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.     

Cyclomaltodextrin glucanotransferase-producing moderate thermophile,Bacillus coagulans

A new CGTase-producing moderate thermophile was isolated from soil, and was identified as Bacillus coagulans which have not previously been listed as cyclodextrin producing bacteria. The culture filtrate of the isolate as the CGTase source converted about 60% of soluble starch to CD's in 20 h at 50°C, and the ratio of α-: β-: γ-CD produced was 1.0: 0.9: 0.3.     

Purification and partial characterisation of three endo-glucanases from dichomitus squalens

Three endo-glucanases (En I-III) were obtained by chromatofocusing fractionation of a culture supernatant from the white-rot fungus Dichomitus squalens. They were purified further on Phenyl-Sepharose CL-4B, DEAE-Trisacryl, and Ultrogel AcA 54; ∼21-, ∼16-, and ∼9-fold purifications were obtained for En I-III, respectively. The enzymes appeared as homogeneous proteins on disc gel electrophoresis with and without SDS (sodium dodecyl sulphate), and on isoelectric focusing; the respective mol. wts. were 42,000, 56,000, and 47,000, and the isoelectric points 4.8, 4.3, and 4.1. Optimum conditions for the hydrolysis of CM-cellulose were pH 4.8 and 55° for each enzyme, and each was stable over the pH range 4.0–8.5 but inactivated completely within 30 min at 70°. None of the purified enzymes exhibited β-d-glucosidase or cellobiohydrolase activity, but En II was weakly active towards laminaran and xylan. En I and En II acted more randomly on CM-cellulose than did En III. Cellotetraose was degraded by each endo-glucanase, whereas only En III could hydrolyse cellotriose.     

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.     

Physiological and fermentation properties of <Emphasis Type="Italic">Bacillus coagulans</Emphasis> and a mutant lacking fermentative lactate dehydrogenase activity

Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50–55°C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55°C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35°C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55°C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.     

β-cyclodextrin production by the cyclodextrin glucanotransferase from Paenibacillus illinoisensis ZY-08: cloning, purification, and properties

The gene encoding the cyclodextrin glucanotransferase (CGTase, EC2.4.1.19) of Paenibacillus illinoisensis was isolated, cloned, sequenced and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34-residues. The deduced amino acid sequence of the CGTase from P. illinoisensis ZY-08 exhibited highest identity (99 %) to the CGTase sequence from Bacillus licheniformis (P14014). The four consensus regions of carbohydrate converting domain and Ca²⁺ binding domain could be identified in the sequence. The CGTase was purified by using cold expression vector, pCold I, and His-tag affinity chromatography. The molecular weight of the purified enzyme was about 74 kDa. The optimum temperature and pH of the enzyme were 40 °C and pH 7.4, respectively. The enzyme activity was increased by the addition of Ca²⁺ and inhibited by Ba²⁺, Cu²⁺, and Hg²⁺. The K _m and V _max values calculated were 0.48 mg/ml and 51.38 mg of β-cyclodextrin/ml/min. The ZY-08 and recombinant readily converted soluble starch to β-cyclodextrin but ZY-08 did not convert king oyster mushroom powder and enoki mushroom powder. However the recombinant CGTase converted king oyster mushroom powder and enoki mushroom powder to β-cyclodextrin.     

A thermostable leucine dehydrogenase from Bacillus coagulansNL01: Expression,purification and characterization

L-Tert-leucine is the most representative unnatural amino acid and its production is valuable in industry. At present, l-tert-leucine is mainly produced by bioconversion, in which leucine dehydrogenase (LeuDH) plays a major role. In this study, a highly thermo- and pH-stable LeuDH from Bacillus coagulans NL01 (Bc-LeuDH) was reported and successfully expressed in Escherichia coli BL21(DE3). The enzyme was purified and its enzymatic properties were characterized. The specific activity of Bc-LeuDH at optimum condition (pH 8.0 and 50 °C) is 1337.97 U/mg, and the Km and kcat for sodium α-ketoisocaproate was 1.369 mM and 0.125 _S^-1, respectively. Furthermore, Bc-LeuDH possessed excellent thermostability that held nearly 80 % activity after 72 h incubation at temperature 50 °C and 55 °C. Notably, the half-life of Bc-LeuDH activity also was long for near 12 h at a high temperature of 70 °C, which was the longest in previous reports. Based on the sequence and structure analysis, the hydrophobic amino acid residues and hydrophobic patches are considered to have an important contribution to the thermostability of Bc-LeuDH. Furthermore, the Bc-LeuDH demonstrated better pH stability over a wide pH ranging from 7.0-10.0. These results indicate the potential industrial application of Bc-LeuDH in the future.     

Production and characterization of a new cyclodextrin glycosyltransferase from Bacillus firmus isolated from Brazilian soil

A new CGTase was obtained from Bacillus firmus, strain 7B, isolated from oat soil culture, using a high alkaline pH medium containing 1% Na₂CO₃. The enzyme was characterized in soluble form, for pH 5–11, temperature from 30 to 85 °C, using a 1% maltodextrin substrate solution and appropriate buffers. It produced mainly β-CD and the cell-free supernatant had a precipitating activity measured by the trichloroethylene method that is a 100-fold greater than that of the enzyme of Bacillus firmus, strain 37, previously studied by our group. The molecular weight of the pure protein was measured as 56,230 Da with SDS-PAGE. The optimum temperature for the enzyme activity was 50 °C and it was most active at pH 6.0. Thermal deactivation was noticeable above 65 °C and the enzyme was highly stable below 60 °C. The influence of substrate or product concentration on the initial rate of CD production was studied and the kinetic parameters were determined. The enzyme showed cyclization activity on different raw and hydrolyzed starches and hydrolyzed cornstarch gave the highest activity.     

Immobilization of cyclodextrin glucanotransferase on amberlite IRA-900 for biosynthesis of transglycosylated xylitol

Cyclodextrin glucanotransferase (CGTase) fromThermoanaerobacter sp. was adsorbed on the ion exchange resin Amberlite IRA-900. The optimum conditions for the immobilization of the CGTase were pH 6.0 and 600 U CGTase/g resin, and the maximum yield of immobilization was around 63% on the basis of the amount ratio of the adsorbed enzyme to the initial amount in the solution. Immobilization of CGTase shifted the optimum temperature for the enzyme to produce transglycosylated xylitol from 70°C to 90°C and improved the thermal stability of immobilized CGTase, especially after the addition of soluble starch and calcium ions. Transglycosylated xylitol was continuously produced using immobilized CGTase in the column type packed bed reactor, and the operating conditions for maximum yield were 10% (w/v) dextrin (13 of the dextrose equivalent) as the glycosyl donor, 10% (w/v) xylitol as the glycosyl acceptor, 20 mL/h of medium flow rate, and 60°C. The maximum yield of transglycosylated xylitol and productivity were 25% and 7.82 g·L⁻¹·h⁻¹, respectively. The half-life of the immobilized CGTase in a column type packed bed reactor was longer than 30 days.     

Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus

A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the pure protein was estimated to be 78 000 and 82 000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a pH optimum in the range 5.5–8.5. It was stable over the pH range 7–11 at 10 °C, and at pH 7.0 at 60 °C. The optimum temperature for enzyme activity was 65 °C. In the absence of substrate, the enzyme rapidly lost its activity above 30 °C. K _m and k _cat for the pure enzyme were 1.21 mg/ml and 145.17 μM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from 150 g/l starch without using any additives. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.2:9.2:0.6 and 0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation. Received: 25 September 1998 / Received revision: 15 December 1998 / Accepted: 21 December 1998     

Properties of o-diphenol: O2 oxidoreductase from Musa cavendishii

Banana fruits (Musa cavendishii) contain a non-particulate o-diphenol: O₂ oxidoreductase that has been partially purified. The enzyme is inactivated during its catalytic action with a half time of 135 sec. The activation energy for the catalytic process is 4445 cal/mol. Classical thermal denaturation takes place only above 70°, with an entropy change of about 190 cal/mol °K at pH 6·0.     

Purification and characterization of an extremely stable glucose isomerase from Geobacillus thermodenitrificans TH2

The D-glucose/D-xylose isomerase was purified from a thermophilic bacterium, Geobacillus thermodenitrificans TH2, by precipitating with heat shock and using Q-Sepharose ion exchange column chromatography, and then characterized. The purified enzyme had a single band having molecular weight of 49 kDa on SDS-PAGE. In the presence of D-glucose as a substrate, the optimum temperature and pH of the enzyme were found to be 80°C and 7.5, respectively. The purified xylose isomerase of G. thermodenitrificans TH2 was extremely stable at pH 7.5 after 96 h incubation at 4°C and 50°C. When the thermal stability profile was analyzed, it was determined that the purified enzyme was extremely stable during incubation periods of 4 months and 4 days at 4°C and 50°C, respectively. The K _m and V _max values of the purified xylose isomerase from G. thermodenitrificans TH2 were calculated as 32 mM and 4.68 μmol/min per mg of protein, respectively. Additionally, it was detected that some metal ions affected the enzyme activity at different ratios. The enzyme was active and stable at high temperatures and nearly neutral pHs which are desirable for the usage in the food and ethanol industry.     

Purification and properties of an α-amylase produced by a cassava-fermenting strain ofMicrococcus luteus

An extracellular α-amylase produced by a cassava-fermenting strain ofMicrococcus luteus was purified 26-fold by gel filtration and ion-exchange chromatography. The molar mass was estimated to be approximately 56 kDa. The optimum temperature of the enzyme was 30°C, optimum pH 6.0 and optimum substrate concentration was 0.6% (W/V). Treatment of the enzyme at 70°C for 10 min resulted in 70% loss of activity. The activation energy was determined to be 34.8 kJ/mol. The activity of the enzyme was enhanced by Mg²⁺, Ca²⁺, K⁺, Na⁺ and inhibited by EDTA, KCN and citric acid. The enzyme may find some application in local food processing.     

Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungus Scytalidium thermophilum

Glucoamylase produced byScytalidium thermophilum was purified 80-fold by DEAE-cellulose, ultrafiltration and CM-cellulose chromatography. The enzyme is a glycoprotein containing 9.8% saccharide, pI of 8.3 and molar mass of 75 kDa (SDS-PAGE) or 60 kDa (Sepharose 6B). Optima of pH and temperature with starch or maltose as substrates were 5.5/70 °C and 5.5/65 °C, respectively. The enzyme was stable for 1 h at 55 °C and for about 8 d at 4 °C, either at pH 7.0 or pH 5.5. Starch, amylopectin, glycogen, amylose and maltose were the substrates preferentially hydrolyzed. The activity was activated by 1 mmol/L Mg²⁺ (27%), Zn²⁺ (21%), Ba²⁺ (8%) and Mn²⁺ (5%).K _m and {ie11-1} values for starch and maltose were 0.21 g/L, 62 U/mg protein and 3.9 g/L, 9.0 U/mg protein, respectively. Glucoamylase activity was only slightly inhibited by glucose up to a 1 mol/L concentration.     

Highly thermostable neutral protease from Bacillus stearothermophilus

Bacillus stearothermophilus MK232, which produced a highly thermostable neutral protease, was isolated from a natural environment. By several steps of mutagenesis, a hyper-producing mutant strain, YG185, was obtained. The enzyme productivity was twice as much as that of the original strain. This extracellular neutral protease was purified and crystallized. The molecular weight of the enzyme was 34,000 by SDS-polyacrylamide gel electrophoresis and gel filtration. The optimum pH and temperature for the enzyme activity were 7.5 and 70°C, respectively, and the enzyme was stable at pH 5–10 and below 70°C. The thermostability and specific activity of the new protease are around 10% and 40% higher than those of thermolysin (the neutral protease from Bacillus thermoproteolyticus), respectively. The enzyme was inactivated by EDTA, but not by phenylmethylsulfonyl fluoride. These results indicate that the enzyme is a highly thermostable neutral-(metallo)protease.     

Immobilisation of CGTase for continuous production of long-carbohydrate-chain alkyl glycosides: Control of product distribution by flow rate adjustment

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) was covalently immobilised on Eupergit C and used in a packed-bed reactor to investigate the continuous production of long-carbohydrate-chain alkyl glycosides from α-cyclodextrin (α-CD) and n-dodecyl-(1,4)-β-maltopyranoside (C₁₂G₂β). The effects of buffer ion strength and pH, and enzyme loading on the immobilisation yield and the enzyme activity were evaluated. Approximately 98% of the protein and 33% of the total activity were immobilised. At pH 5.15, the enzymatic half-life was 132 min at 60 °C and 18 min at 70 °C. The immobilised enzyme maintained 60% of its initial activity after 28 days storage at 4 °C. The degree of conversion was controlled by simple regulation of the flow rate through the reactor, making it possible to optimise the product distribution. It was possible to achieve a yield of the primary coupling product n-dodecyl-(1,4)-β-maltooctaoside (C₁₂G₈β) of about 50%, with a ratio between the primary and the secondary coupling product of about 10. Thermoanaerobacter sp. CGTase (Toruzyme 3.0 L) immobilised on Eupergit C had good operational stability at 60 and 70 °C thus showing the advantages of using more thermostable enzymes in biocatalysis. However, this enzyme was unsuitable for the production of C₁₂G₈β due to extensive disproportionation reactions, giving a broad product range.     

The Effect of Oidium lactis on the Sporulation of Bacillus coagulans in Tomato Juice

Oidium lactis can raise the pH of tomato juice in thin layers to a pH range optimum for sporulation of Bacillus coagulans in 24 hr at 25 C and in 48 hr at 35 C. The percentage of sporulation of B. coagulans was greater in tomato juice where O. lactis had grown than in the thermoacidurans broth. As high as 61.7% sporulation occurs within 24 hr in the tomato juice in which Oidium lactis had grown when this juice was incubated at 52 C. When tomato juice was adjusted to pH 5.0 and B. coagulans or a mixture of O. lactis and the vegetative cells of B. coagulans were added as an inoculum, only in the juice in which O. lactis was growing were spores produced within 72 hr at 35 C.     

Production and purification of a hyperthermostable chitinase from Brevibacillus formosus BISR-1 isolated from the Great Indian Desert soils

A strain of Brevibacillus formosus, capable of producing a high level of chitinase, was isolated and characterized for the first time from the Great Indian Desert soils. The production of extracellularly secreted chitinase was analyzed for its biocontrol potential and optimized by varying media pH, temperature, incubation period, substrate concentrations, carbon and nitrogen sources, etc. A twofold increase in chitinase production (798 IU/mL) was achieved in optimized media containing (g l^?1) chitin 2.0, malt extract 1.5, glycerol 1.0, ammonium nitrate 0.3 %, T-20 (0.1 %) and media pH 7.0 at 37 °C. The produced enzyme was purified using a three-step purification procedure involving ultra-filtration, ammonium sulphate precipitation and adsorption chromatography. The estimated molecular weight of the purified enzyme was 37.6 kDa. The enzyme was found thermostable at higher temperatures and showed a t _½ of more than 5 h at 100 °C. Our results show that the chitinase produced by B. formosus BISR-1 is thermostable at higher temperatures.     

Purification and properties of an acidic β-glucosidase from Acidobacterium capsulatum

Acidobacterium capsulatum, an acidophilic, mesophilic and chemoorganotrophic bacterium, produced an inducible, acidic β-glucosidase in the cellobiose medium. The enzyme was successively purified 109 times by CM-Sepharose, Sephacryl S-200 chromatography and preparative discontinuous polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the purified enzyme gave a single band at pH 4.3. The enzyme had an optimum pH of 3.0 and optimum reaction temperature of 55°C, being stable from pH 1.5 to 6.0 and at temperatures from 20 to 45°C. No activity was detected above pH 6.5 or above 65°C. The molecular weight of 90,000 was estimated by gel filtration and the enzyme had an isoelectric point of 7.0. The enzyme hydrolyzed aryl-β-glycosides and β-linked disaccharides.     

A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52 % amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55 % of the maximum activity when assayed at 40–75 °C, 23 % at 20 °C, 16 % at 85 °C, and even 8 % at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62 % xylanase activity and stability at the concentration of 3–30 % (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5–19.0, 15.3–19.0, 21.9–27.7, and 28.2–31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.