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.     

Cyclodextrins are likely to induce cyclodextrin glycosyltransferase production inBacillus macerans

Cyclodextrin glycosyltransferase (CGTase) activity was monitored inBacillus macerans culture fluids up to 56 h incubation time using glucose (G₁), maltose (G₂), maltotriose (G₃), maltoheptaose (G₇), α-,β-,γ-cyclodextrins (CDs) and soluble starch as carbon sources. Highest maximum specific growth rates (μ_max) were observed with glucose, γ-CD and soluble starch (μ_max values were 0.86, 0.74 and 0.69/h, respectively) while the maximum viable cell numbers were always within the range of 2.3–7.1×10¹¹ CFU/mL independently of the carbon source used. Highest CGTase production was found in the presence of soluble starch and G₇ (55.0 and 35.4 nkat/mL, respectively), these saccharides being easily transformed to CDs by CGTase. Moreover, when culture media were supplemented with cyclic malto-oligosaccharides the CGTase activities were about twice higher (19.6–20.6 nkat/mL) than those obtained with the linear G₂ and G₃ saccharides (8.9 and 11.3 nkat/mL, respectively) which give rise only to negligible quantities of CDs. CDs, which are the major end products of the action of CGTase, are regarded thus as the likely physiological inducers of the enzyme.     

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 biochemical characterization of a novel SDS and surfactant stable,raw starch digesting,and halophilic α-amylase from a moderately halophilic bacterium,Nesterenkonia sp. strain F

An extracellular halophilic α-amylase from Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100 kDa and 106 kDa by SDS–PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 °C. The amylase was active in a wide range of salt concentrations (0–4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺ strongly inhibited the enzyme, whereas Ca²⁺ stimulated the amylase activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. K_m value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an α-amylase activity.     

Study of the formation and properties of the amylolytic system ofCandida japonica

In the logarithmic phase of growth,Candida japonica produced into media containing soluble starch, maltose or glucose, an amylase hydrolysing starch to glucose, oligosaccharides of the maltose series and dextrins which coloured red with iodine. The presence of calcium carbonate in the medium promoted growth of the culture, but markedly lowered the amylolytic activity of the culture fluid. Paper electrophoretic separation, ionex chromatography on DEAE cellulose and study of substrate specificity and the relationship of activity to the temperature and pH of the medium showed only one enzyme in the crude enzyme preparation. This was a dextrinogenic amylase of the α-amylase type, with greater capacity for breaking down lower homologues of substances of the amylose series, including maltose, than other known α-amylases. The optimum temperature was 55°C, with 20 minutes’ incubation, and the optimal pH was between 5 and 6. The reaction rate of hydrolytic reactions rose with the length of the chain of the substrate. Isomaltotetraose was hydrolysed about 20 times more slowly than maltotetraose. No formation of isomaltose or panose transglucosidation products was found.     

Transformation of maltose into prebiotic isomaltooligosaccharides by a novel α-glucosidase from Xantophyllomyces dendrorhous

The transglycosylation activity of a novel α-glucosidase from the basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) was studied using maltose as glucosyl donor. The enzyme synthesized oligosaccharides with α-(1 → 2), α-(1 → 4) and α-(1 → 6) bonds. Using 200 g/l maltose, the yield of oligosaccharides was 53.8 g/l, with prebiotic oligosaccharides containing at least one α-(1 → 6) linkage (panose, 6-O-α-glucosyl-maltotriose and 6-O-α-isomaltosyl-maltose) being the major products (47.1 g/l). The transglycosylatying yield was 3.6 times higher than the observed with the α-glucosidase from Saccharomyces cerevisiae (53.8 vs. 14.7 g/l). Moreover, when increasing the maltose concentration up to 525 g/l, the maximum production of tri- and tetrasaccharides reached 167.1 g/l, without altering the percentage of oligosaccharides in the mixture. Compared with other microbial α-glucosidases in which the main transglycosylation product is a disaccharide, the enzyme from X. dendrorhous yields a final product enriched in trisaccharides and tetrasaccharides.     

The effect of cyclodextrins on the ethanol tolerance of microorganisms suggests potential application

Cyclodextrins (CDs) are used in food, pharmaceutical, and chemical industries, as well as agriculture and environmental engineering. Cyclodextrin glucanotransferase (CGTase) is an important industrial extracellular enzyme which is used to produce CDs and oligosaccharides. We previously developed a novel yeast-surface CGTase expression system which was used for the production of CDs from starch. In the present study, we showed that the presence of CDs may increase the ethanol tolerance of microorganisms. The cell numbers of Saccharomyces cerevisiae and Escherichia coli in the presence of β-cyclodextrin and ethanol were 1,000-fold and 10-fold higher than that without CDs. The yeast strain with the immobilized CGTase produced 13 g CDs/l and 1.8 g ethanol/l when it was incubated in yeast medium supplemented with 4% starch. The effect of CDs on microorganisms suggests a potential application for the co-production of CDs and ethanol.     

Starch degradation by the mould Trichoderma viride II. Regulation of enzyme synthesis

The synthesis of amylolytic enzymes by the maltose not-utilizing Trichoderma viride strain CBS 354.44 requires the presence of starch or dextrins. Several readily utilizable carbon sources such as glucose and glutamic acid were shown to exert a strong catabolite repression which completely inhibited enzyme induction by starch or dextrins.Enzyme synthesis occurs in the exponential and in the stationary growth phase. In the latter, the ratio between saccharifying and dextrinizing enzyme activity is invariably high. In the exponential growth phase this ratio depends on the nature of the inducing substrate. Growth on starch results in an initially high production of dextrinizing activity, the saccharifying one becoming predominant in the course of exponential growth. The latter activity in dextrin DE 30 cultures is predominant from the very beginning. Thus, the amylolytic enzyme system of T. viride consists of at least two different enzymes, the synthesis of each being controlled specifically. The careful regulation of the synthesis of the dextrinizing enzyme is discussed with special reference to the production of non-utilizable maltose by the latter.     

A novel thermophilic anaerobic bacteria producing cyclodextrin glycosyltransferase

A novel thermophilic anaerobic, rod-shaped, non-spore forming, gram positive bacterium was isolated from an oil field in Turkey, that produces cyclodextrin glycosyltransferase (CGTase) from starch. According to the some morphological, biochemical and 16S rRNA analysis, the strain belongs to the genus Thermoanaerobacter. The strain mainly utilizes starch and derivatives, glucose and fructose as carbon source between 45 and 75 °C, optimally at 65 °C. Optimum pH for growth is 7.5. 16S RNA studies indicate that the bacterium has a similarity of 98.3% to homoacetogenic Thermoanaerobacter kivui and the main fermentation product is acetic acid as in the case with homoacetogenic bacteria. The main difference between the bacterium and T. kivui concerns the utilization of starch. Based on the phylogenetic and biochemical analysis, it is proposed that the species are a new member of the genus Thermoanaerobacter. The strain has CGTase activity optimum at 80 °C and pH 7.0–8.0.     

Production of chitosan oligosaccharides by chitosanase directly immobilized on an agar gel-coated multidisk impeller

An immobilized enzyme bioreactor consisting of an agar gel-coated multidisk impeller was developed for the hydrolysis of highly viscous chitosan solutions, and the operating conditions for the production of physiologically active chitosan oligosaccharides (pentamers and hexamers) were investigated. Chitosanase was directly immobilized on the agar gel-coated multidisk impeller by a multipoint attachment method. The high stability of the immobilized enzyme was confirmed by means of five repetitions of a batch hydrolysis reaction. When the enzyme activity at the support surface was relatively high, the yield of the target products was higher at an impeller speed of 2 s⁻¹ than at a speed of 1 s⁻¹. However, no significant increase in yield was observed at impeller speeds higher than 2 s⁻¹ in reactions at either of the two substrate concentrations tested (5 and 20 kg/m³). When the surface enzyme activity was low, the impeller speed did not affect the yield of the target products. The maximum yield of pentamers and hexamers increased as the surface enzyme activity decreased, and high yields (>30%) were obtained at activities below 160 U/m². From the viewpoint of productivity, the optimal surface-enzyme activity was about 340 U/m², and at that activity, the yield of target products was 22%. This yield was higher than that reported for conventional acid hydrolysis. To maximize both the productivity and the yield of the target products, the surface area for the immobilized enzyme should be increased. Our results suggest that it may be possible to obtain high yields of pentamers and hexamers of chitosan oligosaccharides from highly viscous chitosan solutions with this reactor.     

The effect of glucose and maltose concentrations on pyruvate and ascorbate production,antioxidant enzyme activities and LPO levels in Fusarium equiseti

Changes in pyruvate and ascorbate production and antioxidant enzyme activities together with lipid peroxidation levels in Fusarium equiseti were investigated in relation to changes in the concentrations of glucose and maltose as carbon sources in the range of 5–25 g/l in Armstrong Fusarium Medium (AFM). The highest pyruvate concentration obtained at 20 g/l maltose was 67.5 ± 0.69 μg/ml while ascorbic acid reached a maximum value at 25 g/l glucose of 1866±26.1 μg/ml The maximum superoxide dismutas (SOD) activities related to increased pyruvate production were determined in AFM medium containing 20 g/l glucose as 41.49±0.65 and maltose as 61.12±0.8 IU/mg. Catalase (CAT) activity variations showed coherence with SOD activity in a medium containing maltose and reached 219.11±2.8 IU/mg while they were decreased with increasing glucose concentration. glutathione peroxidase (GSH-Px) activities in F. equiseti did not change significantly with glucose and maltose concentration and were determined to be 1.21±0.22 and 1.67±0.15 IU/mg, respectively. Minimum lipid peroxidation levels for each carbon source were determined in both 20 g/l maltose and glucose concentrations as 0.9 and 1.62 nmol MDA/g wet weight.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Regioselective synthesis of neo-erlose by the β-fructofuranosidase from Xanthophyllomyces dendrorhous

The β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous (Xd-INV) catalyzes the synthesis of neo-fructooligosaccharides (neo-FOS of the ⁶G-series), which contain a β(2 → 6) linkage between a fructose and the glucosyl moiety of sucrose. In this work we demonstrate that the enzyme is also able to fructosylate other carbohydrates that contain glucose, in particular disaccharides (maltose, isomaltulose, isomaltose, trehalose) and higher oligosaccharides (maltotriose, raffinose, maltotetraose), but not monosaccharides (glucose, fructose, galactose). With maltose as acceptor, the reaction in the presence of Xd-INV proceeded with high regioselectivity; the product was purified and chemically characterized, and turned out to be 6′-O-β-fructosylmaltose (neo-erlose). Using 100 g/L sucrose as fructosyl donor and 300 g/L maltose as acceptor, the maximum concentration of neo-erlose was 38.3 g/L. Thus, novel hetero-fructooligosaccharides with potential applications in the functional food and pharmaceutical industries can be obtained with Xd-INV.     

Production of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. TS1-1: Optimization of carbon and nitrogen concentration in the feed medium using central composite design

Optimisation of nutrient feeding was developed to overcome the limitation in batch fermentation and to increase the CGTase production from Bacillus sp. TS1-1 in fed batch fermentation. Optimisation of the C/N ratio in the feed stream was conducted in a 5 l fermenter, where feeding was initiated at constant rate of 0.02 h⁻¹. In our initial screening process, the addition of nitrogen source boosted the growth of the microbes, but on the other hand reduced the CGTase production. The amount of tapioca starch and yeast extract was optimised in order to obtain a sufficient growth and thus, increased the CGTase production. Results were analysed using three-dimensional response surface plot, and the optimised values of carbon and nitrogen concentration of 3.30% (w/v) and 0.13% (w/v) were obtained, respectively. CGTase activity increased up to 80.12 U/ml, which is 13.94% higher as compared to batch fermentation (70.32 U/ml). This also led to 14.54% increment of CGTase production in fed batch culture as compared to the production before the optimisation. The CGTase activity obtained was close to the predicted value, which is 78.05 U/ml.     

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.     

Selective and stable production of physiologically active chitosan oligosaccharides using an enzymatic membrane bioreactor

We investigated the production of chitosan oligosaccharides by continuous hydrolysis of chitosan in an enzyme membrane bioreactor, with the goal of improving the yield of physiologically active oligosaccharides (pentamers and hexamers) and achieving operational stability. The bioreactor was a continuous-flow stirred-tank reactor equipped with an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, and the hydrolysis was accomplished with chitosanase from Bacillus pumilus. After optimization of the reaction parameters, such as the amount of enzyme, the yield of the target oligosaccharides produced in the membrane bioreactor with free chitosanase reached 52% on the basis of the fed concentration of chitosan. An immobilized chitosanase prepared by the multipoint attachment method was used to improve the operational stability of the membrane bioreactor. Under the optimized conditions, pentameric and hexameric chitosan oligosaccharides were steadily produced at 2.3 g/L (46% yield) for a month. The half-life of the productivity of the reactor was estimated to be 50 d under the conditions examined.     

Cyclodextrin glucanotransferase synthesis by semicontinuous cultivation of magnetic biocatalysts from cells of Bacillus circulans ATCC 21783

Cells of the alkalotolerant producer of cyclodextrin glucanotransferase (CGTase) Bacillus circulans ATCC 21783 were used as a model for preparing of magnetic biocatalysts applied for CGTase synthesis in batch and semicontinuous processes. The cell immobilization was carried out with four types of magnetic nano- and microparticles: magnetite microparticles (1–5 μm), entrapped in agar gel beads with bacterial cells (AM-biocatalyst); silanized magnetite (20–40 nm) covalently bound on the cell surface (SM-biocatalyst); and alkaline and citrate ferrofluids (10–20 nm), attached on the cell wall by an ionic interaction (FF-alkaline and FF-citrate biocatalyst). The highest CGTase production was achieved after 96 h of semicontinuous process using SM-biocatalysts (particularly, these composed of 80 mg silanized magnetite and 140 mg bacterial cells) when the specific enzyme activity was 8.4-fold higher compared to that of free cells. Cells modified with magnetic alkaline and citrate ferrofluids exhibited 2.19- and 1.55-fold increase of the specific CGTase activities. Magnetic nanoparticles linked on the cell walls by ionic interactions were partially released during the cultivation, while the covalent bond between the activated magnetite and the cells was very stable. The data obtained demonstrate convincingly the effect of the magnetic technologies for an effective enzyme production.     

Purification and characterization of a thermostable raw starch digesting amylase from a Streptomyces sp. isolated in a milling factory

A raw starch utilizing microbe was isolated from mud in a milling factory. The 16S ribosomal DNA (rDNA) sequencing and morphological properties of the strain indicated that it belongs to the genus Streptomyces. A strongly raw starch digesting amylase was purified from the culture supernatant of the strain by chromatographic procedures. The specific activity of the enzyme was 11.7 U/mg, molecular mass 47 kDa, optimum pH 6.0, and optimum temperature 50 to 60 degrees C. The enzyme showed sufficient activity even at 70 degrees C. It was activated by calcium, cobaltous, and magnesium ions, and inhibited by copper, nickel, zinc, and ferrous ions. It formed maltose mainly from raw and gelatinized starch, and glycogen. No products were formed from glucose, maltose, maltotriose, pullulan, or cyclodextrins (CDs). The enzyme digested raw wheat, rice, and waxy rice starch rapidly, and raw corn, waxy corn, sweet potato, tapioca, and potato starch normally.     

Constitutive expression of a novel isoamylase from Bacillus lentus in Pichia pastoris for starch processing

Isoamylase is essential to saccharifying starch by cleavage of 1,6-glucoside linkages in starch molecules. In this study, a novel isoamylase gene from Bacillus lentus JNU3 was cloned. The open reading frame of the gene was 2412 base pairs long and encoded a polypeptide of 804 amino acids with a calculated molecular mass of 90 kDa. The deduced amino acid sequence shared less than 40% homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. A constitutive GAP promoter was used to express the recombinant isoamylase in the yeast Pichia pastoris by continuous high cell-density fermentation to avoid the use of methanol, which resulted in 318 U/mL extracellular isoamylase activity after 72 h in a 10 L fermenter. The recombinant enzyme was purified and characterized. It had an estimated molecular mass of 90 kDa, with its optimal activity at 70 °C, pH 6.5 and was quite stable between 30 °C and 70 °C. The recombinant isoamylase proves to be superior to pullulanase as an auxiliary enzyme in maltose production from starch. Therefore it will contribute significantly to the starch debranching process.     

Purification and characterization of cyclodextrinase from Paenibacillus sp. A11

Paenibacillus sp. A11 produced an intracellular cyclodextrinase (CDase), its presence was confirmed by activity detection on an agar plate with specific screening medium containing β-cyclodextrin (β-CD) and phenolphthalein. The CDase was purified up to 22-fold with a 28% yield. The enzyme was a single polypeptide with a molecular weight of 80 kDa. Optimum activity was at pH 7.0 and 40 °C. The enzyme had an isoelectric point of 5.4 and N-terminal sequence was M F L E A V Y H R P R K N W S. When relative hydrolytic activities of the CDase on different substrates were compared, it was found that high specificity was exerted by β-CD while maltoheptaose, its linear counterpart, was only 40% as active. The enzyme recognized α-1,4-glucose units and the hydrolysis depended on the size of oligosaccharides. Highly branched carbohydrates such as glycogen or dextran or other heteropolymers as glucomannan could not be hydrolyzed. This enzyme was different from other CDases in its ability to hydrolyze maltose and trehalose, though with very low hydrolytic activity. The major product from all substrates was maltose. The k_cat/K_m value for β-CD was 8.28 × 10⁵ M⁻¹min⁻¹. The enzyme activity was completely inactivated by 1 mM N-bromosuccinimide and diethylpyrocarbonate suggesting the crucial importance of Trp and His for its catalytic activity. Essential Trp was confirmed to be at enzyme active site by substrate protection experiment. Partial inactivation by 5 mM phenylglyoxal suggests the involvement of Arg, which has never been reported in other CDases.