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.     

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.     

Isolation of Novel Alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> Strains for Cyclodextrin Glucanotransferase Production

New alkaliphilic Bacillus producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) were isolated from 17 Bulgarian alkaline and normal habitats (springs and soils) by three steps of a selection. None of the isolates obtained, producing CGTase, appeared to be thermophilic in character. One hundred and thirty-seven strains were estimated for CGTase activity by batch cultivation in a liquid alkaline medium. Twenty-seven of them had a detectable CGTase activity in their culture supernatants under the enzyme assay conditions, despite of the significant growth of all isolates. The phenotypic properties of three selected strains (20RF, 8SB and 24WE) were determined. They were aerobic endospore-forming Bacillus strains: two of them were obligated alkaliphiles (20RF and 8SB) and one, alkalitolerant (24WE). Both obligated alkaliphiles were further characterised by 16S rRNA analysis. According to the full 16S rRNA gene sequences obtained and deposited to the NCBI GenBank database, both isolated obligated alkaliphiles 20RF and 8SB were clustered into the group of alkaliphilic Bacillus species. The exhibited CGTase production by them (230–250 U ml⁻¹ for 20RF and 130–160 U ml⁻¹ for 8SB) defined these new isolates as promising producers of the enzyme, especially Bacillus sp. 8SB synthesising thermostable alkaline β-CGTase. Both new enzymes from 20RF and 8SB Bacillus strains formed only two types of cyclodextrins, beta and gamma, which could be of interest for their easy separation and industrial production.     

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.     

Cellulose production based on hemicellulose hydrolysate from steam-pretreated willow

The production cost of cellulolytic enzymes is a major contributor to the high cost of ethanol production from lignocellulosics using enzymatic hydrolysis. The aim of the present study was to investigate the cellulolytic enzyme production ofTrichoderma reesei Rut C 30, which is known as a good cellulase secreting micro-organism, using willow as the carbon source. The willow, which is a fast-growing energy crop in Sweden, was impregnated with 1–4% SO₂ and steam-pretreated for 5 min at 206°C. The pretreated willow was washed and the wash water, which contains several soluble sugars from the hemicellulose, was supplemented with fibrous pretreated willow and used for enzyme production. In addition to sugars, the liquid contains degradation products such as acetic acid, furfural, and 5-hydroxy-methylfurfural, which are inhibitory for microorganisms. The results showed that 50% of the cellulose can be replaced with sugars from the wash water. The highest enzyme activity, 1.79 FPU/mL and yield, 133 FPU/g carbohydrate, was obtained at pH 6.0 using 20 g/L carbon source concentration. At lower pHs, a total lack of growth and enzyme production was observed, which probably could be explained by furfural inhibition.     

Xanthan gum production from cassava bagasse hydrolysate with Xanthomonas campestris using alternative sources of nitrogen

Cassava bagasse was hydrolyzed using HCl and the hydrolysate was used for the production of xanthan gum using a bacterial culture of Xanthomonas campestris. Cassava bagasse hydrolysate with an initial concentration of approx 20 g of glucose/L proved to be the best substrate concentration for xanthan gum production. Among the organic and inorganic nitrogen sources tested to supplement the medium—urea, yeast extract, peptone, potassium nitrate, and ammonium sulfate—potassium nitrate was most suitable. Ammonium sulfate was the least effective for xanthan gum production, and it affected sugar utilization by the bacterial culture. In media with an initial sugar concentration of 48.6 and 40.4 g/L, at the end of fermentation about 30 g/L of sugars was unused. Maximum xanthan gum (about 14 g/L) was produced when fermentation was carried out with a medium containing 19.8 g/L of initial reducing sugars supplemented with potassium nitrate and fermented for 72 h, and it remained almost the same until the end of fermentation (i.e., 96 h).     

Purification and Properties of a New Thermostable Cyclodextrin Glucanotransferase from Bacillus pseudalcaliphilus 8SB

A new cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was studied in respect to its γ-cyclizing activity. An efficient conversion of a raw corn starch into only two types of cyclodextrins (β- and γ-CD) was achieved by the purified enzyme. Crude enzyme obtained by ultrafiltration was purified up to fivefold by starch adsorption with a recovery of 62% activity. The enzyme was a monomer with a molecular mass 71 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The CGTase exhibited two pH optima, at pH 6.0 and 8.0, and was at most active at 60 °C and pH 8.0. The enzyme retained more than 80% of its initial activity in a wide pH range, from 5.0 to 11.0. The CGTase was strongly inhibited by 15 mM Cu(2+), Fe(2+), Ag(+), and Zn(2+), while some metal ions, such as Ca(2+), Na(+), K(+), and Mo(7+), exerted a stimulating effect in concentration of 5 mM. The important feature of the studied CGTase was its high thermal stability: the enzyme retained almost 100% of its initial activity after 2 h of heating at 40-60 °C; its half-life was 2 h at 70 °C in the presence of 5 mM Ca(2+). The achieved 50.7% conversion of raw corn starch into 81.6% β- and 18.4% γ-CDs after 24 h enzyme reaction at 60 °C and pH 8.0 makes B. pseudalcaliphilus 8SB CGTase industrially important enzyme for cyclodextrin production.     

Purification and Refolding of Cyclodextrin Glycosyltransferase Expressed Escherichia coli

Recombinant DNA technology and protein engineering are currently utilized in the cost-effective production of pharmaceutical and industrial proteins with native conformation. Escherichia coli retains its dominant position as the first choice of host for speed, simplicity and well-established production protocols. However, protein production using recombinant E. coli occasionally encounters complex purification and refolding steps. This paper introduces an efficient scheme for purification andin vitro refolding of industrially important proteins including cyclodextrin glycosyltransferase (CGTase) expressed in recombinant E. coli employing a polycationic amino acid fusion system. Fusion of polycationic amino acids to CGTase allowed purification and refolding of CGTase to be simple and efficient. A novel CGTase production strategy will be discussed by considering recent progress in protein purification and refolding techniques.     

A technique for mycelial development of ectomycorrhizal fungi on agar media

A technique was established to study ectomycorrhizal fungi on agar media. Petri dishes, 60 mm in diameter, containing 10 mL of culture medium covered with a cellophane disk were used for easy collection of the mycelium after growth. For analysis of fungal biomass production, a sterilized cellophane sheet was placed on the medium’s surface. Inoculation was achieved by placing a mycelial block onto the center of the cellophane sheet and then incubating at 25°C in the dark. Colony radial growth was measured and biomass dry wt was determined. Fresh mycelia were homogenized with 10 mL of acetate buffer (pH 5.5) for enzyme analysis. A crude extract was obtained by adding all culture medium to 90 mL of distilled water and homogenizing in a Potter. Reducing sugars, enzyme concentration, and pH were determined. Three fungal strains, Suillus collinitus, Pisosithus arrhizus, and Hebeloma cylindrosporum, were grown in different culture media (potato dextrose agar or Pintro’s medium). Parameters measured over time included glucose concentration, phosphatase activity, biomass, and pH.     

Production of Cyclodextrins by CGTase from Bacillus clausii Using Different Starches as Substrates

Cyclodextrins (CDs) are cyclic oligasaccharides composed by d-glucose monomers joined by α-1,4-d glicosidic linkages. The main types of CDs are α-, β- and γ-CDs consisting of cycles of six, seven, and eight glucose monomers, respectively. Their ability to form inclusion complexes is the most important characteristic, allowing their wide industrial application. The physical property of the CD-complexed compound can be altered to improve stability, volatility, solubility, or bio-availability. The cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an enzyme capable of converting starch into CD molecules. In this work, the CGTase produced by Bacillus clausii strain E16 was used to produce CD from maltodextrin and different starches (commercial soluble starch, corn, cassava, sweet potato, and waxy corn starches) as substrates. It was observed that the substrate sources influence the kind of CD obtained and that this CGTase displays a β-CGTase action, presenting a better conversion of soluble starch at 1.0%, of which 80% was converted in CDs. The ratio of total CD produced was 0:0.89:0.11 for α/β/γ. It was also observed that root and tuber starches were more accessible to CGTase action than seed starch under the studied conditions.     

β-Cyclodextrin production by simultaneous fermentation and cyclization

Production of β-cyclodextrin (CD) with high-dextrose equivalent (DE) starch hydrolysates by simultaneous fermentation and cyclization (SFC) gives higher yields than using only the enzyme CGTase, because fermentation eliminates glucose and maltose that inhibit CD production, while at the same time, produces ethanol that increases yield. A 10% (w/v) solution of cassava starch, liquefied with α-amylase, was incubated with CGTase using: only the enzyme, added ethanol (from 1 to 5%), and added yeast,S. cerevisiae (12% w/v), plus nutrients, the latter being the SFC process. Reaction conditions were: 38αC, pH 6.0, DE from 2 to 25, and 3.3 mL of CGTase/L. The yield of β-CD has decreased with an increase in DE, and maximum reaction yields were found for DE equal to 3.54, reaching 5.6, 14.7, and 11.5 mM β-CD, respectively. For an increase of DE, of approx 6 times (from 3.54 to 23.79), β-CD yield decreased 6 times for the first, and second reaction media with 3% (v/v) ethanol, and only approx 3 times for SFC (from 11.5 to 3.73 mM), showing that this process is less sensitive to variations in the DE     

Effect of ethanol on the synthesis of large-ring cyclodextrins by cyclodextrin glucanotransferases

Cyclodextrin glucanotransferase (EC 2.4.1.19, CGTase) synthesizes cyclodextrins (CD) composed of 6 to more than hundred glucose units from amylose by an intramolecular transglycosylation reaction. The addition of ethanol to the reaction medium resulted in an increase of the yield of large-ring CD obtained with a CGTase from Bacillus sp. BT3-2 and Bacillus macerans. The presence of 15% ethanol in the reaction mixture with the CGTase from Bacillus sp. BT3-2 resulted in a 30% increase of the amounts of CD₁₀–CD₁₃ synthesized after 5 h of reaction. The addition of 20% ethanol increased the yield of CD₁₄–CD₂₁ up to 1000%. The hydrolysis of the large-ring CD by the CGTases was suppressed in the presence of ethanol. The ring-opening coupling cyclization reactions of the CGTase were effected differently by the organic solvent which may contribute to the observed increase of the yield and size of the CD obtained in the synthesis reactions.     

Lipase-catalyzed production of short-chain acids terpenyl esters of interest to the food industry

The production of low molecular weight esters as flavor compounds by biotechnological processes has a potential interest for the food industry. The use of natural available substrates and enzymes is an essential part of the process design, because the products may obtain natural label. In this study, direct esterification of citronellol and geraniol with short-chain fatty acids catalyzed by free lipase from Mucor miehei was performed with high yields in n-hexane. The effects of the acid:alcohol ratio on the bioconversion rate of increasing chain length esters was investigated. To reach the optimum yield, substrates and enzyme concentration were determined. The inhibiting effects of acid are strongly attenuated by reducing the quantity of acid and increasing the amount of enzyme in media following the optimum values. Improvements have been made to increase the ester purity. The consumption of excess substrate by adding calculated amounts of acid gives a 10% yield enhancement, and leads to 100% pure terpenyl esters. The first steps to a scale-up application were attempted using a reactor that allowed us to produce ester quantities up to 100 cm³. Separation and purification of the products were treated with success, underlining the lipase stability and efficiency under the conditions of this study. The ability to recover the enzyme, and reusing it in bioconversions, plays a major role in reducing the cost of the overall process.     

Methods and Supports for Immobilization and Stabilization of Cyclomaltodextrin Glucanotransferase from Thermoanaerobacter

Thermoanaerobacter cyclomaltodextrin glucanotransferase (CGTase) was immobilized using different supports and immobilization methods to study the effect on activity recovery. The enzyme covalently attached into glyoxyl-silica showed low activity recovery of 1.5%. The hydrophobic adsorption of the enzyme on Octadecyl-Sepabeads yielded also low activity recovery, 3.83%, and the enzyme could easily leak from the support at low ionic strength, although the immobilization yield was satisfactory, approximately 76%. The CGTase encapsulated in a sol–gel matrix gave an activity recovery of 6.94% and maximum cyclization activity at 60 °C, at pH 6.0. The half-time life at 60 °C, pH 6.0, in the presence of substrate was 100 min, which was lower than that of the free enzyme. The best activity recovery in this work (6.94%) is approximately five times smaller than that obtained previously using glyoxyl-agarose as support and covalent immobilization. Thus, the best support and method we tested so far for immobilization of CGTase is covalent attachment on glyoxyl-agarose.     

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.     

Production of insoluble exopolysaccharide of Agrobacterium sp. (ATCC 31749 and IFO 13140)

Agrobacterium isolated from soil samples produced two extracellular polysaccharides: succinoglycan, an acidic soluble polymer, and curdlan gum, a neutral, insoluble polymer. Maize glucose, cassava glucose, and maize maltose were used in fermentation medium to produce insoluble polysaccharide. Two Agrobacterium sp. strains which were used (ATCC 31749 and IFO 13140) in the production of insoluble exopolysaccharide presented equal or superior yields compared to the literature. The strain ATCC 31749 yielded better production when using maize maltose, whose yield was 85%, whereas strain IFO 13140 produced more when fed maize glucose, producing a yield of 50% (on reducing sugars).     

Continuous production of extracellular l-glutaminase by ca-alginate-immobilized marine Beauveria bassiana BTMF S-10 in packed-bed reactor

l-Glutamine amidohydrolase (l-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on l-glutaminase. In this article, we report the continuous production of extracellular l-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized in the order-support (Na-alginate) concentration, concentration of CaCl₂ for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production medium, temperature of incubation, and retention time. Parameters optimized under batch mode for l-glutaminase production were incorporated into the continuous production studies. Beads with 12×10⁸ spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous production of l-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mL·h), which indicates that continuous production of the enzyme by Ca-alginate-immobilized spores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of l-glutaminase.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.     

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.     

Evaluation of Cashew Apple Juice for the Production of Fuel Ethanol

A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentrations. Maximal ethanol, cell, and glycerol concentrations were obtained when 103.1 g L⁻¹ of initial sugar concentration was used. Cell yield (Y _X/S) was calculated as 0.24 (g microorganism)/(g glucose + fructose) using cashew apple juice medium with 41.3 g L⁻¹ of initial sugar concentration. Glucose was exhausted first, followed by fructose. Furthermore, the initial concentration of sugars did not influence ethanol selectivity. These results indicate that cashew apple juice is a suitable substrate for yeast growth and ethanol production.