Optimisation of growth medium for the production of cyclodextrin glucanotransferase from Bacillus stearothermophilus HR1 using response surface methodology

Cyclodextrin glucanotransferase (CGTase) activity was observed when the bacterium was grown in the medium at various initial pH values, containing carbon, nitrogen, phosphorus and mineral salt sources at 50 °C for 24 h in the shake flasks. The optimisation of this growth medium was carried out using response surface methodology. The design contains a total of 32 experimental trials involving 10 star points and 6 replicates at the centre points. The design was employed by selecting sago starch, peptone from casein, K₂HPO₄, CaCl₂ and initial pH as five independent variables in this study. The optimal calculated values of tested variables for maximal production of CGTase were found to be comprised of: sago starch, 16.02 g/l; peptone from casein, 20 g/l; K₂HPO₄, 1.4 g/l; CaCl₂, 0.2 g/l and initial pH, 7.54 with a predicted CGTase activity of 14.20 U/ml. These predicted optimal parameters were tested in the laboratory and the final CGTase activity obtained was very close to the predicted value at 14.80 U/ml.     

Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001

A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) alpha-cyclomaltodextrin (alpha-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991--1997, 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (cgtB), the gene encoding CGTase (cgtA), a gene homolog for a CD-binding protein (CBP) (cgtC), and a putative CBP-dependent ABC transporter involved in uptake of CDs (cgtDE). The CDase was expressed in Escherichia coli and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95 degrees C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed beta-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to alpha-CD. However, the CBP exhibited higher thermostability in the presence of alpha-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.     

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.     

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     

Heterologous expression of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> thermostable alpha-amylase in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and its application in boiling stable resistant sago starch preparation

A gene encoding the thermostable α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP _w of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch increased from 8.3 to 18.1%. The starch recovery rate was 71%.     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.     

Itaconic acid production using sago starch hydrolysate by Aspergillus terreus TN484-M1

Sago starch was hydrolyzed using either chemical agents, or enzymes at various pH and concentrations. Hydrolysis using 5000 AUN/ml (0.5%, w/v) glucoamylase exhibited the highest itaconic acid yield up to 0.36 g/g sago starch, whereas hydrolysis using nitric acid at pH 2.0 yielded 0.35 g/g sago starch. The medium was optimized and the composition was (g/l) 140 sago starch, 1.8 corn steep liquor, 1.2 MgSO(4).7H(2)O and 2.9 NH(4)NO(3). When the optimal conditions of hydrolysis and medium composition were applied to itaconic acid production in a 3-l jar fermentor, the itaconic acid production was 48.2 g/l with a yield of 0.34 g/g sago starch. This was filtered from the cultured broth and 37.1g of itaconic acid was recovered with a purity of 97.2%. This result showed that sago starch could be converted to a value-added product with only a simple pretreatment.     

Effect of C/N ratio and starch concentration on ethanol production from sago starch using recombinant yeast

Recombinant Saccharomyces cerevisiae YKU 131 (capable of expressing glucoamylase) was used to produce ethanol from sago starch. The optimum C/N ratio for ethanol production by the recombinant yeast was 7.9, where 4.7 and 10.1 g/l ethanol was produced from 20 and 40 g/l sago starch, respectively. At sago starch concentration higher than 40 g/l and C/N ratio higher than 10.4, glucoamylase production and rate of starch hydrolysis were reduced, which in turn, reduced ethanol production significantly. The theoretical yield of ethanol based on sago starch consumed in fermentation using 40 g/l was 72.6%. This yield was slightly lower than those obtained in fermentation using soluble starch such as potato and corn starch, which ranged from 80–90% as reported in the literature. However, S. cerevisiae YKU 131 could only utilize 62% of the total amount of starch added to a medium.     

Direct fermentation of gelatinized sago starch to acetone–butanol–ethanol by Clostridium acetobutylicum

Direct fermentation of gelatinized sago starch into solvent (acetone–butanol–ethanol) by Clostridium acetobutylicum P262 was studied using a 250 ml Schott bottle anaerobic fermentation system. Total solvent production from fermentation using 30 g sago starch/l (11.03g/l) was comparable to fermentation using corn starch and about 2-fold higher than fermentation using potato or tapioca starch. At the range of sago starch concentration investigated (10–80 g/l), the highest total solvent production (18.82 g/l) was obtained at 50 g/l. The use of a mixture of organic and inorganic nitrogen source (yeast extract + NH₄NO₃) enhanced growth of C. acetobutylicum, starch hydrolysis and solvent production (24.47 g/l) compared to the use of yeast extract alone. This gave the yield based on sugar consumed of 0.45 g/g. Result from this study also showed that the individual concentrations of nitrogen and carbon influenced solvent production to a greater extent than did carbon to nitrogen (C/N) ratio.     

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.     

Cloning,extracellular expression and characterization of a predominant β-CGTase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. G1 in <Emphasis Type="Italic">E. coli</Emphasis>

The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60°C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% β-cyclodextrin (CD) and 10% γ-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of β-CD.     

Enhancing the cyclodextrin production by synchronous utilization of isoamylase and α-CGTase

Cyclodextrins (CD) are cyclic α-1,4-glucans composed of glucose units, and they have multiple applications in food, pharmaceuticals, cosmetics, agriculture, chemicals, etc. CD are usually produced by cyclodextrin glycosyltransferase (CGTase) from starch. In the present study, a simultaneous conversion approach was developed to improve the yield of CD from starch by conjunction use of isoamylase with α-CGTase. The isoamylase of Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The biochemical characterization of the enzyme showed that the optimum temperature and pH of the recombinant enzyme was 50 °C and 5.5, respectively, and it maintained 60 %, 85 % and 78 % relative activity at 30 °C, 40 °C and 60 °C, respectively. When the recombinant isoamylase and α-CGTase were used simultaneously to convert potato starch (15 %, w/v) into CD, the optimum conditions were found to be: 10 U of α-CGTase and 48 U of isoamylase per gram of substrate, with reaction temperature of 30 °C and pH 5.6. On the optimum condition, the total yield of CD reached 84.6 % (w/w) after 24 h, which was 31.2 % higher than transformation with α-CGTase alone. This is the first report of synchronous bioconversion of CD by both α-CGTase and isoamylase, and represents the highest efficiency of CD production reported so far.     

Continuous production of cyclodextrins in an ultrafiltration membrane reactor,catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrins (CDs) are cyclic oligosaccharides of wide industrial application, whose synthesis is catalyzed by Cyclodextrin glycosyltransferase (CGTase) from starch. Here, CDs were produced using CGTase from Bacillus circulans DF 9R in continuous process and an ultrafiltration membrane reactor. The batch process was conducted as a control. This method allowed increasing the yield from 40 to 55.6% and the productivity from 26.1 to 99.5 mg of CD per unit of enzyme. The method also allowed obtaining a high‐purity product. The flow rate remained at 50% of its initial value after 24 h of process, improving the results described in the literature for starch hydrolysis processes. CGTase remained active throughout the process, which could be explained by the protective effect of the substrate and reaction products on CGTase stability. In addition, batch processes were developed using starches from different sources. We concluded that any of the starches studied could be used as substrate for CD production with similar yields and product specificity. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:695–699, 2015     

Purification and characterization of cyclodextrin β-glucanotransferase from novel alkalophilic bacilli

The discovery of novel bacterial cyclodextrin glucanotransferase (CGTase) enzyme could provide advantages in terms of its production and relative activity. In this study, eight bacterial strains isolated from soils of a biodiversity-rich vegetation in Egypt based on their hydrolyzing activity of starch, were screened for CGTase activity, where the most active strain was identified as Bacillus lehensis. Optimization process revealed that the using of rice starch (25 %) and a mixture of peptone/yeast extract (1 %) at pH 10.5 and 37 °C for 24 h improved the bacterial growth and enzyme activity. The bacterial CGTase was successively purified by acetone precipitation, gel filtration chromatography in a Sephadex G-100 column and ion exchange chromatography in a DEAE-cellulose column. The specific activity of the CGTase was increased approximately 274-fold, from 0.21 U/mg protein in crude broth to 57.7 U/mg protein after applying the DEAE-cellulose column chromatography. SDS-PAGE showed that the purified CGTase was homogeneous with a molecular weight of 74.1 kDa. Characterization of the enzyme exhibited optimum pH and temperature of 7 and 60 °C, respectively. CGTase relative activity was strongly inhibited by Mg²⁺, Zn²⁺, Al³⁺ and K⁺, while it was slightly enhanced by 5 and 9 % with Cu²⁺ and Fe²⁺ metal ions, respectively.     

Chemical modification of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.

Counting of integral numbers of cysteine residues of the reduced and denaturated form of cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans var. alkalophilus (ATCC 21783) showed two cysteine residues per enzyme molecule. Titrations of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) led to the same result. No free SH-group was detected in denatured form of CGTase, indicating that the two cysteine residues are linked by one disulfide bridge. Cyclizing activity of the GdmCl-denaturated and reduced enzyme was 13% of that of the native one. Incubation of CGTase with diethylpyrocarbonate (DEP) showed a pseudo-first-order inhibition with second-order rate constant of 3.2 M-1 s-1. Reaction with hydroxylamine and spectroscopic studies implied that inactivation of CGTase by DEP is due to modification of one histidine residue concomitantly with a 50% decrease in the cyclizing activity (t1/2 = 10.8 min). The inhibition was partially reversible. CGTase was protected against inactivation by alpha- and beta-cyclodextrins suggesting that the modified histidine residue is at or near the active site. Conversion of starch with DEP-modified enzyme resulted in a decreased formation of cyclodextrins while the relative amount of reducing sugars increased. Preliminary results on modification of CGTase with other reagents, e.g., Woodward's reagent K, 2,3-butanedione and carbodiimide are included.     

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.     

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.     

Isolation of a kojic acid-producing fungus capable of using starch as a carbon source

A fungal strain (S33-2), able to grow on cooked starch and produce a substantially high level of kojic acid, was isolated from morning glory flower ( Bixa orellana ). The fungus was characterized and identified as Aspergillus flavus. The effect of different types of starch (sago, potato and corn starch) on growth of strain S33-2 and kojic acid production was examined using shake flasks. It was found that strain S33-2 grew well on all types of starch investigated. However, kojic acid production was highest when corn starch was used, with the maximum kojic acid obtained being comparable to fermentation using glucose. The highest kojic acid production (19·2 g l⁻¹) was obtained when 75 g l⁻¹ corn starch was used. This gave a yield, based on starch consumed, and an overall productivity of 0·256 g g⁻¹ and 0·04 g l⁻¹ h⁻¹, respectively.     

Sago starch as a low-cost carbon source for exopolysaccharide production by Lactobacillus kefiranofaciens

Low-cost sago starch was used as a carbon source for production of the exopolysaccharide kefiran by Lactobacillus kefiranofaciens. A simultaneous saccharification and fermentation process of sago starch for kefiran production was evaluated. Factors affecting the process such as an initial pH, temperature, starch concentration, including a mixture of α-amylase and glucoamylase were determined. The highest kefiran concentration of 0.85 g/l was obtained at the initial pH of 5.5, temperature of 30 °C, starch concentration of 4% and mixed-enzymes with activity of 100 U/g-starch. The use of a mixture of α-amylase and glucoamylase could enhance the productivity compared to the use of α-amylase alone. The optimal ratio of α-amylase to glucoamylase of 60:40 gave the highest kefiran production rate of 11.83 mg/l/h. This study showed that sago starch could serve as a low-cost substrate for kefiran production.     

Optimization of fermentation conditions for the production of ethanol from sago starch using response surface methodology

The quantitative effects of temperature, pH and time of fermentation were investigated on simultaneous saccharification and fermentation (SSF) of ethanol from sago starch with glucoamylase (AMG) and Zymomonas mobilis ZM4 using a Box–Wilson central composite design protocol. The SSF process was studied using free enzyme and free cells and it was found that with sago starch, maximum ethanol concentration of 70.68 g/l was obtained using a starch concentration of 140 g/l, which represents an ethanol yield of 97.08%. The optimum conditions for the above yield were found to be a temperature of 36.74 °C, pH of 5.02 and time of fermentation of 17 h. Thus by using the central composite design, it is possible to determine the accurate values of the fermentation parameters where maximum production of ethanol occurs.