of GalCer and SM4s are present also in mammalian kidney, we analyzed the composition of lipids in the kidney of Cgt(-/-) and, as a control, Cgt(+/-) and wild-type mice. The homozygous mutant mice lacked GalCer, galabiaosylceramide (Ga(2)Cer), and SM4s. Yet, they did not show any major morphological or functional defects in the kidney. A slight increase in GlcCer containing 4-hydroxysphinganine was evident among neutral glycolipids. Intriguingly, more polar sulfoglycolipids, that is, lactosylceramide II(3)-sulfate (SM3) and gangliotetraosylceramide II(3),IV(3)-bis-sulfate (SB1a), were expressed at 2 to 3 times the normal levels in Cgt(-/-) mice, indicating upregulation of biosynthesis of SB1a from GlcCer via SM3. Given that SM4s is a major polar glycolipid constituting renal tubular membrane, the increase in SM3 and SB1a in the mice deficient in CGT and thus SM4s appears to be a compensatory process, which could partly restore kidney function in the knockout mice.

Twitcher mice with only a single active galactosylceramide synthase gene exhibit clearly detectable but therapeutically minor phenotypic improvements.

Cross-breeding of mouse mutants, each defective in either synthesis (CGT knockout) or degradation (twitcher) of galactosylceramide, generates hybrids with a genotype of galc -/-, cgt +/-, in addition to doubly deficient mice. They are ideally suited to test the potential usefulness of limiting synthesis of the substrate as a treatment of genetic disorders due to degradative enzyme defects. The rate of accretion of galactosylceramide in the brain of CGT knockout carrier mice (cgt +/-) is approximately two-thirds of the normal, suggesting a gene-level compensation for the reduced gene dosage. Phenotype of twitcher mice with a single dose of normal cgt gene was indeed milder with statistical significance, albeit only slightly. Compared among 10 paired littermates, the difference in the life span was 7+/-3.9 days (S.D.) and the difference in the maximum attained body weight was 1.9+/-1.2 g (S.D.). Neuropathologists were able to distinguish blindly galc -/-, cgt +/- mice from galc -/-, cgt +/+ mice. The brain psychosine level in galc -/-, cgt +/- mice was also approximately two-thirds of the galc -/-, cgt +/+ mice. These observations indicate that reduction of galactosylceramide synthesis to two-thirds of the normal level results in minor but clearly detectable phenotypic improvements. Because of the detrimental consequences of drastic reduction in galactosylceramide synthesis that may be required for pragmatically meaningful improvements, this approach by itself is unlikely to be useful as the sole treatment but may be helpful as a supplement to other therapies.

Transcriptional regulation of the Bacillus ohbensis cyclodextrin glucanotransferase gene in B. subtilis.

The expression of the cyclodextrin glucanotransferase (CGTase) gene (cgt) of Bacillus ohbensis, when introduced into an alpha-amylase-defective strain of B. subtilis on a multicopy plasmid, pHY300PLK, was induced in the presence of starch and was subject to catabolite repression by glucose as well as in the original strain, B. ohbensis. We constructed a cgt'::'lacZ translational fusion to study the expression in B. subtilis, and this construct was confirmed to be subject to both starch induction and catabolite repression. In order to define the region involved in the regulation of the cgt gene, a series of cgt'::'lacZ gene with various lengths of deletion in the promoter region was constructed on pHY300PLK. DNA regions responsible for starch induction and catabolite repression by glucose could be separated in the deletion experiment. Primer extension analysis showed that the catabolite repression was controlled at the initiation of transcription, while the starch induction is likely to be controlled by a transcriptional termination-antitermination mechanism.

Myelin glycolipids and their functions

During myelination, oligodendrocytes in the CNS and Schwann cells in the PNS synthesise myelin-specific proteins and lipids for the assembly of the axon myelin sheath. A dominant class of lipids in the myelin bilayer are the glycolipids, which include galactocerebroside (GalC), galactosulfatide (sGalC) and galactodiglyceride (GalDG). A promising approach for unravelling the roles played by various lipids in the myelin membrane involves knocking out the genes encoding important enzymes in lipid biosynthesis. The recent ablation of the ceramide galactosyltransferase ( cgt) gene in mice is the first example. The cgt gene encodes a key enzyme in glycolipid biosynthesis. Its absence causes glycolipid deficiency in the lipid bilayer, breakdown of axon insulation and loss of saltatory conduction. Additional knock-out studies should provide important insights into the various functions of glycolipids in myelinogenesis and myelin structure.

Regulation of cyclodextrin glucanotransferase synthesis in Bacillus ohbensis

The production of cyclodextrin glucanotransferase (CGTase) by Bacillus ohbensis is dependent on the presence of starch and inhibited by glucose in the medium. Northern blot analysis revealed that the CGTase gene ( cgt) was transcribed to almost the same level irrespective of the presence or absence of starch, but glucose completely repressed the transcription. Furthermore, a relatively high amount of CGTase protein was detected on Western blotting only in the medium with starch, showing the lack of posttranslational control of the CGTase activity. These findings suggest some starch induction mechanism for the cgt gene, possibly at the posttranscriptional level, besides negative transcriptional regulation by glucose.

Purification of Cyclodextrin Glycosyltransferase by Immunochromatography

AbstractCyclodextrin glycosyltransferase (CGTase) was produced in recombinant Escherichia coli which contains the cgt gene of Bacillus macerans in the pTCGT1 plasmid. Antiserum against CGTase was prepared in rabbits. Anti‐CGTase IgG in Serum was purified using an immobilized Protein A affinity column and an immobilized E. coli cell lysate affinity column. The purified anti‐CGTase IgG was coupled to Sepharose 4B (5.3mg IgG/mL gel), which was activated with cyanogen bromide. Crude CGTase was purified with this immunoadsorbent with CGTase binding capacity of 0.27 mg/mL and eluted with 0.1M glycine buffer, pH 10.6. Approximately 65% of enzyme activity was recovered from the crude CGTase preparation by this method. The purified CGTase was electrophoretically and antigenically homogeneous, appearing as a single band. After 10 cycles, the recovery yield was not reduced significantly. There was no cross reaction between anti‐CGTase IgG and other tested amylolytic enzymes. This immunoaffinity column is, therefore, effective, efficient, and convenient to use, and can be used widely to purify CGTase from various sources.