Cloning, Purification, and Characterization of a Heat- and Alkaline-Stable Endoglucanase B from <em>Aspergillus niger</em> BCRC31494

Endoglucanase B (EGLB) derived from Aspergillus niger BCRC31494 has been used in the food fermentation industry because of its thermal and alkaline tolerance. It was cloned and expressed in Pichia pastoris. According to sequence analysis, the gene open reading frame comprises 1,217 bp with five introns (GenBank GQ292753). According to sequence and protein domain analyses, EGLB was assigned to glycosyl hydrolase family 5 of the cellulase superfamily. Several binding sites were found in the promoter region. The purified recombinant enzyme was induced by 0.5% methanol, and it exhibited optimal activity at 70 °C and pH 4. EGLB was stable for 3 h at temperatures below 60 °C, with more than 90% of its activity remaining. The enzyme was specific for substrates with β-1,3 and β-1,4 linkages. In Lineweaver-Burk plot analysis, the K_m and V_max values of EGLB for β-D-glucan were 134 mg/mL and 4.68 U/min/mg, respectively. The enzyme activity was increased by 1.86-fold by Co²⁺ and by 2-fold by Triton X-100 and Tween 80. These favorable properties make EGLB a potential candidate for use in laundry and textile industrial applications.     

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.     

Production of Cyclodextrins from Cornstarch Granules in a Sequential Batch Mode and in the Presence of Ethanol

The influence of Toruzyme? cyclomaltodextrin glucanotransferase concentration and the presence of ethanol have been studied for the production of α-, β-, and γ-cyclodextrins (CDs) from 15% (w/v) cornstarch, at 65 °C and pH 6, with the aim of increasing CD yield. The selected concentrations for a single batch reactor were 10% (v/v) ethanol and 0.1% (v/v) enzyme, yielding after 12 h, 37% total CDs, of which 52.2% was α-CD, 38.8% β-CD, and 9.0% γ-CD. The enzyme specific activities per unit mass of protein for producing α-, β-, and γ-CD were 37.25, 19.61, and 8.63 U mg(-1), respectively. Total CD yield per milliliter of enzyme was 55 g. To increase CD yield per enzyme charge and thus reduce costs, the production of CDs was tested with two sequential batches in which a single enzyme charge was used. At the end of the first batch, the enzyme was adsorbed either on 65 °C pretreated starch granules or on raw starch, and a second batch was run with this material. The best result, in this case, was obtained for pretreated starch, increasing total CD produced by 57.4%, with 53.2% α-CD, 36.1% β-CD, and 10.7% γ-CD. CD yield per milliliter of enzyme was then 87 g.     

Evaluation of supports and methods for immobilization of enzyme cyclodextringlycosyltransferase

An experimental design with factorial planning was used for the immobilization of the enzyme cyclodextringlycosyltransferase (CGTase) from Bacillus firmus (strain no.37) to select the best combination of support, method of immobilization, and conditions that gives primarily higher average values for the specific immobilized enzyme activity, and secondarily, higher average values for the percentage of protein fixation. The experimental design factors were as follows: supports—controlled-pore silica, chitosan, and alumina; immobilization methods—adsorption, and two covalent bonding methods, either with γ-aminopropyltriethoxysilane or hexamethylenediamine (HEMDA); conditions—7°C without agitation and 26°C with stirring. The best combination of factors that lead to higher average values of the response variables was obtained with immobilization of CGTase in silica with HEMDA at 7°C. However, immobilization in chitosan at 7°C gave the highest immobilized CGTase specific activity, 0.25 μmole of β-CD/(min·mg protein). Physical adsorption gave low specific enzyme activities, and, in general, a high load of enzyme leads to lower specific enzyme activity.     

Statistical Optimization of Alpha-Amylase Production with Immobilized Cells of <Emphasis Type="Italic">Streptomyces erumpens</Emphasis> MTCC 7317 in <Emphasis Type="Italic">Luffa cylindrica</Emphasis> L. Sponge Discs

The purpose of this investigation was to study the effect of Streptomyces erumpens cells immobilized in various matrices, i.e., agar–agar, polyacrylamide, and luffa (Luffa cylindrica L.) sponge for production of α-amylase. Luffa sponge was found to be 21% and 51% more effective in enzyme yield than agar–agar and polyacrylamide, respectively. Response surface methodology was used to evaluate the effect of three main variables, i.e., incubation period, pH, and temperature on enzyme production with immobilized luffa cells. The experimental results showed that the optimum incubation period, pH, and temperature were 36h, 6.0, and 50 °C, respectively. The repeated batch fermentation of immobilized cells in shake flasks showed that S. erumpens cells were more or less equally physiologically active on the support even after three cycles of fermentation (3,830–3,575 units). The application of S. erumpens crude enzyme in liquefying cassava starch was studied. The maximum hydrolysis of cassava starch (85%) was obtained with the application of 4ml (15,200 units) of crude enzyme after 5 h of incubation.     

Assessment of the complexation degree of camptothecin derivatives and cyclodextrins using spectroscopic and separative methodologies

The complexation of camptothecin and homocamptothecin derivatives, topoisomerase I inhibitors, with two cyclodextrins (CDs) of pharmaceutical interest (native and hydroxypropylated β-CD) was studied at pH 3.5 and 6. In a first step, the affinity order of the six compounds studied for the β-CD and HP-β-CD was evaluated in HPLC using immobilized stationary phases [Cyclobond I 2000 (β-CD) and Cyclobond I 2000 RSP (HP-β-CD)]. In a second step, the apparent binding constants of the 12 complexes studied were determined at both pH by HPLC using Scott’s method with CD as a chiral additive. The 1:1 stoichiometry of the complex formed between HP-β-CD and the homocamptothecin derivative elomotecan (R)-6 was established by fluorescence spectroscopy using the continuous variation method developed by Job and ESI-MS. Complementary investigations were achieved for topotecan (S)-3 and elomotecan (R)-6 using CE. Further studies provided similar conclusions concerning affinity of all the derivatives studied for both CDs: that is, a slightly larger affinity was observed for HP-β-CD with respect to β-CD, except for (S)-3. For (S)-3, this affinity increase with pH, in the range studied.     

Bioethanol Production from Uncooked Raw Starch by Immobilized Surface-engineered Yeast Cells

Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.     

Chitosan sponge matrices with β-cyclodextrin for berberine loading

Biocompatible polymer sponge materials based on biodegradable natural polymer chitosan, which can be loaded with clinician-selected drugs are still in the centre of interest for their wide use in clinical practice. This study shows possibilities of the technology which combines simple addition of β-cyclodextrin (β-CD), with dialdehyde starch (DAS) as a cross-linking agent of chitosan, to chitosan solutions for subsequent formation of sponge matrix. The advantage of such system is in avoiding chemical modifications and working only with natural substances. It is shown that, in matrix formation during lyophilisation, β-CD molecules tend to accumulate on the surface of the porous matrix structure. This was confirmed by a study of the known inclusion complex of β-CD and salicylic acid (SA) in heptane. The same study was applied to berberine (BER) which can also form an inclusion complex with β-CD in a water solution. Moreover, adsorption of drugs on the surface of the porous structure has to be also taken into account. This enables the production of sponge topical medical preparations useful for sustained release of BER.     

Preparation of Dry Extract of<em> Mikania glomerata</em> Sprengel (Guaco) and Determination of Its Coumarin Levels by Spectrophotometry and HPLC-UV

Guaco (Mikania glomerata Sprengel) syrup is one of the most popular herbal medicines used to treat the symptoms of asthmatic bronchitis, cough and hoarseness. The coumarin 2H-1-benzopyran-2-one, is one of the major constituents of Guaco and contributes to its pharmacological effects. The pharmaceutical capsule form of dry extract of Guaco is recommended by the Brazilian Program of Medicinal Plants and Herbal Medicines and used in primary health care. In order to identify a new protocol to obtain the raw material for Guaco capsule production we evaluated two methods, including a freeze-drying process (lyophilization) and the spray-dryer technique, as well as the use of two adjuvants, Maltodextrins and Aerosil^?, in different concentrations. The coumarin levels of the dried extracts were analyzed by UV-spectrophotometry and HPLC-UV/DAD. The adjuvant Aerosil^? 8% showed better dry powder physical appearance. Lyophilization was observed to be the best process to obtain the dry extract of Guaco based on the measured coumarin levels.     

Influences of <em>Dryopteris crassirhizoma</em> Extract on<em> </em>the Viability, Growth and Virulence Properties of <em>Streptococcus </em><em>m</em><em>utans</em><em></em>

Dryopteris crassirhizoma is traditionally used as an herbal remedy for various diseases, and has been identified in a previous study as a potential anti-caries agent. In this study, the effect of a methanol extract of D. crassirhizoma on the viability, growth and virulence properties of Streptococcus mutans, a cariogenic dental pathogen, was investigated. In addition, the phytochemical composition of the extract was analyzed. The extract showed bactericidal and bacteriostatic activity against oral bacteria (MIC and MBC of S. mutans: 62.5 and 250 μg/mL, respectively). At two times the MBC, the extract significantly eliminated S. mutans up to 99.9% after 1 h incubation. The extract also dose-dependently reduced growth rates of S. mutans at sub-MIC levels. Furthermore, at sub-MIC levels, virulence properties (acid production, acid tolerance, glucosyltransferase activity and sucrose-dependent adherence) of S. mutans were also inhibited in a dose-dependent manner. GC-MS analysis revealed the presence of mono and disaccharides (44.9%), fatty acids (12.3%) and sugar alcohols (6.8%) in the extract. These data indicate that the extract might be useful for the control of dental caries.     

Transformation of geniposide into genipin by immobilized β-glucosidase in a two-phase aqueous-organic system

Genipin is the bioactive compound of geniposide and a natural cross-linking agent. In order to improve the preparation process of genipin, the hydrolysis of geniposide to genipin by immobilized β-glucosidase in an aqueous-organic two-phase system was studied. β-glucosidase was immobilized by the crosslinking-embedding method using sodium alginate as the carrier. The optimum reaction temperature, pH value and time were 55 °C, 4.5 and 2.5 h, respectively. To reduce genipin hydrolysis and byproduct production the reaction was carried out in an aqueous-organic two-phase system comprising ethyl acetate and sodium acetate buffer. The product was analyzed by HPLC, UV, IR, and NMR. The yield of genipin was 47.81% and its purity was over 98% (HPLC).     

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.     

Antibacterial modification of cellulose fibers by grafting β-cyclodextrin and inclusion with ciprofloxacin

Antibacterial-modified cellulose fiber was prepared by covalently bonding β-cyclodextrin (β-CD) with cellulose fiber via citric acid (CA) as crosslinking agent, followed by the inclusion of ciprofloxacin hydrochloride (CipHCl) as antibiotic. Effects of reaction time, temperature, concentration of β-cyclodextrin citrate (CA-β-CD) and pH on the grafting reaction were investigated, and the grafting ratio of β-CD onto cellulose fibers was 9.7 % at optimal conditions; the loading and releasing behaviors of CipHCl into/from β-CD grafted cellulose fibers were also revealed, the load amount of CipHCl into grafted cellulose fibers increased remarkably, and the release of CipHCl from the grafted cellulose fibers was prolonged. The microstructure, phase and thermal stability of modified cellulose fibers were characterized by FT-IR, ¹³C CPMAS NMR, X-ray diffraction and TGA. Considerably longer bacterial activity against E. coli and S. aureus was observed for grafted fibers loading CipHCl compared to virgin ones. Optical and mechanical properties of the paper sheets decreased generally with more antibacterial-modified fibers added.     

<em>In Vitro </em>and <em>in </em><em>Vivo</em> Antitumor Effect of Trachylobane-360, a Diterpene from<em> Xylopia langsdorffiana</em>

Trachylobane-360 (ent-7α-acetoxytrachyloban-18-oic acid) was isolated from Xylopia langsdorffiana. Studies have shown that it has weak cytotoxic activity against tumor and non-tumor cells. This study investigated the in vitro and in vivo antitumor effects of trachylobane-360, as well as its cytotoxicity in mouse erythrocytes. In order to evaluate the in vivo toxicological aspects related to trachylobane-360 administration, hematological, biochemical and histopathological analyses of the treated animals were performed. The compound exhibited a concentration-dependent effect in inducing hemolysis with HC₅₀ of 273.6 μM, and a moderate in vitro concentration-dependent inhibitory effect on the proliferation of sarcoma 180 cells with IC₅₀ values of 150.8 μM and 150.4 μM, evaluated by the trypan blue exclusion test and MTT reduction assay, respectively. The in vivo inhibition rates of sarcoma 180 tumor development were 45.60, 71.99 and 80.06% at doses of 12.5 and 25 mg/kg of trachylobane-360 and 25 mg/kg of 5-FU, respectively. Biochemical parameters were not altered. Leukopenia was observed after 5-FU treatment, but this effect was not seen with trachylobane-360 treatment. The histopathological analysis of liver and kidney showed that both organs were mildly affected by trachylobane-360 treatment. Trachylobane-360 showed no immunosuppressive effect. In conclusion, these data reinforce the anticancer potential of this natural diterpene.     

Cellulase Production by Aspergillus japonicus URM5620 Using Waste from Castor Bean (Ricinus communis L.) Under Solid-State Fermentation

The activity of β-glucosidase (βG), total cellulase (FPase) and endoglucanase (CMCase), produced by Aspergillus japonicus URM5620, was studied on solid-state fermentation using castor bean meal as substrate. The effect of the substrate amount, initial moisture, pH, and temperature on cellulase production was studied using a full factorial design (2(4)). The maximum βG, FPase, and CMCase activity was 88.3, 953.4, and 191.6 U/g dry substrate, respectively. The best enzyme activities for all three enzymes were obtained at the same conditions with 5.0 g of substrate, initial moisture 15% at 25 °C and pH 6.0 with 120 h of fermentation. The optimum activity for FPase and CMCase was found at pH 3.0 at an optimum temperature of 50 °C for FPase and of 55 °C for CMCase. The cellulases were stable in the range of pH 3.0-10.0 at 50 °C temperature. The enzyme production optimization demonstrated clearly the impact of the process parameters on the yield of the cellulolytic enzymes.     

Optimized Conditions of Enantioseparation of β-Blockers by CZE Using Carboxymethyl-β-Cyclodextrin as Chiral Selector

Capillary zone electrophoresis was used for the enantiomeric separation of six β-blocking drug substances with β-cyclodextrin (β-CD) and its derivatives as chiral selectors employing an uncoated capillary. The effects of pH value and composition of the background electrolyte (BGE), the capillary temperature and running voltage have been investigated. The results showed that β-CD type, concentration and pH value have a strong influence on the efficiency of the chiral separation. Carboxymethyl-β-cyclodextrin (CM-β-CD) gave a baseline enantiomeric separation for six β-blocking drug substances under optimal conditions, whereas the β-CD, hydroxypropyl-β-cyclodextrin (HP-β-CD) showed no chiral recognition. The potential and capillary temperature did not have a great effect on enantiomer resolution.     

Study on the complexation of isoquercitrin with β-cyclodextrin and its derivatives by spectroscopy

The inclusion complexes of isoquercitrin (IQ) with cyclodextrins (CDs) including β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD) and dimethyl-β-cyclodextrin (DM-β-CD) have been investigated using the methods of steady-state fluorescence, UV-vis absorption and induced circular dichroism. The stoichiometric ratio of the three complexes was found to be 1:1 and the stability constants (K) were estimated from spectrofluorometric titrations, as well as the thermodynamic parameters. Maximum inclusion ability was measured in the case of DM-β-CD due to the increased hydrophobicity of the host cavity, followed by HP-β-CD and β-CD. The effect of pH on the complexation process was also quantitatively assessed. IQ exists in different molecular forms depending on pH and β-CDs were most suitable for inclusion of the neutral form of IQ. The phase-solubility diagrams obtained with β-CD, HP-β-CD and DM-β-CD were all classical A_L type. And DM-β-CD provided the best solubility enhancement, 12.3-fold increase compared to 2.8- and 7.5-fold increase for β-CD and HP-β-CD. The apparent stability constants obtained from the solubility data at 25 °C were comparable with those obtained from the fluorescence assays. Moreover, ¹H NMR was carried out, which revealed that the IQ favorably inserted into the inner cavity from the chromone part instead of the phenyl part, which was in agreement with molecular modeling studies.     

Enantioselective resolution of chiral aromatic acids by biphasic recognition chiral extraction

This paper reports a new chiral separation technology—biphasic recognition chiral extraction for the separation of aromatic acid enantiomers such as α-cyclohexyl-mandelic acid (CHMA) and naproxen (NAP). The biphasic recognition chiral extraction system was established by adding hydrophobic d(l)-isobutyl tartrate in 1,2-dichloroethane organic phase and hydrophilic β-cyclodextrin (β-CD) derivative in aqueous phase, which preferentially recognize the (R)-enantiomer and (S)-enantiomer, respectively. These studies involve an enantioselective extraction in a biphasic system, where aromatic acid enantiomers form complexes with the β-cyclodextrin derivative in the aqueous phase and d(l)-isobutyl tartrate in the organic phase, respectively. Factors affecting the extraction mechanism are analyzed, namely the influence of the concentrations of the extractants and aromatic acid enantiomers, the types of the extractants, pH, and temperature. The experimental results show that the biphasic recognition chiral extraction is of much stronger chiral separation ability than the monophasic recognition chiral extraction, which utilizes the cooperations of the forces of the tartrate and the β-CD derivative. Hydroxypropyl-β-cyclodextrin (HP-β-CD), hydroxyethyl-β-cyclodextrin (HE-β-CD), and methyl-β-cyclodextrin (ME-β-CD) have stronger recognition abilities for the (S)-aromatic acid enantiomers than those for (R)-aromatic acid enantiomers, among which HP-β-CD has the strongest ability. d-Isobutyl tartrate preferentially recognizes (R)-CHMA and (S)-NAP, while l-isobutyl tartrate preferentially recognizes (S)-CHMA and (R)-NAP. The maximum enantioselectivities of CHMA and NAP are 2.49 and 1.65, under conditions at which the pH values of the aqueous phases are 2.7 and 2.5, at the ratio of 2:1 of [isobutyl tartrate] to [HP-β-CD].     

Cytotoxic Podophyllotoxin Type-Lignans from the Steam Bark of <em>Bursera fagaroides </em>var. <em>fagaroides</em>

The hydroalcoholic extract of the steam bark of B. fagaroides var. fagaroides displayed potent cytotoxic activity against four cancer cell lines, namely KB (ED₅₀ = 9.6 × 10^-2 μg/mL), PC-3 (ED₅₀ = 2.5 × 10^-1 μg/mL), MCF-7 (ED₅₀ = 6.6 μg/mL), and HF-6 (ED₅₀ = 7.1 × 10^-3 μg/mL). This extract also showed anti-tumour activity when assayed on mice inoculated with L5178Y lymphoma cells. Bioactivity-directed isolation of this extract, afforded seven podophyllotoxin-type lignans identified as podophyllotoxin (1), β-peltatin-A-methylether (2), 5'-desmethoxy-β-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7) by 1D and 2DNMR and FAB-MS analyses, and comparison with reported values. All the isolated compounds showed potent cytotoxic activity in the cell lines tested, especially compound 3, which exhibited greater activity than camptothecin and podophyllotoxin against PC-3 (ED₅₀ = 1.0 × 10^-5 μg/mL), and KB (ED₅₀ = 1.0 × 10^-5 μg/mL). This is the first report of the isolation of podophyllotoxin and its acetate in a Bursera species.