Expression of Bacillus macerans cyclodextrin glucanotransferase gene in Saccharomyces cerevisiae

Cyclodextrin glucanotransferase (CGTase) gene of Bacillus macerans was subcloned down-stream of yeast ADH1 promoter and expressed in Saccharomyces cerevisiae. Most of the CGTase expressed was in the extracellular medium with a maximum activity of about 0.28 unit ml^–1 after 48 h cultivation. The recombinant CGTase was secreted as an N-linked-glycosylated form and predominantly produced -cyclodextrin from starch.     

来源于软化芽孢杆菌的环糊精葡萄糖基转移酶在毕赤酵母和枯草杆菌中的表达

通过PCR扩增软化芽孢杆菌α-CGT酶基因,将基因片段分别克隆到毕赤酵母表达载体pPIC9K和大肠杆菌-枯草杆菌穿梭载体pMA5中,分别转化毕赤酵母KM71和枯草杆菌WB600。结果表明,重组毕赤酵母发酵上清液中α-CGT酶活性仅0.2U/mL,重组枯草杆菌产酶达到1.9U/mL。对重组枯草杆菌发酵条件进行了优化,当以TB为出发培养基,初始pH6.5,温度为37oC时,摇瓶培养24h后α-CGT酶环化活性达到4.5U/mL(水解活性为3200IU/mL),是野生菌株软化芽孢杆菌表达量的9.8倍。     

Cyclodextrins are likely to induce cyclodextrin glycosyltransferase production inBacillus macerans

Cyclodextrin glycosyltransferase (CGTase) activity was monitored inBacillus macerans culture fluids up to 56 h incubation time using glucose (G₁), maltose (G₂), maltotriose (G₃), maltoheptaose (G₇), α-,β-,γ-cyclodextrins (CDs) and soluble starch as carbon sources. Highest maximum specific growth rates (μ_max) were observed with glucose, γ-CD and soluble starch (μ_max values were 0.86, 0.74 and 0.69/h, respectively) while the maximum viable cell numbers were always within the range of 2.3–7.1×10¹¹ CFU/mL independently of the carbon source used. Highest CGTase production was found in the presence of soluble starch and G₇ (55.0 and 35.4 nkat/mL, respectively), these saccharides being easily transformed to CDs by CGTase. Moreover, when culture media were supplemented with cyclic malto-oligosaccharides the CGTase activities were about twice higher (19.6–20.6 nkat/mL) than those obtained with the linear G₂ and G₃ saccharides (8.9 and 11.3 nkat/mL, respectively) which give rise only to negligible quantities of CDs. CDs, which are the major end products of the action of CGTase, are regarded thus as the likely physiological inducers of the enzyme.     

Domain E of Bacillus macerans cyclodextrin glucanotransferase: An independent starch-binding domain

The starch-binding domains of glucoamylase I (SBD of GA-I) from Aspergillus awamori and of cyclodextrin glucanotransferase (domain E of CGTase) from Bacillus macerans were fused to the C-terminus of beta-galactosidase (beta-gal) The majority of the fusion proteins produced in Escherichia coli were found as inclusion bodies. Active fusion proteins were purified by partial solubilization of the inclusion bodies with 2 M urea followed by affinity chromatography. Adsorption isotherms of purified fusion proteins on corn starch and cross-linked amylose were generated. The beta-gal fusion proteins had similar affinities for cross-linked amylose and corn starch but significantly different saturation capacities on corn starch. The adsorption and elution data from the potato starch column as well as the adsorption isotherms of p-gal-domain E fusion protein (BDE109) on corn starch and cross-linked amylose demonstrated that domain E of CGTase is an independent domain, which retained its starch-binding activity when separated from the other four (A-D) domains in CGTase. (c) 1995 John Wiley & Sons Inc.     

Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli

Coexpression of folding accessory proteins, molecular chaperones, and human peptidyl-prolyl cis-trans isomerase (PPIase) increased production of active cyclodextrin glycosyltransferase (CGTase) of Bacillus macerans, which is otherwise mainly expressed as inclusion body in recombinant Escherichia coli. The best partner for soluble expression of CGTase was found to be human PPIase followed by coexpression of DnaK-DnaJ-GrpE together with GroEL-GroES. Such a significant enhancement by human PPIase coexpression seemed to be due to dual functions of chaperone and peptidyl-prolyl cis-trans isomerization. Coexpression of GroEL-GroES or minichaperone alone did not influence the specific CGTase activity. For production of active CGTase in large amounts, a high cell density culture was achieved using a pH-stat fed-batch strategy. The optimized fed-batch fermentation resulted in dry cell weight of 103.4 g/L and CGTase activity of 1200 U/mL. Combination of human PPIase expression at a gene level and cell culture optimization at a process scale exerted a synergistic effect on the product yield of soluble CGTase expression in recombinant E. coli.     

Investigation of the active site of Bacillus macerans cyclodextrin glucanotransferase by use of modified maltooligosaccharides.

The active site of Bacillus macerans cyclodextrin glucanotransferase (CGTase) was examined by use of derivatives of phenyl alpha-maltopentaoside and phenyl alpha-glucoside as the substrates and acceptors, respectively. The active site of this enzyme is considered to be composed of tandem subsites (S4, S3, S2, S1, S1', S2', etc.) geometrically complementary to several glucose residues, and the alpha-1,4-glycosidic linkage of a substrate is split between S1 and S1'. The features of subsites S3 and S4 of the glycon binding site were estimated from the modes of the enzymatic action on phenyl alpha-maltopentaoside (G-G-G-G-G-phi; G, glucose residue; phi, phenyl residue; -, alpha-1,4-glycosidic bond) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2I (IG-G-G-G-G-phi; IG, 6-deoxy-6-iodo-D-glucose residue), CH2NH2 (AG-G-G-G-G-phi; AG, 6-amino-6-deoxy-D-glucose residue), or COOH (CG-G-G-G-G-phi; CG, glucuronic acid residue). p-Nitrophenyl alpha-glucopyranoside (G-P; P, p-nitrophenyl residue) was used as an acceptor. HPLC analysis of the digests revealed that the CG residue of CG-G-G-G-G-phi was excluded from subsite S3, while it was accommodated in subsite S4. The Km and Vmax values for CG-G-G-G-G-phi were remarkably larger and smaller, respectively, than those for any other substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of cyclodextrins in ultrafiltration membrane reactor containing cyclodextrin glycosyltransferase from Bacillus macerans

An enzyme reactor installed with ultrafiltration membrane was developed to produce alpha-, beta-, and gamma-cyclodextrins (CDs) from soluble starch by Bacillus macerans cyclodextrin glycosyltransferase (CGTase) tagged with 10 lysines at its C-terminus (CGTK10ase). Ultrafiltration membrane YM10 with 10,000 of molecular cutoff was chosen for membrane modification and CD production. A repeated-batch type of the enzyme reaction with free CGTK10ase resulted in a alpha-CD yield of 24.0 (+/-1.5)% and a productivity of 4.68 (+/-0.88) g/l-h, which were 7 times higher that those for CGTK10ase immobilized on modified YM10 membrane. Addition of 1- nonanol increased CD yields by 30% relative to the control, which might be due to prevention of the reversible hydrolysis of CDs.     

Reaction conditions for maximal cyclodextrin production by cyclodextrin glucanotransferase from Bacillus megaterium

The effect of the reaction conditions (substrate concentration, enzyme dosage, and pH) on cyclodextrin production by cyclodextrin glucanotransferase from Bacillus megaterium was investigated by applying mathematical modeling methods. Adequate models were developed and they were used for determination of the optimal conditions for maximal formation of beta-cyclodextrins at minimal concentrations of a- and gamma-cydclodextrins. The main factor affecting the ratio of the products was pH of the reaction mixture. At pH 9 the enzyme formed mainly beta- and y-cyclodextrins and the ratio a:beta:gamma was 2.6:83.5:13.9; at pH 5 the ratio changed to 8.6:84.6:6.8. Mathematical models were used for determination of the conditions for maximal conversion of the substrate into cyclodextrins. 45.88% conversion of starch was achieved at 5% substrate concentration, 3.5 U/g enzyme dosage, and pH 7.4.     

Fermentation of 6-Deoxyhexoses by Bacillus macerans

Under anaerobic conditions Bacillus macerans ATCC 7068 fermented 6-deoxyhexoses (l-rhamnose, l-fucose, and d-fucose) to a mixture of 1,2-propanediol (PD), acetone, H(2), CO(2), and ethanol. The final PD concentration was proportional to the amount of l-rhamnose fermented ( approximately 0.9 mol of PD per mol of rhamnose). PD was not produced from hexoses (e.g., d-glucose or l-mannose), despite active fermentation of these substrates. Relative to the fermentation of d-glucose, the fermentation of l-rhamnose was accompanied by a twofold reduction in yield of H(2), CO(2), and cell mass. Exposure of cell extracts to l-rhamnose resulted in the transient appearance of an aldehyde intermediate. Cell extracts contained a pyridine nucleotide-linked lactaldehyde reductase activity which converted synthetic d- or l-lactaldehyde to PD. The data suggest an Embden-Meyerhof pathway for 6-deoxyhexose catabolism, with the formation of lactaldehyde by a conventional aldolase cleavage reaction and subsequent reduction to PD.     

Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with gamma-cyclodextrin

Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of cyclodextrins from starch by an intramolecular transglycosylation reaction. To manipulate the product specificity of the Paenibacillus sp. A11 and Bacillus macerans cyclodextrin glycosyltransferases towards the preferential formation of gamma-cyclodextrin (CD(8)), crosslinked imprinted proteins of both cyclodextrin glycosyltransferases were prepared by applying enzyme imprinting and immobilization methodologies. The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD(8) showed pH and temperature optima similar to those of the native and immobilized cyclodextrin glycosyltransferases. However, the pH and temperature stability of the immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were higher than those of the native cyclodextrin glycosyltransferases. When the catalytic activities of the native, immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were compared, the efficiency of the crosslinked imprinted enzymes for CD(8) synthesis was increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased. Comparison of the product ratios by high-performance anion exchange chromatography showed that the native cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced CD(6) : CD(7) : CD(8) : > or = CD(9) ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of reaction at 40 degrees C with starch substrates. In contrast, the crosslinked imprinted cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and 17 : 14 : 49 : 20, respectively. The size of the synthesis products formed by the crosslinked imprinted cyclodextrin glycosyltransferases was shifted towards CD(8) and > or = CD(9), and the overall cyclodextrin yield was increased by 12% compared to the native enzymes. The crosslinked imprinted cyclodextrin glycosyltransferases also showed higher stability in organic solvents, retaining 85% of their initial activity after five cycles of synthesis reactions.     

Catabolism of protocatechuate by Bacillus macerans.

An aerobic endospore-forming bacterium, tentatively identified as a strain (JJ-lb) of Bacillus macerans, was isolated by enrichment on 4-hydroxybenzoate (4HBA), using as an inoculum soil taken from a 50 degrees C Iadho hot spring. Enzymatic analyses of cells grown on succinate and 4HBA indicated that strain JJ-1b degrades 4HBA by way of the novel protocatechuate (PCA) 2,3-dioxygenase pathway. Purification of the PCA 2,3-dioxygenase by affinity chromatography allowed the first observation of the immediate ring fission product of PCA, namely, 5-carboxy-2-hydroxymuconic semialdehyde (CHMS; labmda max at pH 7.0 = 348 nm). An affinity column fraction was obtained that decarboxylated CHMS to 2-hydroxymuconic semialdehyde (HMS; lambdamax at pH 7.0 = 375 nm). Thus, conversion of PCA to HMS is accomplished in two steps, 2,3-fission of the PCA ring followed by enzymatic decarboxylation of the ring fission product, forming HMS.     

Catabolism of protocatechuate by Bacillus macerans.

An aerobic endospore-forming bacterium, tentatively identified as a strain (JJ-lb) of Bacillus macerans, was isolated by enrichment on 4-hydroxybenzoate (4HBA), using as an inoculum soil taken from a 50 degrees C Iadho hot spring. Enzymatic analyses of cells grown on succinate and 4HBA indicated that strain JJ-1b degrades 4HBA by way of the novel protocatechuate (PCA) 2,3-dioxygenase pathway. Purification of the PCA 2,3-dioxygenase by affinity chromatography allowed the first observation of the immediate ring fission product of PCA, namely, 5-carboxy-2-hydroxymuconic semialdehyde (CHMS; labmda max at pH 7.0 = 348 nm). An affinity column fraction was obtained that decarboxylated CHMS to 2-hydroxymuconic semialdehyde (HMS; lambdamax at pH 7.0 = 375 nm). Thus, conversion of PCA to HMS is accomplished in two steps, 2,3-fission of the PCA ring followed by enzymatic decarboxylation of the ring fission product, forming HMS.     

New cyclomaltodextrin-glucanotransferases, produced by Bacillus macerans

For the first time, microorganisms producing cyclomaltodextrin glucan transferases (CGT, EC 2.4.1.19) were isolated from soil samples of various ecogeographical regions. These microorganisms were identified as Bacillus macerans. The enzymes were purified by affinity chromatography on a alpha-cyclodextrin polymer and gel filtration on Biogel P-450 and proved to be electrophoretically homogeneous. Some of their physicochemical and biochemical properties are reported.     

Molecular cloning, DNA nucleotide sequencing, and expression in Bacillus subtilis cells of the Bacillus macerans cyclodextrin glucanotransferase gene.

The gene for cyclodextrin glucanotransferase from Bacillus macerans was cloned in an Escherichia coli bacteriophage, lambda D69, and was recloned in a Bacillus subtilis plasmid, pUB110. Starting from an ATG initiation codon, a unique reading frame was shown to extend for 2,142 base pairs (714 amino acids). The nucleotide sequence revealed that the enzyme is composed of two identical subunits.     

Cyclodextrin production by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrins (CD) are cyclic oligosaccharides with multiple applications in the food, pharmaceutical, cosmetic, agricultural and chemical industries. In this work, the conditions used to produce CD with cyclodextrin glycosyltransferase from Bacillus circulans DF 9R were optimized using experimental designs. The developed method allowed the partial purification and concentration of the enzyme from the cultural broth and, subsequently, the CD production, using the same cassava starch as enzyme adsorbent and as substrate. Heat-treatment of raw starch at 70 degrees C for 15 min in the presence of adsorbed cyclodextrin glycosyltransferase allowed the starch liquefaction without enzyme inactivation. The optimum conditions for CD production were: 5% (w/v) cassava starch, 15 U of enzyme per gram of substrate, reaction temperature of 56 degrees C and pH 6.4. After 4h, the proportion of starch converted to CD reached 66% (w/w) and the weight ratio of alpha-CD:beta-CD:gamma-CD was 1.00:0.70:0.16.     

Germination requirements of Bacillus macerans spores

2-Phenylacetamide is an effective germinant for spores of five strains of Bacillus macerans, particularly in the presence of fructose. Benzyl penicillin, the phenyl acetamide derivative of penicillin, and phenylacetic acid are also good germinants. l-Asparagine is an excellent germinant for four strains. alpha-Amino-butyric acid is moderately effective. Pyridoxine, pyridoxal, adenine, and 2,6-diaminopurine are potent germinants for NCA strain 7X1 only. d-Glucose is a powerful germinant for strain B-70 only. d-Fructose and d-ribose strongly potentiate germination induced by other germinants (except l-asparagine) but have only weak activity by themselves. Niacinamide and nicotinamide-adenine dinucleotide, inactive by themselves, are active in the presence of fructose or ribose. Effects of pH, ion concentration, and temperature are described.     

Construction of chimeric cyclodextrin glucanotransferases from Bacillus circulans A11 and Paenibacillus macerans IAM1243 and analysis of their product specificity

Three DNA fragments of 7919 base pairs containing genes for beta-cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), an iron III dicitrate transport protein-like protein and a partial coding sequence for putative ferrichrome ABC transporter from Bacillus circulans A11 were cloned and sequenced (GenBank Accession AF302787). The DNA sequence contained a CGTase open reading frame of 2139 base pairs, which encoded a polypeptide of 713 amino acid residues. The signal peptide constituted the N-terminal 27 amino acid residues. The amino acid sequence was highly homologous to that of Bacillus sp. 1011 with 98.7% identity. The cloned CGTase gene contained its own promoter that directed the expression of the gene in Escherichia coli host cells. Chimeric construction against the alpha-CGTase from B. macerans IAM1243 was carried out by means of three created restriction sites, XhoI, SpeI, and MfeI, introduced by mutagenesis in between domains A/B and C, C and D, and D and E, respectively, and the NdeI site within the domains A/B. The various chimeras with different combinations of domains and part of domains A/B were analyzed for their dextrinizing and CD-forming activities. Their activities were of three groups: chimeras with no dextrinizing and cyclization activities, chimeras with only dextrinizing activity, and chimeras with both dextrinizing and cyclization activities. Two chimeras in the latter group showed altered product specificity. The results located the amino acid segment essential for the product specificity at the C-terminal half of domains A/B. Further, the function of domains C and D in positioning domain E in the correct orientation and proximity to domains A/B is implicated.