Effect of organic solvents on enzymatic production of cyclodextrins from unliquefied corn starch in an attrition bioreactor

Cyclodextrins were produced from unliquefied corn starch in the presence of water-miscible organic solvents using cyclodextrin glycosyltransferase (CGTase) in an attrition bioreactor. The production yield was singnificatly increased by isopropanol and tertiary butanol, and maximum enhancement was observed to be about 40% by 5% tertiary butnol. Increase in the production of cyclodextrins by organic solvents seems to be due to the fact that organic solvents decreased the product inhibition of CGTase by forming an inclusion complex with cyclodextrins.     

Enzymatic production of cyclodextrins from milled corn starch in an ultrafiltration membrane bioreactor

Cyclodextrin glycosyltransferase (CGTase) was found to be severely inhibited by cyclodextrins. In order to increase the conversion yield by reducing product inhibition and reuse the CGTase in the production of cyclodextrins from milled corn starch, an ultrafiltration membrane bioreactor system was employed. In a batch operation with ultrafiltration, the conversion yield was increased 57% compared with that without ultrafiltration. Operating conditions for the continuous production of cyclodextrins in the membrane bioreactor were optimized by taking into consideration the filtration rate and the conversion yield as follows: initial starch concentration, 7% (w/v); starch feeding rate, 240 mg/h; CGTase loading, 350 units/initial gram starch. When cyclodextrins were continuously produced in the membrane bioreactor under optimized conditions, 340 units of CGTase was require to produce 1 g of cyclodextrins for 48 h, while in the case of conventional batch operation, 1 g of cyclodextrins was produced for 24 h by 1410 units of CGTase. (c) 1993 John Wiley & Sons, Inc.     

Characterisation of cyclodextrin glucanotransferase fromBacillus circulans ATCC 21783 in terms of cyclodextrin production

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) fromBacillus circulans ATCC 21783 was purified by ultrafiltration and a consecutive starch adsorption. Total enzyme yield of 75.5% and purification factor of 13.7 were achieved. CGTase was most active at 65°C, possessed two clearly revealed pH-optima at 6.0 and 8.6 and retained from 75 to 100% of its initial activity in a wide range of pH, between 5.0 and 11.0. The cyclising activity was enhanced by 1 mM CaCl₂ or 4 mM CoCl₂. The enzyme was thermostable up to 70°C, and 64% of the original activity remained at 70°C after 30 min heat treatment. Up to 41% conversion into cyclodextrins was obtained from 40 g l^?1 starch without using any additives. This CGTase produced two types of cyclodextrins, beta and gamma, in a ratio 73:27 after 4 h reaction time at 65°C. This feature of the enzyme could be of interest for industrial cyclodextrin production.     

一株产环糊精葡萄糖基转移酶的地衣芽孢杆菌的选育、产酶条件及酶学特性

从土壤分离物中筛选到一株环糊精葡萄糖基转移酶 (CGTase)产生菌 4 0 3,96h发酵酶活为 0 95U mL。经紫外辐射和硫酸二乙酯复合诱变而获得突变株CLS4 0 3,96h发酵酶活达 1 36U mL ,提高 4 3%。该突变菌株被鉴定为地衣芽孢杆菌 (Bacilluslicheniformis) ,产CGTase的最佳碳源为可溶性淀粉 ,最佳氮源为硝酸铵 ,最适初始pH为 6 5 ,最适培养温度为 35℃ ,发酵期间CGTase的产生高峰 (第 96h)滞后于菌体生物量高峰 (第 4 8h) 2d。菌株所产CGTase的最适反应pH为 6 0 ,最适温度为 5 5℃ ,在pH 6 0～ 7 5间和 5 0℃下保持 1h后的剩余酶活均达 90 %以上 ;酶液中适量添加Ca2 能大幅提高CGTase在 5 5℃下的稳定性。经高效液相色谱分析 ,CGTase作用于淀粉后的产物以α 环糊精为主 ,β 环糊精为次 ,二者比例为 2 4 7∶1,环糊精总产率达 2 9 8% ,但产物中不含γ 环糊精     

Studies on enzymatic continuous production of cyclodextrins in an ultrafiltration membrane bioreactor

Studies on simultaneous hydrolysis of starch and synthesis of cyclodextrins by Thermo-aerobacter cyclodextrin glucosyltransferase were conducted in an ultrafiltration membrane bioreactor, allowing enzyme recovery and reduction of product inhibition. The influence of various reaction parameters like starch concentration, enzyme dosage and residence time on cyclodextrin composition was tested. A comparison of batch and continuous cyclodextrin production indicates that employing an ultrafiltration membrane bioreactor increases process efficiency.     

Secretion of cyclodextrin glucanotransferase in E. coli using Bacillus subtilis lipase signal peptide and optimization of culture medium

The cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132 was fused to the secretive lipase signal peptide of B. subtilis. This leads to an efficient secretion of the recombinant enzyme into the culture medium of E. coli as an active and soluble form contrasting with the native construction leading to a periplasmic production. In order to enhance the yield of CGTase production, an experimental design methodology was applied for the optimization of the culture composition. Hence, the media components were submitted to preliminary screening using a Plakett-Burman design. The concentrations of the major operating ones were then optimized to enhance the secretion of CGTase using response surface methodology. The findings revealed that concentrations of 0.5% potato starch, 3% yeast extract, 3% tryptone, 1.5% casein hydrolysate, 0.5% NaCl, 0.2% KH2PO4, and 0.02% MgSO4 were the optimal conditions for CGTase production. The experimental value (9.43 U/mL) obtained for CGTase activity was very close to the predicted value (9.27 U/mL).     

Engineering of Cyclodextrin Glucanotransferase on the Cell Surface of Saccharomyces cerevisiae for Improved Cyclodextrin Production

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.     

A single step purification process for cyclodextrin glucanotransferase from a Bacillus sp. isolated from soil

Cyclodextrin glucanotransferase (CGTase) is a commercially important enzyme which catalyses the formation of cyclodextrins (CDs) from starch. A CGTase producing bacterium was isolated from soil which gave a fairly high enzyme activity of 7.5 U mL(-1) after 24 h of growth which was further increased to 22 U mL(-1) by proper media design. The enzyme was purified to homogeneity by a novel single affinity precipitation step which resulted in a very high recovery of more than 90%. The molecular weight, as determined by SDS-PAGE, was found to be 68 kDa. The pH optimum was found to be 6.6 while a temperature optimum of 65 degrees C was observed. The enzyme exhibited a fairly high degree of functional stability with little loss of activity, even after 8 h of incubation at 65 degrees C. Ca++ had little effect on the activity of the enzyme while urea at 10 mM concentration increased the activity of the enzyme by more than 200%, suggesting that it is a unique enzyme.     

Bioconversion of waste cellulose by using an attrition bioreactor

A new type of reactor, an attrition bioreactor, was tested to achieve a higher rate and extent of enzymatic saccharification of cellulose than is possible with conventional methods. The reactor consisted of a jacketted stainless-steel vessel with shaft, stirrer, and milling media, which combined the effect of the mechanical action of wet milling with cellulose hydrolysis. The substrates tested were newsprint and white-pine heartwood. The performance of the reactor was excellent. The extent and rate of enzymatic hydrolysis could be markedly improved over other methods. The power consumption of the attrition bioreactor was also measured. The cellulase enzyme deactivation during attrition milling was not significant.     

亚位点?7处突变对碱性芽胞杆菌CGT酶产物特异性的影响

为了研究来源于碱性芽胞杆菌的γ-环糊精葡萄糖基转移酶(CGT酶)具有较高产物特异性的作用机理,对其氨基酸序列和模拟结构进行了分析,确定其亚位点7处氨基酸的缺失可能影响其产物特异性。运用重叠PCR的方法,在其亚位点7处添加缺失的6个氨基酸,造成插入突变。将突变基因与pET-20b(+)连接并在大肠杆菌BL21(DE3)中表达。以可溶性淀粉为底物进行酶转化,HPLC分析转化产物中的环糊精含量。结果表明,相对于野生型γ-CGT酶,突变酶转化生成的3种环糊精中,γ-环糊精所占的比例从76.0%降至12.5%,α-、β-环糊精分别从8.7%和15.2%提高至37.5%和50%。分析其可能机理为:与α-、β-CGT酶相比,野生型γ-CGT酶的亚位点7处缺失6个氨基酸,该构象为葡萄糖的结合提供了更大的空间,从而更适合γ-环糊精的生成;而在其亚位点7处插入6个氨基酸,造成插入突变后,葡萄糖链结合的空间变小,这种构象不利于γ-环糊精的生成。     

Optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae AS-22 in batch, fed-batch, and continuous cultures

Production of a novel cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS-22 strain, which converts starch predominantly to alpha-CD at high conversion yields, in batch, fed-batch, and continuous cultures, is presented. In batch fermentations, optimization of different operating parameters such as temperature, pH, agitation speed, and carbon-source concentration resulted in more than 6-fold increase in CGTase activity. The enzyme production was further improved by two fed-batch approaches. First, using glucose-based feed to increase cell density, followed by starch-based feed to induce enzyme production, resulted in high cell density of 76 g dry cell weight/L, although the CGTase production was low. Using the second approach of a single dextrin-based feed, 20-fold higher CGTase was produced compared to that in batch fermentations with media containing tapioca starch. In continuous operation, more than 8-fold increase in volumetric CGTase productivity was obtained using dextrin-based media compared to that in batch culture using starch-based media.     

Cyclodextrin glucanotransferase: from gene to applications

Cyclodextrin glucanotransferase (CGTase) is an important industrial enzyme which is used to produce cyclodextrins. CGTase genes from more than 30 bacteria have been isolated and several of the enzymes have been identified and biochemically characterized. For a better understanding of the reaction mechanism and function of CGTase, the enzyme has been analyzed at gene level and protein level with regard to its structure and the similarity of different CGTase subgroups. The biological role of the enzyme is proposed based on the genetic and enzymatic analyses. Methods to enhance the production of active CGTase by bacteria are compared. The enzyme can be applied in biotechnology for the production of cyclodextrins and oligosaccharides with novel properties.     

Addition of polar organic solvents can improve the product selectivity of cyclodextrin glycosyltransferase. Solvent effects on cgtase

Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) is an enzyme that produces cyclodextrins from starch via an intramolecular transglycosylation reaction. Addition of small amounts (10% v/v) of polar organic solvents can affect both the overall production yield and the type of cyclodextrin produced from a maltodextrin substrate under simulated industrial process conditions. Using CGTase from Thermoanaerobacter sp. all solvents produced an increase in cyclodextrin yield when compared with a control, the greatest increase being obtained with addition of ethanol (26%). In addition product selectivity was affected by the nature of the organic solvent used: beta-cyclodextrin was favoured in the absence of any solvent and on the addition of dimethylsulphoxide, t-butanol and dimethylformanide while alpha-cyclodextrin was favoured by addition of acetonitrile, ethanol and tetrahydrofuran. With CGTase from Bacillus circulans strain 251 relatively smaller increases in overall cyclodextrin production were achieved (between 5-10%). Addition of t-butanol to a B. circulans catalysed reaction however did produce the largest selectivity for beta-cyclodextrin of any solvent-enzyme combination (82%). The effect of solvent addition was shown not to be related to the product inhibition of CGTase, but may be related to reduced competition from the intermolecular transglycosylation reaction that causes degradation of cyclodextrin products. This rate of this reaction was shown to be dependent on the nature of the organic solvent used.     

Production of cyclodextrin glycosyltransferase by alkaliphilic <Emphasis Type="Italic">Bacillus circulans</Emphasis> in submerged and solid-state cultivation

Cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19) is an industrially important enzyme, which is used to produce cyclodextrins (CDs). In this research, we report the use of experimental factorial design to find the best conditions of pH and temperature for CGTase production by Bacillus circulans var. alkalophilus. The optimized calculated values for the tested variables were, respectively, pH 9.7 and temperature 36^oC, with a CGTase activity of 615 U mL⁻¹. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC) and solid-state cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on SC was 1,155 U mL⁻¹ under aerobic conditions. Cell growth and CGTase synthesis in SSC using SIFR as substrate was excellent, with CGTase activity of 32,776 U g_(SIFR) ⁻¹. These results strongly support the use of SIFR for CGTase production since it is a non-expensive residue.     

Ultrafiltration system for cyclodextrin production in repetitive batches by CGTase from <Emphasis Type="Italic">Bacillus firmus</Emphasis> strain 37

This study aimed to improve the yield of cyclodextrins (CDs) production in repetitive batches. An innovative ultrafiltration system was used to remove the inhibitory products that accumulated in the medium and to recover the enzyme. The assays were performed with the CGTase from Bacillus firmus strain 37 in purified, semi-purified, and crude extract forms. Maltodextrin (10 % w/v) and corn starch (5 % w/v) were used as substrates. After eight repetitive 24-h batches, the yield of β-CD obtained with the purified enzyme and the corn starch substrate was 0.54 mmol/L/h, which was 36 % greater than that observed with the 10 % maltodextrin substrate. The crude CGTase extract with the corn starch substrate showed a productivity of 0.38 mmol/L/h, which was 29 % lower than using the purified enzyme and the corn starch substrate but 7 % higher than using the purified enzyme and the maltodextrin substrate. The crude extract, assayed with the corn starch substrate in the presence of 10 % ethanol reached 0.43 mmol/L/h productivity, which was 12 % higher compared to the assay without ethanol. The semi-purified enzyme was assayed with the corn starch substrate in the presence of 10 % ethanol for eight batches lasting 12 h and an excellent selectivity for the β-CD was obtained, reaching a mean percentage of 96.0 %. Therefore, this ultrafiltration system enabled several batches of CD production, with efficient removal of products inhibitory to the CGTase and recovery of the enzyme. The possibility of industrial application of this system is promising.     

Engineering of cyclodextrin glucanotransferase on the cell surface of Saccharomyces cerevisiae for improved cyclodextrin production

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.     

Bacterial cyclodextrin glucanotransferase

Cyclodextrin glucanotransferase (CGTase, Ec 2.4.1.19) is an enzyme which catalyze intramolecular (cyclizing) and intermolecular (coupling, disproportionation) transglycosylation as well as having a hydrolytic action on starch and cyclodextrins. By a cyclizing reaction, the enzyme converts starch and related -1, 4-glucans to cyclodextrins which are widely utilized in food, pharmaceutical, and chemical industries. The present review attempts to summarize the reported data concerning the bacterial producers of CGTase, growth cultural conditions providing optimal enzyme biosynthesis in batches, repeated batch and continuous cultivation of free and immobilized cells, as well as some physicochemical and biochemical characteristics of the enzyme, CGTase immobilization, and enzyme structure.     

Optimization of cyclodextrin production from sago starch

Cyclodextrin (CD) is synthesized by bacterial cyclodextrin glycosyltransferase (CGTase) and is widely used in food, pharmaceutical, cosmetic, and agricultural industries. In this study, Bacillus circulans CGTase was partially purified by ammonium sulfate precipitation at 50-70% saturation. The optimum pH and temperature for CD production from sago starch were found to be in the ranges of 4.5-5.0 and 55-60 degrees C, respectively. beta-CD was the predominant product, constituting 65% of all CD products. The beta-CD produced using partially purified and crude CGTase were compared and found to have no significant difference in yield and productivity. The appropriate proportion of CGTase to sago starch for beta-CD production was determined by response surface methodology. The most appropriate enzyme:substrate ratio was 50 U g sago starch(-1) CGTase and 60 g l(-1) sago starch.     

Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic bacilli

Cells of obligated alkaliphiles Bacillus pseudalcaliphilus 20RF and Bacillus pseudalcaliphilus 8SB isolated from Bulgarian habitats, producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), were immobilized by three different techniques: on two types of polysulphone membranes; entrapped in agar-gel beads containing magnetite and by nano-particles of silanized magnetite covalently bound on the cell surface. The biocatalysts obtained demonstrated the opportunity for a significantly enhanced CGTase production compared to free cells for a long period of time (10 days semicontinuous cultivation) without impact on their mechanical stability. The cell membrane-biocatalysts exhibited the highest enzyme activity after 240 h repeated batch cultivation and retained 1.3–2.3-fold increase of the CGTase yield compared to free cells at the end of the process. Membrane biocatalysts were applied for a direct cyclodextrin (CD) production. The results obtained demonstrated the possibility of starch conversion into cyclodextrins by immobilized cells without using of crude or purified enzyme. The membrane biocatalysts of both obligated alkaliphiles formed mainly β- and γ-CDs after 6 h enzyme reaction at pH 9.0 of the reaction mixture. Under these conditions, the quantity of γ-CDs was a relative high, to 35–37% of the total CD amount.     

环糊精葡萄糖基转移酶的结构特征与催化机理

随着环糊精在食品、医药等领域的应用越来越广,生产环糊精所必需的环糊精葡萄糖基转移酶(CGT酶)已经成为当今研究的热点。特别是近二十年来,国外对该酶进行了比较深入的研究。首先介绍了CGT酶的功能特性与结构特征。CGT酶是一种多功能型酶,能催化三种转糖基反应(歧化、环化和耦合反应)和水解反应,其中,能将淀粉转化为环糊精的环化反应是特征反应;作为α-淀粉酶家族的成员,CGT酶除了具有与α-淀粉酶相同的A、B、C结构域外,还存在D和E结构域。另外,对CGT酶的催化机理包括底物结合方式、转糖苷反应机理以及环化机理等进行了详细的讨论。