Enzymatic synthesis of capric acid-rich structured lipids and their effects on mice with high-fat diet-induced obesity.

The objective of this study was to produce structured lipids (SLs) by enzymatic acidolysis using Rhizopus oryzae lipase covalently immobilized in a low-cost material. Grape seed oil was used to synthesize SLs containing the medium-chain fatty acid (C10:0) capric acid. SL synthesis led to 38.8% medium-chain fatty acid incorporation with 5 reuses of the enzymatic derivative. The reaction conditions for the synthesis of MLM-TAGs (triacylglycerols with one long- and two medium-chain acyl residues) were at a molar ratio of fatty acid:oil of 3:1, performed at 40 °C and lipase immobilized load of 5% (w/w). The in vivo effects of SLs were studied in Swiss mice fed premade diets: control (C) diet, high-fat diet (HFD) with 100% lipid content as lard, HFD with 50% lipid content as grape seed oil (HG) or HFD with 50% lipid content as capric acid-containing SLs produced from grape seed oil (HG-MCT). Mice from HG and HG-MCT groups had decreases in body weight gain and reductions in the weights of white adipose tissues. In addition, HG and HG-MCT mice had low plasma levels of glucose and total cholesterol, and improvements in the glucose tolerance. HG and HG-MCT diets have remarkable antioxidant properties, since low plasma levels of TBARS (thiobarbituric acid reactive substances, biomarkers of lipid peroxidation) were found in mice fed these diets. Interestingly, TBARS levels in HG-MCT mice were further decreased than values of HG mice. Mice fed HG and HG-MCT diets also showed preservation in the activity of the antioxidant enzyme paraoxonase 1. Both HG and HG-MCT diets promoted reduction of IL-6 and IL-10 production by splenocytes. The capric acid-containing SLs produced from grape seed oil emerges as a functional oil capable to mitigate obesity complications resulting from oxidative stress and inflammation.

Extraction and Characterization of Hemicellulose from Eucalyptus By-product: Assessment of Enzymatic Hydrolysis to Produce Xylooligosaccharides.

Eucalyptus wood is the primary source of fibers to produce paper and cellulose in South American countries. The major by-product generated in the cellulose industry is sawdust derived from chip wood production, which is designated as Eucalyptus by-product (EB). The xylooligosaccharides (XOS) are xylose-based oligomers with proven effects over maintenance and stimulation of beneficial human gut bacteria. This study reported the EB extraction and characterization along with an assessment of hemicellulose hydrolysis using commercial xylanases to produce XOS. Hemicellulose derived from extracted and NaClO2 pretreated (HEEBPT) presented xylan content of 55%, which was similar to 58.5% found in commercial Birchwood hemicellulose (CBH). The enzymatic hydrolysis of HEEBPT and CBH presented 30% as maximum conversion of xylan into XOS without significant difference among the enzymatic extracts evaluated. The XOS production from EB was proven as a technically feasible alternative to recover a value-added product from hemicellulosic fraction generated in the cellulose industry. However, lignin removal with NaClO2 from EB affects the feasibility of an industrial process because they generate toxic compounds in the pretreatment step. Thus, further studies with alternative reagents, such as ionic liquids, are required to asses selectively lignin removal from EB. Graphical Abstract.

Chemical analysis and biorefinery of red algae Kappaphycus alvarezii for efficient production of glucose from residue of carrageenan extraction process.

BACKGROUND: Biorefineries serve to efficiently utilize biomass and their by-products. Algal biorefineries are designed to generate bioproducts for commercial use. Due to the high carbohydrate content of algal biomass, biorefinery to generate biofuels, such as bioethanol, is of great interest. Carrageenan is a predominant polysaccharide hydrocolloid found in red macroalgae and is widely used in food, cosmetics, and pharmaceuticals. In this study, we report the biorefinery of carrageenan derived from processing of experimental strains of the red macroalgae Kappaphycus alvarezii. Specifically, the chemical composition and enzymatic hydrolysis of the residue produced from carrageenan extraction were evaluated to determine the conditions for efficient generation of carbohydrate bioproducts. RESULTS: The productivity and growth rates of K. alvarezii strains were assessed along with the chemical composition (total carbohydrates, ash, sulfate groups, proteins, insoluble aromatics, galacturonic acid, and lipids) of each strain. Two strains, brown and red, were selected based on their high growth rates and productivity and were treated with 6 % KOH for extraction of carrageenan. The yields of biomass from treatment with 6 % KOH solution of the brown and red strains were 89.3 and 89.5 %, respectively. The yields of carrageenan and its residue were 63.5 and 23 %, respectively, for the brown strain and 60 and 27.8 %, respectively, for the red strain. The residues from the brown and red strains were assessed to detect any potential bioproducts. The galactan, ash, protein, insoluble aromatics, and sulfate groups of the residue were reduced to comparable extents for the two strains. However, KOH treatment did not reduce the content of glucan in the residue from either strain. Glucose was produced by enzymatic hydrolysis for 72 h using both strains. The glucan conversion was 100 % for both strains, and the concentrations of glucose from the brown and red strains were 13.7 and 11.5 g L(-1), respectively. The present results highlight the efficiency of generating a key bioproduct from carrageenan residue. CONCLUSIONS: This study demonstrates the potential for glucose production using carrageenan residue. Thus, the biorefinery of K. alvarezii can be exploited not only to produce carrageenan, but also to generate glucose for future use in biofuel production.

Immobilization of Trypsin in Lignocellulosic Waste Material to Produce Peptides with Bioactive Potential from Whey Protein.

In this study, trypsin (Enzyme Comission 3.4.21.4) was immobilized in a low cost, lignocellulosic support (corn cob powder-CCP) with the goal of obtaining peptides with bioactive potential from cheese whey. The pretreated support was activated with glyoxyl groups, glutaraldehyde and IDA-glyoxyl. The immobilization yields of the derivatives were higher than 83%, and the retention of catalytic activity was higher than 74%. The trypsin-glyoxyl-CCP derivative was thermally stable at 65 °C, a value that was 1090-fold higher than that obtained with the free enzyme. The trypsin-IDA-glyoxyl-CCP and trypsin-glutaraldehyde-CCP derivatives had thermal stabilities that were 883- and five-fold higher, respectively, then those obtained with the free enzyme. In the batch experiments, trypsin-IDA-glyoxyl-CCP retained 91% of its activity and had a degree of hydrolysis of 12.49%, while the values for trypsin-glyoxyl-CCP were 87% and 15.46%, respectively. The stabilized derivative trypsin-glyoxyl-CCP was also tested in an upflow packed-bed reactor. The hydrodynamic characterization of this reactor was a plug flow pattern, and the kinetics of this system provided a relative activity of 3.04 ± 0.01 U·g-1 and an average degree of hydrolysis of 23%, which were suitable for the production of potentially bioactive peptides.

Buffalo Cheese Whey Proteins, Identification of a 24 kDa Protein and Characterization of Their Hydrolysates: In Vitro Gastrointestinal Digestion.

Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.

Descoloração de azul de bromofenol utilizando peroxidase imobilizada em pó de sabugo de milho altamente ativado / Bromofhenol blue discoloration using peroxidase immobilized on highly activated corncob powder

The aim of the present study was to evaluate the efficacy of peroxidase immobilized on corncob powder for the discoloration of dye. Peroxidase was extracted from soybean seed coat, followed by amination of the surface of the tertiary structure. The aminated peroxidase was immobilized on highly activated corncob powder and employed for the discoloration of bromophenol blue. Amination was performed with 10 or 50 mmol.L-1 carbodiimide and 1 mol.L-1 ethylenediamine. The amount of protein in the extract was 0.235 ± 0.011 mg.mL-1 and specific peroxidase activity was 86.06 ± 1.52 μmol min-1. mg -1 , using 1 mmol.L-1 ABTS as substrate. Ten mmol.L-1 and 50 mmol.L-1 aminated peroxidase retained 88 and 100% of the initial activity. Following covalent immobilization on a corncob powder-glyoxyl support, 10 and 50 mmol.L-1 aminated peroxidase retained 74 and 86% of activity, respectively. Derivatives were used for the discoloration of 0.02 mmol.L-1 bromophenol blue solution. After 30 min, 93 and 89% discoloration was achieved with the 10 mmol.L-1 and 50 mmol.L-1 derivatives, respectively. Moreover, these derivatives retained 60% of the catalytic properties when used three times. Peroxidase extracted from soybean seed coat immobilized on a low-cost corncob powder support exhibited improved thermal stability.

Nesta pesquisa a enzima peroxidase foi extraída do tegumento de sementes de soja, e a superfície da estrutura terciária foi aminada. A peroxidase aminada foi imobilizada em suporte pó de sabugo de milho altamente ativado e utilizado na descoloração de azul de bromofenol. A aminação da peroxidase foi realizada com carbodiimida em concentrações de 10 e 50 mmol.L-1 , e 1 mol.L-1 de etilenodiamina. A quantidade de proteínas no extrato foi de 0,235 ± 0,011 mg.mL-1 , e a atividade específica da peroxidase foi 86,06 ± 1,52 μmol min-1 .mg-1, usando 1 mmol.L-1 de ABTS como substrato. A peroxidase aminada a 10 mmol.L-1 reteve 88% e a aminada a 50 mmol.L-1 reteve 100% da atividade inicial. As peroxidases aminadas a 10 ou 50 mmol.L-1 foram covalentemente imobilizadas em suporte glioxil-pó de sabugo de milho com atividade recuperada de 74% e 86%, respectivamente. Os derivados obtidos foram utilizados na descoloração de solução de azul de bromofenol 0,02 mmol.L-1 . Após 30 min 93% de descoloração foram alcançados com o derivado glioxil-pó de sabugo de milho com a peroxidase aminada 10 mmol.L-1 e 89% com a aminada 50 mmol.L-1 . Estes derivados mantiveram 60% das propriedades catalíticas, quando utilizado por três vezes. A peroxidase extraída do tegumento da semente de soja imobilizada em suporte de baixo custo pó de sabugo de milho apresentou melhoria na estabilidade térmica da enzima.

Produção de glicose e frutose por invertase de Saccharomyces cerevisiae imobilizada em suporte MANAE-Agarose / Glucose and Fructose Production by Saccharomyces cerevisiae Invertase Immobilized on MANAE-Agarose Support

Invertase from Saccharomyces cerevisiae was immobilized on agarose beads, activated with various groups (glyoxyl, MANAE or glutaraldehyde), and on some commercial epoxy supports (Eupergit and Sepabeads). Very active and stable invertase derivatives were produced by the adsorption of the enzyme on MANAE-agarose, MANAE-agarose treated with glutaraldhyde and glutaraldehyde-agarose supports. At pH 5.0, these derivatives retained full activity after 24h at 40 ºC and 50 ºC. When assayed at 40 °C and 50 °C, with the pH adjusted to 7.0, the invertase-MANAE-agarose derivative treated with glutaraldehyde retained 80% of the initial activity. Recovered activities of the derivatives produced with MANAE, MANAE treated with glutaraldehyde and glutaraldehyde alone were 73.5%, 44.4% and 36.8%, respectively. These three preparations were successfully employed to produce glucose and fructose in 3 cycles of sucrose hydrolysis.

Invertase de Saccharomyces cerevisiae foi imobilizada em agarose ativada com diferentes grupos (glioxil, MANAE ou glutaraldeído) e suportes epóxidos comerciais (Eupergit e Sepabeads). Derivados de invertase ativos e estabilizados foram produzidos pela adsorção da enzima em suportes MANAE-agarose, MANAE-agarose tratado com glutaraldeído e glutaraldeído-agarose. Em pH 5,0 estes derivados retiveram total atividade até 24h a 40 ºC e 50 ºC. Quando os ensaios foram a 40 °C e 50 °C com o pH alterado para 7,0, o derivado invertase-MANAE-agarose tratado com glutaraldeído apresentou 80% da atividade inicial. As atividades recuperadas dos derivados foram 73,5%, 44,4% e 36,8%, respectivamente para MANAE, MANAE tratado com glutaraldeído e glutaraldeído. Essas três preparações foram empregadas com sucesso em 3 ciclos de hidrólise da sacarose para produzir glicose e frutose.

Fontes de carbono de baixo custo para a produção de xilanase termoestável por Aspergillus niger / Low-cost carbon sources for the production of a thermostable xylanase by Aspergillus niger

A strain of the filamentous fungus Aspergillus niger was isolated and shown to possess extracellular xylanolytic activity. These enzymes have biotechnological potential and can be employed in various industries. This fungus produced its highest xylanase activity in a medium made up of 0.1% CaCO3, 0.5% NaCl, 0.1% NH4Cl, 0.5% corn steep liquor and 1% carbon source, at pH 8.0. A low-cost hemicellulose residue (powdered corncob) proved to be an excellent inducer of the A. niger xylanolytic complex. Filtration of the crude culture medium with suspended kaolin was ideal for to clarify the extract and led to partial purification of the xylanolytic activity. The apparent molecular mass of the xylanase was about 32.3 kDa. Maximum enzyme activity occurred at pH 5.0 and 55-60ºC. Apparent Km was 10.41 ± 0.282 mg/mL and Vmax was 3.32 ± 0.053 U/mg protein, with birchwood xylan as the substrate. Activation energy was 4.55 kcal/mol and half-life of the crude enzyme at 60ºC was 30 minutes. Addition of 2% glucose to the culture medium supplemented with xylan repressed xylanase production, but in the presence of xylose the enzyme production was not affected.

Uma linhagem do fungo filamentoso Aspergillus niger foi isolada e apresentou atividade xilanolítica extracelular. Estas enzimas possuem grande potencial biotecnológico e podem ser aplicadas em diversas indústrias. O fungo produziu sua maior atividade de xilanase em um meio contendo CaCO3 0,1%, NaCl 0,5%, NH4Cl 0,1%, 0,5% água de maceração de milho e 1% de fonte de carbono, em pH 8,0. Um resíduo lignocelulósico de baixo custo (sabugo de milho em pó) mostrou ser um excelente indutor do complexo xilanolítico em A. niger. A filtração do extrato cru com caulim foi ideal para a clarificação do extrato e levou à purificação parcial da enzima. A massa molecular aparente da xilanase foi de 32,3 kDa. A máxima atividade da enzima ocorreu em pH 5,0 e a 55-60ºC. O Km aparente foi de 10,41 ± 0,282 mg/mL e a Vmax foi de 3,32 ± 0,053 U/mg proteína, utilizando-se xilana birchwood como substrato. A energia de ativação foi de 4,55 kcal/mol, e a meia-vida da enzima a 60ºC foi de 30 minutos. A adição de 2% de glicose ao meio de cultura suplementado com xilana reprimiu a produção de xilanase, mas em presença de xilose a produção da enzima não foi afetada.

Protein hydrolysis by immobilized and stabilized trypsin.

The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pK(a) amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pK(a) amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins.

Effects of culture conditions on the production of inulinase by Kluyveromyces marxianus

The present study was conducted to investigate the influence of initial sucrose concentration, pH and aeration rate on biomass and inulinase production by Kluyveromyces marxianus var. bulgaricus in a stirred batch reactor. Maximum inulinase activity (15.29 UmL-1) was obtained at a sucrose concentration of 10 g L-1, pH 5.0 and aeration rate of 1 vvm. The 20 g L-1 sucrose concentration was suitable for cell growth; however, enzymatic activity at this concentration was inhibited due to catabolic repression. The increase in aeration rate caused a reduction in enzyme activity with no relevant biomass increase.

O estudo foi conduzido para investigar a influência da concentração inicial da sacarose, a taxa da aeração e do pH na biomassa e na produção da inulinase pela Kluyveromyces marxianus var. bulgaricus em um reator em batelada. A máxima atividade de inulinase, 15.29 UmL-1, foi obtida na concentração de 10 g L-1 de sacarose, no pH 5.0 e na taxa da aeração de 1 vvm. A concentração de sacarose de 20g L-1 foi apropriada para o crescimento celular, porém nesta concentração a atividade enzimática foi inibida, devido a repressão catabólica. O aumento na taxa da aeração propiciou redução da atividade enzimática, ao mesmo tempo em que não houve aumento considerável do biomassa.

Cyclodextrin glycosyltransferase production by new Bacillus sp. strains isolated from brazilian soil / Produção de ciclodextrina glicosiltransferase por novas cepas de Bacillus sp. isoladas de solo brasileiro

Three strains of Bacillus sp. (BACRP, BACNC-1 and BACAR) were isolated from soil adhered to cassava husk. CGTase specific activity for the three isolated strains was higher when cultivated at 40¨¬C. Potato starch, cassava starch, maltodextrin and glucose were used as carbon source and growth temperatures varied from 25 to 55¨¬C. The three isolates presented higher CGTase specific activity when cultivated with potato starch at 40¨¬C. Isolated BACRP and BACAR presented specific activity of 4.0x10-3 and 2.2x10-3 U/mg prot at pH 7.0, respectively, when cultivated in mediums added with NaCl 2 percent; at pH 10,0 their activities were of 3.4x10-3 and 3.0x10-3 U/mg prot, respectively, in the same concentration of NaCl. On the other hand, the isolated BACNC-1 presented activity specific of 2.4x10-3 U/mg prot when cultivated at pH 7.0 added of NaCl 1 percent, and at pH 10.0 the specific activity was of 3.4x10-3 U/mg prot without NaCl addition. This work also showed the presence of cyclodextrins formed during fermentation process and that precipitation with acetone or lyophilization followed by dialysis was efficient at removing CDs (cyclodextrins), thus, eliminating interference in the activity assays. The enzyme produced by the BACAR strain was partially purified and ¥â-CD was liberated as a reaction product.

Três linhagens de Bacillus sp (BACRP, BACNC- 1 e BACAR) foram isoladas a partir de solo aderido em casca de mandioca. Foram utilizados amido de batata, amido de mandioca, maltodextrina e glicose como fonte de carbono, e temperaturas de crescimento de 25-55¨¬C, sendo que os três isolados apresentaram maior atividade específica de CGTase quando cultivados com amido de batata a 40¨¬C. Em pH 7,0 os isolados BACRP e BACAR apresentaram atividade específica de 4,0x 10-3 e 2,2x10-3 U/mg prot, respectivamente, quando cultivados em meios acrescidos de 2 por cento de NaCl; em pH 10,0 suas atividades foram de 3,4x10-3 e 3,0x10-3 U/mg prot na mesma concentração de NaCl. Por outro lado, o isolado de BACNC-1 apresentou atividade específica 2,4x10-3 U/mg prot quando cultivado em pH 7,0 acrescido de 1 por cento de NaCl, e em pH 10,0 sua atividade específica foi de 3,4x10-3 U/mg prot sem adição de NaCl. Também foi demonstrada neste trabalho que ciclodextrinas são formadas durante o processo fermentativo, e que a precipitação com acetona ou liofilização seguida de diálise foram eficientes na remoção destas CDs, eliminando sua interferência nos ensaios enzimáticos. A enzima produzida pela cepa BACAR foi purificada parcialmente liberando b-CD como produto da reação.

Studies on productivity and characterization of polygalacturonase from Aspergillus giganteus submerged culture using citrus pectin and orange waste.

Polygalacturonases are part of the group of enzymes involved in pectin degradation. The aim of this work was to investigate some of the factors affecting polygalacturonase production by an Aspergillus giganteus strain and to characterize this pectinolytic activity. Several carbon sources, both pure substances and natural substrates, were tested in standing cultures, and the best results were obtained with orange bagasse and purified citrus pectin. On citrus pectin as sole carbon source, the highest extracellular activity (9.5 U/ml and 40.6 U/mg protein) was obtained in 4.5-day-old cultures shaken at 120 rpm, pH 3.5 and 30 degrees C, while on orange bagasse, the highest extracellular activity (48.5 U/ml and 78.3 U/mg protein) was obtained in 3.5-day-old cultures shaken at 120 rpm, pH 6.0 and 30 degrees C. Optimal polygalacturonase activity was observed in assays conducted at pH 5.5-6.5 and 55-60 degrees C. The activity showed good thermal stability, with half-lives of 90 and 30 min when incubated at 55 and 60 degrees C, respectively. High stability was observed from pH 4.5 to 8.5; more than 90% of the activity remained after 24 h in this pH range.

Cyclodextrin glycosyltransferase production by new Bacillus sp. strains isolated from brazilian soil.

Three strains of Bacillus sp. (BACRP, BACNC-1 and BACAR) were isolated from soil adhered to cassava husk. CGTase specific activity for the three isolated strains was higher when cultivated at 40°C. Potato starch, cassava starch, maltodextrin and glucose were used as carbon source and growth temperatures varied from 25 to 55°C. The three isolates presented higher CGTase specific activity when cultivated with potato starch at 40°C. Isolated BACRP and BACAR presented specific activity of 4.0×10(-3) and 2.2×10(-3) U/mg prot at pH 7.0, respectively, when cultivated in mediums added with NaCl 2%; at pH 10,0 their activities were of 3.4×10(-3) and 3.0×10(-3) U/mg prot, respectively, in the same concentration of NaCl. On the other hand, the isolated BACNC-1 presented activity specific of 2.4×10(-3) U/mg prot when cultivated at pH 7.0 added of NaCl 1%, and at pH 10.0 the specific activity was of 3.4×10(-3) U/mg prot without NaCl addition. This work also showed the presence of cyclodextrins formed during fermentation process and that precipitation with acetone or lyophilization followed by dialysis was efficient at removing CDs (cyclodextrins), thus, eliminating interference in the activity assays. The enzyme produced by the BACAR strain was partially purified and ß-CD was liberated as a reaction product.

Cyclodextrin glycosyltransferase from Bacillus licheniformis: optimization of production and its properties

Cyclodextrin glycosyltransferase (EC 2.4.1.19) is an enzyme that produces cyclodextrins from starch via an intramolecular transglycosylation reaction. An alkalophilic Bacillus strain, isolated from cassava peels, was identified as Bacillus licheniformis. CGTase production by this strain was better when potato starch was used as carbon source, followed by cassava starch and amylopectin. Glucose and amylose, on the other hand, acted as synthesis repressors. When the cultivation was supplemented with sodium ions and had the pH adjusted between 6.0 and 9.0, the microorganism maintained the growth and enzyme production capacity. This data is interesting because it contradicts the concept that alkalophilic microorganisms do not grow in this pH range. After ultrafiltration-centrifugation, one protein of 85.2 kDa with CGTase activity was isolated. This protein was identified in plates with starch and phenolphthalein. Determination of the optimum temperature showed higher activities at 25°C and 55°C, indicating the possible presence of more than one CGTase in the culture filtrate. Km and Vmax values were 1.77 mg/mL and 0.0263 U/mg protein, respectively, using potato starch as substrate.

Ciclodextrina glicosiltransferase (EC 2.4.1.19) é uma enzima que produz ciclodextrinas a partir de amido via transglicosilação intramolecular. Uma cepa de Bacillus alcalofílico, isolada de cascas de mandioca, foi identificada como Bacillus licheniformis. A produção de CGTase por esta cepa foi melhor quando amido de batata foi utilizado como fonte de carbono, seguido por amido de mandioca e amilopectina. Glicose e amilose, por outro lado, atuaram como repressor de síntese desta enzima. Quando o cultivo foi suplementado com íons sódio e teve o pH ajustado entre 6,0 e 9,0, o microrganismo manteve a capacidade de crescimento e de produção da enzima. Este dado é interessante pois contraria o conceito de que microrganismos alcalofílicos não apresentam crescimento nesta faixa de pH. Após ultrafiltração-centrifugação, uma proteína de 85,2 kDa com atividade de CGTase foi isolada. Esta proteína foi identificada em placas contendo amido e fenolftaleína. A determinação da temperatura ótima mostrou atividades mais elevadas em 25°C e 55°C, indicando a possível presença de mais de uma CGTase no filtrado de cultura. Valores de Km e Vmax foram 1,77 mg/mL e 0,0263 U/mg proteína, respectivamente, usando amido de batata como substrato.

Microbial rennet produced by Mucor miehei in solid-state and submerged fermentation

A produção de renina microbiana por Mucor miehei foi estudada através de Fermentação Submersa e em Estado Sólido. O objetivo deste trabalho foi verificar o efeito de diferentes fontes de carbono e nitrogênio, utilizando Fermentação Submersa e da adição de caseína utilizando Fermentação em Estado Sólido na produção da renina microbiana. Os picos de biomassa foram de 6,7; 8,1 e 8 g/L e atividade enzimática de 1.066; 857 e 480 Unidades Soxhlet (U.S.) para as concentrações de glicose: 18, 25 e 35 g/L respectivamente. Em frascos aletados, os picos de biomassa foram de 6,7; 8,3 e 10 g/L e atividade enzimática de 648, 279 e 300 U.S., para a mesma concentração de glicose. Quando se utilizou Proflo (Farinha de semente de algodão, Traders ®) e Água de Maceração de Milho os picos de atividade enzimática foram de 667 e 923 U.S., respectivamente. Nos experimentos utilizando Fermentação em Estado Sólido com a adição de HCl 0,2 N a máxima atividade enzimática foi de 414 U.S. e, quando utilizou-se caseína (1 e 2 gramas), verificou-se valores mais altos de atividade: 966 e 1117 U.S respectivamente. Os resultados sugerem que o aumento na concentração de glicose afeta a síntese da enzima e que a caseína é um importante fator na indução neste processo. Fermentação em Estado Sólido pode ser considerada uma boa opção para a produção de renina.

Partial purification and properties of cellulase-free alkaline xylanase produced by Rhizopus stolonifer in solid-state fermentation

Rhizopus stolonifer foi cultivado em meio de farelo de trigo para produzir uma xilanase alcalina celulase-free. Uma amostra parcialmente purificada desta enzima foi obtida após cromatografia de exclusão molecular em Sephacryl S–200 HR. A temperatura ótima de hidrólise determinada (45º C) está dentro do intervalo citado na literatura (45º C a 60º C) para xilanases microbianas. Quanto ao pH ótimo, a amostra obtida apresentou atividades máximas em pH 6,0 e 9,0. Estes dados diferem da literatura, uma vez que o pH ótimo citado para a maioria das xilanases estudadas varia entre 4,0 e 5,5. De acordo com os estudos cinéticos realizados, a xilanase apresentou maior Vmax em pH 9,0 (0,87 µmol/mg proteína) e menor Km em pH 6,0 (7,42 mg/mL). Os dois pHs ótimos determinados podem indicar a presença de isoformas desta enzima. Estes dados são interessantes pelo fato de que enzimas xilanolíticas alcalinas celulase-free podem ser utilizadas para o biobranqueamento da polpa na indústria de papel.

Isolation of natural inhibitors of papain obtained from Carica papaya latex

Este trabalho apresenta novos dados sobre inibidores naturais de papaína. O látex fresco de frutos verdes de Carica papaya foi coletado pela manhã em plantações da região de Araraquara, SP, Brasil e imediatamente transportado ao laboratório em banho de gelo. Três frações com efeito inibitório da atividade esterásica da papaína foram isoladas a partir do látex fresco, através de diálise, filtração em Sephadex G-25 e cromatografia em SP-Sephadex C-25. As frações isoladas identificadas como inibidores I e II, mostraram reação negativa à ninidrina; entretanto, a fração identificada como P-III mostrou reação positiva. Dados cinéticos revelaram inibição não-competitiva (inibidor I) e incompetitiva (inibidores II e P-III).

Production of CGTase by a Bacillus alkalophilic CGII strain isolated from wastewater of a manioc flour industry

Estudou-se a produção de CGTase por uma nova cepa de Bacillus alkalophilic CGII, isolada de água residuária de uma fecularia de mandioca, durante cultivo em meio composto de 1,5% de amido, 1,5% de fonte de nitrogênio e 1% Na2CO3. A atividade enzimática foi alta quando se utilizou amido, maltodextrina e galactose como fontes de carbono. Quando se utilizou glicose no meio de cultivo não se observou produção da enzima. Atividade enzimática alta (88,6 U/mL) e melhor crescimento foram obtidos quando se aumentou a aeração. A caracterização da enzima mostrou um pH ótimo de 8,0 e temperatura ótima de 55ºC sendo que a enzima sofreu uma pequena ativação por Mg+ e Ca++. A enzima foi fortemente inibida por Hg+ e Cu+.

Immobilization of lactase from Kluyveromyces lactis greatly reduces the inhibition promoted by glucose. full hydrolysis of lactose in milk.

The kinetic constants (Km, Vmax, and inhibition constants for the different products) of soluble and different immobilized preparations of beta-galactosidase from Kluyveromyces lactis were determined. For the soluble enzyme, the Km was 3.6 mM, while the competitive inhibition constant by galactose was 45 mM and the noncompetitive one by glucose was 758 mM. The immobilized preparations conserved similar values of Km and competitive inhibition, but in some instances much higher values for the noncompetitive inhibition constants were obtained. Thus, when glyoxyl or glutaraldehyde supports were used to immobilize the enzyme, the noncompetitive inhibition was greatly reduced (Ki approximately 15,000 and >40,000 mM, respectively), whereas when using sugar chains to immobilize the enzyme the behavior had an effect very similar to the soluble enzyme. These results presented a great practical relevance. While using the soluble enzyme or the enzyme immobilized via the sugar chain as biocatalysts in the hydrolysis of lactose in milk only around 90% of the substrate was hydrolyzed, by using of these the enzyme immobilized via the glyoxyl or the glutaraldehyde groups, more than 99% of the lactose in milk was hydrolyzed.

Production and purification of an Endoter1.4-beta-Xylanase from Humicola grisea var. thermoidea by electroelution

Humicola grisea var. thermoidea produces two forms of extracellular xylanase. The component form 1 was purified using the electroelution method, due to the very small production of this extracellular enzyme. The apparent molecular mass was 61.8 kDa by SDS-PAGE.