Evaluation of solid and submerged fermentations for the production of cyclodextrin glycosyltransferase by Paenibacillus campinasensis H69-3 and characterization of crude enzyme

Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins from starch by an intramolecular transglycosylation reaction. Cyclodextrins have been shown to have a number of applications in the food, cosmetic, pharmaceutical, and chemical industries. In the current study, the production of CGTase by Paenibacillus campinasensis strain H69-3 was examined in submerged and solid-state fermentations. P. campinasensis strain H69-3 was isolated from the soil, which grows at 45°C, and is a Gramvariable bacterium. Different substrate sources such as wheat bran, soybean bran, soybean extract, cassava solid residue, cassava starch, corn starch, and other combinations were used in the enzyme production. CGTase activity was highest in submerged fermentations with the greatest production observed at 48–72 h. The physical and chemical properties of CGTase were determined from the crude enzyme produced from submerged fermentations. The optimum temperature was found to be 70–75°C, and the activity was stable at 55°C for 1 h. The enzyme displayed two optimum pH values, 5.5 and 9.0 and was found to be stable between a pH of 4.5 and 11.0.     

Biochemical properties and cyclodextrin production profiles of isoforms of cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch to cyclodextrin (CD), an important host molecule for the study of host?Cguest interactions. CGTase from Paenibacillus sp. RB01 and its recombinant form showed the same isoform pattern. The three isoforms, two major (isoforms I and II) and one minor (isoform III), all had a different net charge but the same molecular mass. The aim of this work was to characterize the three isoforms, and especially to compare their CD production profiles. Isoforms I and II were separated on a FPLC Mono Q column and showed the same optimum pH (pH 5 for dextrinizing and pH 6?C7 for cyclization activity) and optimum temperature (65?C70 °C for both activities). However, the two isoforms differed in their catalytic efficiency of the coupling reaction with variable concentrations of the ??-CD donor in the presence of a fixed amount of cellobiose acceptor, with k_cat/K_m values of 3.46 × 10^?3 and 2.20 × 10^?3 mM^?1 min^?1, for isoforms I and II, respectively. Both isoforms I and II were found to have ??-CGTase activity and gave a similar CD6:CD7:CD8 product ratio of 0.2:1.0:0.6, with an increase in the ratio of the small-ring to the large-ring CDs from 1.0:0.5 to 1.0:0.3 from a 6 to 24 h reaction time. However, in terms of maximal CD yields, the two isoforms differed in their optimal reaction temperature and time required, the optimal conditions being at 40 °C for 6 h for isoform I and at 60 °C for 24 h for isoform II.     

Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

Characterization of cyclodextrin glycosyltransferase from Bacillus firmus strain No. 37

The enzyme cyclod extringly cosyltransferase (CGTase), EC2.4.1.19, which produces cyclodextrins (CDs) from starch, was obtained from Bacillus firmus strain no. 37 isolated from Brazilian soil and characterized in the soluble form using as substrate 100 g/L of maltodextrin in 0.05 M Tris-HCl buffer, 5 mM CaCl₂, and appropriate buffers. Enzymatic activity and its activation energy were determined as a function of temperature and pH. The activation energy for the production of β- and γ-CD was 7.5 and 9.9 kcal/mol, respectively. The energy of deactivation was 39 kcal/mol. The enzyme showed little thermal deactivation in the temperature range of 35–60°C, and Arrhenius-type equations were obtained for calculating the activity, deactivation, and half-life as a function of temperature. The molecular weight of the enzyme was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, giving 77.6k Da. Results for CGTase activity as a function of temperature gave maximal activity for the production of β-CD at 65°C, pH 6.0, and 7 1.5 mmol of β-CD/(min·mg of protein), whereas for γ-CD it was 9.1 m mol of γ-CD/(min·mg of protein) at 70°C and pH 8.0. For long contact times, the bestuse of the enzymatic activity occurs at 60°C oratalower temperature, and the reaction pH may be selected to increase the vield of a desired CD.     

Enhancement of selectivity for producing γ-cyclodextrin

The production of cyclodextrins (CDs) by cyclodextrin-glycosyl-transferase (CGTase) from Bacillus firmus was studied, with respect to the effect of the source of starch upon CD yield and on the selectivity for producing γ-CD. Cyclodextrin production tests were run for 24 h at 50°C, pH 8.0, and 1 mg/L of CGTase, and substrates were maltodextrin or the starches of rice, potato, cassava, and corn hydrolyzed up to D. E. 10. Cornstarch was the best substrate for producing γ-CD. Later, glycyrrhizin (2.5% [w/v]), which forms a stable complex with γ-CD, was added to the cornstarch reaction medium and increased the yield of γ-CD to about four times that produced with only maltodextrin, but the total yield of CDs remained practically unchanged. Therefore, the results showed that the studied CGTase is capable of giving relatively high yield of γ-CD in the presence of glycyrrhizin as complexant and cornstarch as substrate.     

Characterization of Cyclodextrin Glucanotransferase Produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Cyclodextrin glucanotransferase, produced by Bacillus megaterium, was characterized, and the biochemical properties of the purified enzyme were determined. The substrate specificity of the enzyme was tested with different α-1,4-glucans. Cyclodextrin glucanotransferase displayed maximum activity in the case of soluble starch, with a K _m value of 3.4 g/L. The optimal pH and temperature values for the cyclization reaction were 7.2 and 60 °C, respectively. The enzyme was stable at pH 6.0–10.5 and 30 °C. The enzyme activity was activated by Sr²⁺, Mg²⁺, Co²⁺, Mn²⁺, and Cu²⁺, and it was inhibited by Zn²⁺and Ag⁺. The molecular mass of cyclodextrin glucanotransferase was established to be 73,400 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, 68,200 Da by gel chromatography, and 75,000 Da by mass spectrometry. The monomer form of the enzyme was confirmed by the analysis of the N-terminal amino acid sequence. Cyclodextrin glucanotransferase formed all three types of cyclodextrins, but the predominant product was β-cyclodextrin.     

A recombinant cyclodextrin glycosyltransferase cloned from Paenibacillus sp. strain RB01 showed improved catalytic activity in coupling reaction between cyclodextrins and disaccharides

Recombinant cyclodextrin glycosyltransferase (CGTase) was obtained by cloning the PCR gene fragment from thermotolerant Paenibacillus sp. strain RB01 screened from hot spring area in Thailand and cloned into the Escherichia coli expression vector. The nucleotide sequence was analyzed and aligned. Nucleotide sequence of the recombinant CGTase contained an open reading frame of 2139 bp encoding 713 amino acid residues. The recombinant required one-third of culture time and neutral pH to produce CGTase compared to wild type. CGTases from both wild type and transformant were purified in parallel by starch adsorption and DEAE cellulose column. Their biochemical properties such as molecular weight, optimum pH and temperature were quite similar. However, the recombinant enzyme showed improved catalytic activity in the coupling reaction between cyclodextrins (CDs) and some disaccharides. Among several sugars tested with excess βCD, cellobiose was the best substrate followed by leucrose. Very low activity was observed with trehalose, lactose and mellibiose. Sucrose and raffinose showed no activity. The K _m and other kinetic parameters of recombinant enzyme were determined for cellobiose and several cyclodextrin derivatives. Recombinant CGTase showed lower K _m for βCD and its derivatives, with improved activity compared to wild type enzyme.     

Synthesis of Cyclodextrin Glucosyl Transferase byBacillus cereus for the production of cyclodextrins

A potent indigenous bacillus isolate identified asBacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly. Process optimization of various fermentation parameters has been established for optimal growth of bacillus and the maximum enzyme synthesis. The organism had the highest specific growth rate (0.7μ) with a generation time of 1 h in glucose containing medium at the conditions of pH 7.0, 37°C at 300 rpm, 1.5 vvm of agitation, and aeration. At these conditions, it exhibited the maximum activity of 54 U/mL at the synthesis rate of 2.7 U/L/h. CGTase was produced from the early exponential growth and peaked during the midsporulating stage of about 16 h thereafter maintained at the same level of 50 U/mL. Saccharides containing media were better inducers than starch, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose (65 U/mL) and, more remarkably, by the supplementation of wheat bran extract in glucose medium (106 U/mL). This organism produced CGTase stably in a chemostat culturing over a period of 400 h with a maximum productivity of 5.4 kU/L/h (threefold higher than obtained in batch culturing [1.75 kU/L/h]). Comparatively, CGTase was produced by immobilized cells in a continuous fluidized bed reactor for over approx 360 h, at a relatively high dilution rate of 0.88 h⁻¹ resulting in the productivity of 23.0 kU/L/h.     

Characterization of novel neopullulanase fromBacillus polymyxa

Bacillus polymyxa CECT 155 produces an extracellular neopullulanase activity that degrades pullulan to panose. This activity was stimulated by the presence of pullulan in the culture, and repressed by glucose. The apparent mol wt determined for the enzyme was 58 kDa. The optimum pH and temperature for neopullulanase activity were pH 6.0 and 50°C, respectively. The enzyme was stable in a pH range of 4.0–8.0, and temperatures up to 60°C. These properties make it suitable for the saccharification processes in the starch industries.     

Effect of organic solvents on the yield and specificity of cyclodextrins by recombinant cyclodextrin glucanotransferase (CGTase) from Anaerobranca gottschalkii

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an enzyme that degrades starch and starch related glucans into cyclodextrins (CDs) by intramolecular transglycosylation reaction. The biochemical activity of recombinant CGTase from Anaerobranca gottschalkii for the yield and product specificity of cyclodextrins was investigated in the presence of organic solvents. Compared with the control of starch bioconversion, addition of various organic solvents generally increased the total CD and product specificity by affecting product inhibition and/or intermolecular transglycosylation reaction. The highest conversion (45 %) of starch to CDs was obtained in the presence of ethanol, while the simultaneous addition of two organic solvents, decanol-ethanol, comparatively showed a reduced total yield of 39 %. Despite this, the highest product ratio of 91 % α-CD, and 64 % β-CD was obtained in the presence of decanol and cyclohexane respectively. The effect of organic solvents on the yield and specificity of CD was attributed mainly to their effect on product inhibition and transglycosylation reaction. Although the use of two organic solvents showed almost a significant increase in total yield of CDs, it resulted in a comparatively lower specific product yield compared to their respective individual effect. Generally, normal enzyme activity was favoured at higher temperature of 65 °C, but the addition of organic solvents, in most cases, was found to decrease the bioconversion. Thus, the preferred optimal condition was reduced to 40 °C, where the maximal conversion of starch to CDs in general and α-CD in particular was achieved.     

Characterization of Thermo-stable Endoinulinase from a New Strain <Emphasis Type="Italic">Bacillus Smithii</Emphasis> T7

A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH₄)H₂PO₄ 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively. Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application.     

Gamma-Cyclodextrin Production Using Cyclodextrin Glycosyltransferase from Bacillus clarkii 7364

The production of cyclodextrins (CDs) by cyclodextrin glycosyltransferase (CGTase) from Bacillus clarkii 7364 was studied. Forty-seven percent (w/w) conversion rate to ??-CD was obtained in the process performed by reacting 5 U per gram of starch CGTase with 15?% (w/v) soluble starch in 0.025?M sodium phosphate?CNaOH buffer (pH 12) at 55?°C in the presence of 2?% (w/v) glycyrrhizic acid. Meanwhile, the ratio of ??:??-CD was 89:11, with negligible formation of ??-CD. Under these conditions, there is a significant increase in overall production of CDs and a marked change in product selectivity for ??-CD. The possible mechanisms were discussed upon different product profiles with respect to the size and amount of CDs synthesized at different reaction conditions. The approach described here can be easily applied to an enzymatic process for the production of ??-CD on an industrial scale, and such high selectivity, at high conversions, is especially attractive from a commercial perspective.     

Influence of Different Substrates on the Production of a Mutant Thermostable Glucoamylase in Submerged Fermentation

Three mutations, Ser54→Pro, Thr314→Ala, and His415→Tyr, were identified in Aspergillus awamori glucoamylase gene expressed by Saccharomyces cerevisiae. The mutant glucoamylase (GA) was substantially more thermostable than a wild-type GA at 70 °C, with a 3.0 KJ mol⁻¹ increase in the free energy of thermo-inactivation. The effect of starch from different botanical sources on the production of this GA was measured in liquid fermentation using commercial soluble starch, cassava, potato, and corn as the carbon source. The best substrate for GA production was the potato starch showing an enzymatic activity of 6.6 U/mL. The commercial soluble starch was also a good substrate for the enzyme production with 6.3 U/mL, followed by cassava starch and corn starch with 5.9 and 3.0 U/mL, respectively. These results showed a significant difference on GA production related to the carbon source employed. The mutant GA was purified by acarbose–Sepharose affinity chromatography; the estimated molecular mass was 100 kDa. The mutant GA exhibited optimum activity at pH 4.5 and an optimum temperature of 65 °C.     

Comparison of catalytic properties of free and immobilized cellobiase Novozym 188

The enzyme cellobiase from Novo was immobilized in controlled pore silica particles by covalent binding with the silane-glutaraldehyde method with protein and activity yields of 67 and 13.7%, respectively. The activity of the free enzyme (FE) and immobilized enzyme (IE) was determined with 2 g/L of cellobiose, from 40 to 75°C at pH 3.0–7.0 for FE and from 40 to 70°C at pH 2.2–7.0 for IE. At pH 4.8 the maximum specific activity for the FE and IE occurred at 65°C: 17.8 and 2.2 micromol of glucose/(min·mg of protein), respectively. For all temperatures the optimum pH observed for FE was 4.5 whereas for IE it was shifted to 3.5. The energy of activation was 11 kcal/mol for FE and 5 kcal/mol for IE at pH 4.5–5, showing apparent diffusional limitation for the latter. Thermal stability of the FE and IE was determined with 2 g/L of cellobiose (pH 4.8) at temperatures from 40 to 70°C for FE and 40 to 75°C for IE. Free cellobiase maintained its activity practically constant for 240 min at temperatures up to 55°C. The IE has shown higher stability, retaining its activity in the sametest up to 60°C. Half-life experimental results for FE were 14.1, 2.1, and 0.17 h at 60, 65, and 70°C, respectively, whereas IE at the same temperatures had half-lives of 245, 21.3, and 2.9 h. The energy of thermal deactivation was 80.6 k cal/mol for the free enzyme and 85.2 k cal/mol for the IE, suggesting stabilization by immobilization.     

Modeling cellobiose hydrolysis with integrated kinetic models

The enzyme cellobiase Novozym 188, which is used for improving hydrolysis of bagasse with cellulase, was characterized in its commercial available form and integrated kinetic models were applied to the hydrolysis of cellobiose. The specific activity of this enzyme was determined for pH values from 3.0–7.0, and temperatures from 40–75°C, with cellobiose at 2 g/L. Thermal stability was measured at pH 4.8 and temperatures from 40–70°C. Substrate inhibition was studied at the same pH, 50°C, and cellobiose concentrations from 0.4–20 g/L. Product inhibition was determined at 50°C, pH 4.8, cellobiose concentrations of 2 and 20 g/L, and initial glucose concentration nearly zero or 1.8 g/L. The enzyme has shown the greatest specific activity, 17.8 U/mg, at pH 4.5 and 65°C. Thermal activation of the enzyme followed Arrhenius equation with the Energy of Activation being equal to 11 kcal/mol for pH values 4 and 5. Thermal deactivation was adequately modeled by the exponential decay model with Energy of Deactivation giving 81.6 kcal/mol. Kinetics parameters for substrate uncompetitive inhibition were: Km=2.42 mM, V _max=16.31 U/mg, Ks=54.2 mM. Substrate inhibition was clearly observed above 10 mM cellobiose. Product inhibition at the concentration studied has usually doubled the time necessary to reach the same conversion at the lower temperature tested.     

Immobilization of Naringinase in PVA–Alginate Matrix Using an Innovative Technique

A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA–alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (<80 °C). The combination of alginate and bead treatment with sodium sulfate not only prevented agglomeration but produced beads of high gel strength and conferred enzyme protection from inactivation by boric acid. Naringinase from Penicillium decumbens was immobilized in PVA (10%)–alginate beads with three different sizes (1–3 mm), at three different alginate concentrations (0.2–1.0%), and these features were investigated in terms of swelling ratio within the beads, enzyme activity, and immobilization yield during hydrolysis of naringin. The pH and temperature optimum were 4.0 and 70 °C for the PVA–alginate-immobilized naringinase. The highest naringinase activity yield in PVA (10%)–alginate (1%) beads of 2 mm was 80%, at pH 4.0 and 70 °C. The Michaelis constant (K _Mapp) and the maximum reaction velocity (V _maxapp) were evaluated for both free (K _Mapp = 0.233 mM; V _maxapp = 0.13 mM min⁻¹) and immobilized naringinase (K _Mapp = 0.349 mM; V _maxapp = 0.08 mM min⁻¹). The residual activity of the immobilized enzyme was followed in eight consecutive batch runs with a retention activity of 70%. After 6 weeks, upon storage in acetate buffer pH 4 at 4 °C, the immobilized biocatalyst retained 90% of the initial activity. These promising results are illustrative of the potential of this immobilization strategy for the system evaluated and suggest that its application may be effectively performed for the entrapment of other biocatalysts.     

Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers

Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K _m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V _max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.     

Characterization of Sol-gel bioencapsulates for ester hydrolysis and synthesis

Candida rugosa lipase was entrapped in silica sol-gel particles prepared by hydrolysis of methyltrimethoxysilane and assayed by p-nitrophenyl palmitate hydrolysis, as a function of pH and temperature, giving pH optima of 7.8 (free enzyme) and 5.0–8.0 (immobilized enzyme). The optimum temperature for the immobilized enzyme (50–55°C) was 19°C higher than for the free enzyme. Thermal, operational, and storage stability were determined with n-butanol and bytyric acid, giving at 45°C a half-life 2.7 times greater for the immobilized enzyme; storage time was 21 d at room temperature. For ester synthesis, the optimum temperature was 47°C, and high esterification conversions were obtained under repeated batch cycles (half-life of 138 h).     

Isolation and characteristics of highly active α-amylase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>-150

Partially purified enzyme preparation with specific activities of 153.7 U/mg for α-amylase and 0.15 U/mg for protease was produced by selective adsorption on starch. Enzymes were purified until homogeneous electrophoretically by gel-filtration over HW-55 TSK-gel with specific activities of 245 U/mg for α-amylase and 1.44 U/mg for protease. The optimum temperature and pH for purified α-amylase activity are 40–50°C and pH 6.0. The effects of various metal ions on the activity and stability of the enzyme were studied. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 374–376, July–August, 2007.     

Lipase from a Brazilian strain ofPenicillium citrinum

A lipases (glycerol ester hydrolases E. C. 3.1.1.3) from a brazilian strain ofPenicillium citrinum has been investigated. When the microorganism was cultured in the simple medium (1.0% olive oil and 0.5% yeast extract), using olive oil in as carbon source in the inocula, the enzyme extracted showed maximum activity (409 IU/mL). In addition, decrease of yeast extract concentration also reduces the lipase activity. Nevertheless, when yeast extract was replaced by ammonium sulfate, no activity was detected. Purification by precipitation with ammonium sulfate showed best activity in the 40–60% fraction. The optimum temperature for enzyme activity was found in the range of 34–37°C. However, after 30 min at 60°C, the enzyme was completely inactivated. The enzyme showed optimum at pH 8.0. The dried concentrated fraction (after dialysis and lyophilization) maintained its lipase activity at room temperature (28°C) for 8 mo. This result in lipase stability suggests an application of lipases fromP. citrinum in detergents and other products that require a high stability at room temperature.