Abstract This study reports the use of statistical mixture design as a tool for the simultaneous production of lipase, CMCase, α-amylase, and β-glucosidase by Aspergillus niger under solid-state fermentation. Wheat bran, soybean meal, cottonseed meal, and orange peel were used as substrates, either individually or combined in different formulations, to study their synergistic or antagonistic effects on production of the enzymes. The highest lipase (323 U g-1) and CMCase (10 U g-1) activities were detected after 48 h, while the maximum activities of α-amylase (18 U g-1) and β-glucosidase (15 U g-1) occurred at 72 and 96 h, respectively. Considering the substrate formulation, the ternary mixture of wheat bran (1/3), soybean meal (1/3), and cottonseed meal (1/3) was the most versatile, showing production of CMCase (>5 U g-1) and α-amylase (>8 U g-1) at 24 h, lipase (>320 U g-1) at 72 h, and β-glucosidase (>10 U g-1) at 48 h.

ABSTRACT Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296 bp and G + C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria.

O presente trabalho avaliou os efeitos da embalagem sob atmosfera modificada na inibição do desenvolvimento dos sintomas de danos causados pelo frio, em pêssego 'Douradão' após armazenamento refrigerado, e o possível envolvimento das enzimas da parede celular. Frutos foram colhidos no estádio médio de maturação, acondicionados em bandejas de polipropileno e colocados dentro de sacos de polietileno de baixa densidade (PEBD) com espessuras de 30; 50; 60 e 75 µm e com modificação ativa da atmosfera (10 kPa CO2 + 1,5 kPa O2, balanço de N2). Os seguintes tratamentos foram testados: Controle: pêssegos mantidos em bandejas sem filme plástico; AM30: filme de PEBD - 30 µm; AM50: filme de PEBD - 50 µm; AM60: filme de PEBD - 60 µm; AM75: filme de PEBD - 75 µm. Os frutos foram mantidos em câmara a 1±1ºC e 90±5% de umidade relativa (UR) por 28 dias. Após 14; 21 e 28 dias, as embalagens plásticas foram retiradas, e as amostras foram mantidas em ar a 25±1ºC e 90±5% UR para amadurecimento. No dia da remoção e após 4 dias, os pêssegos foram avaliados quanto à incidência de lanosidade, atividades das enzimas pectinolíticas. A taxa respiratória e a produção de etileno foram monitoradas durante 6 dias de amadurecimento. Os resultados mostraram que os tratamentos AM50 e AM60 tiveram efeito positivo na inibição do desenvolvimento de textura lanosa e reduziram a atividade da pectina metilesterase nos frutos maduros, mantendo a boa qualidade do pêssego 'Douradão' durante 28 dias de estocagem refrigerada. Os tratamentos Controle, AM30 e AM75 apresentaram maior incidência de lanosidade e não apresentaram condições de comercialização após 14 dias de armazenamento refrigerado.

The present study evaluated the effects of modified atmosphere packaging on inhibition of the development of chilling injury symptoms in 'Douradão' peach after cold storage and the possible involvement of cell wall enzymes. Fruits were harvested at the middle stadium of ripening, packed in polypropylene trays and placed inside low density polyethylene (LDPE) bags (30, 50, 60 and 75 µm of thickness) with active modified atmosphere (10 kPa CO2 + 1.5 kPa O2, balance N2). The following treatments were tested: Control: peaches held in nonwrapped trays; MA30: LDPE film - 30 µm; MA50: LDPE film - 50 µm; MA60: LDPE film - 60 µm and MA75: LDPE film - 75 µm. Fruits were kept at 1±1ºC and 90±5% relative humidity (RH) for 28 days. After 14, 21 and 28 days, samples were withdrawn from MAP and kept in air at 25±1ºC and 90±5% RH for ripening. On the day of removal and after 4 days, peaches were evaluated for woolliness incidence, pectolytic enzymes activities. The respiratory rate and ethylene synthesis were monitored during 6 days of ripening. The results showed that MA50 and MA60 treatments had positive effect on the inhibition of the development of woolly texture and reduced pectin methylesterase activity on the ripe fruits, keeping good quality of 'Douradão' peach during 28 days of cold storage. The treatments Control, MA30 and MA75 showed higher woolliness incidence and did not present marketable conditions after 14 days of cold storage.

O amido é encontrado na cana-de-açúcar como um polissacarídeo de reserva. A quantidade depende de diversos fatores. Este trabalho visou a determinação do conteúdo de amido em diferentes variedades de cana-de-açúcar durante o período de Maio a Novembro de 2007; e determinação de algumas características do amido de cana-de-açúcar como: estrutura e tamanho dos grânulos de amido; determinação da temperatura de gelificação; determinação da filtrabilidade da solução de amido de cana-de-açúcar; susceptibilidade do amido de cana-de-açúcar às enzimas glicoamilase, pululanase, -amilases bacterianas e fúngica comerciais. Foi testado também a susceptibilidade do amido de cana-de-açúcar à enzima amilolítica desramificante isoamilase de Flavobacterium sp. O amido de cana-de-açúcar apresentou forma esférica e diâmetro entre 1-3 µm. O amido de cana-de-açúcar complexado com iodo apresentou maior absorção na faixa de 540 a 620 nm. O amido de cana-de-açúcar in natura mostrou maior susceptibilidade à enzima glicoamilase em relação aos outros amidos testados. O amido de cana-de-açúcar mostrou susceptibilidade à enzima amilolítica desramificante pululanase de modo similar ao amido de arroz ceroso. O amido de cana-de-açúcar mostrou susceptibilidade às α-amilases de Bacillus subtilis, Bacillus licheniformis e Aspergillus oryzae de modo similar aos outros amidos testados produzindo glicose; maltose; maltotriose; maltotetraose e α- dextrinas limite.

Starch is found in sugarcane as a storage polysaccharide. Starch concentrations vary widely depending on the country, variety, developmental stage, and growth conditions. The purpose of this study was to determine the starch content in different varieties of sugarcane, between May and November 2007, and some characteristics of sugarcane starch such as structure and granules size; gelatinization temperature; starch solution filterability; and susceptibility to glucoamylase, pullulanase, and commercial bacterial and fungal α-amylase enzymes. Susceptibility to debranching amylolytic isoamylase enzyme from Flavobacterium sp. was also tested. Sugarcane starch had spherical shape with a diameter of 1-3 µm. Sugarcane starch formed complexes with iodine, which showed greater absorption in the range of 540 to 620 nm. Sugarcane starch showed higher susceptibility to glucoamylase compared to that of waxy maize, cassava, and potato starch. Sugarcane starch also showed susceptibility to debranching amylolytic pullulanases similar to that of waxy rice starch. It also showed susceptibility to α-amylase from Bacillus subtilis, Bacillus licheniformis, and Aspergillus oryzae similar to that of the other tested starches producing glucose, maltose, maltotriose, maltotetraose, maltopentose and limit α- dextrin.

Nas últimas décadas, houve um incremento nos estudos da obtenção da isomaltulose, devido às suas propriedades físico-químicas e fisiológicas benéficas à saúde. Propriedades que incluem a baixa cariogenicidade, baixo índice glicêmico e maior estabilidade, propiciam o uso deste adoçante como substituinte da sacarose em alimentos, além de poder ser convertido para isomalte, um açúcar álcool dietético e não cariogênico aplicado na indústria de alimentos e farmacêutica. A isomaltulose (6-O-α-D-glucopyronosyl-1-6-D-fructofuranose) é um dissacarídeo redutor obtido através da conversão enzimática da sacarose, pela enzima α-glicosiltransferase. Foram realizados diferentes tratamentos para a preparação de células íntegras, células lisadas e extrato enzimático bruto da linhagem Erwinia sp. D12 imobilizados em alginato de cálcio. A coluna de leito empacotado de grânulos contendo células de Erwinia sp. sonicadas e imobilizadas em alginato de cálcio (CSI) atingiu máxima conversão de 53-59% de sacarose em isomaltulose e apresentou atividade por 480 horas. O xarope convertido foi purificado e a cristalização da isomaltulose foi realizada por abaixamento de temperatura. Os cristais de isomaltulose apresentaram pureza de 96,5%.

In recent decades, there has been an increase in the studies of isomaltulose obtainment, due to its physicochemical properties and physiological health benefits. These properties, which include low cariogenicity, low glycemic index and greater stability, allow the use of this sweetener as a substitute for sucrose in foods; besides the fact that it can be converted to isomalt, a dietary non-cariogenic sugar alcohol used in pharmaceuticals as well as in the food industry. Isomaltulose (6-O-α-D-glucopyronosyl-1-6-D-fructofuranose) is a disaccharide reducer obtained by the enzymatic conversion of sucrose - the α-glucosyltransferase enzyme. Different treatments were performed for the preparation of whole cells; lysed cells; and crude enzyme extract of Erwinia sp. D12 strain immobilized in calcium alginate. The packed bed column of granules, containing Erwinia sp. cells sonicated and immobilized in calcium alginate (CSI), reached a maximum conversion of 53-59% sucrose into isomaltulose and it presented activity for 480 hours. The converted syrup was purified and the isomaltulose crystallization was performed through the lowering of temperature. The isomaltulose crystals presented purity of 96.5%.

Este estudo foi realizado em uma das mais importantes cultivares produzidas no Estado de São Paulo, Brasil, que tem a preferência dos consumidores devido ao sabor doce, intensa coloração e tamanho grande; entretanto, estes frutos são susceptíveis a danos pelo frio quando estocados sob refrigeração por longos períodos. O uso de atmosfera controlada (elevadas concentrações de CO2 e baixas de O2) previne os sintomas deste distúrbio. Foi avaliado o efeito de três condições de atmosfera controlada (AC1, AC2, AC3 e Controle) para estender a vida de estocagem dos pêssegos 'Douradão'. Após 14, 21 e 28 dias, amostras foram retiradas da AC, mantidas em ar a 25 ± 1 °C e 90 ± 5% UR para completar o amadurecimento. No dia da remoção e após 4 dias, foram avaliadas as características de qualidade dos pêssegos. Os resultados mostraram que a AC reduziu a perda de massa e preveniu a podridão pós-colheita dos frutos. Os tratamentos AC2 e AC3 foram efetivos na manutenção da boa qualidade dos pêssegos 'Douradão' durante 28 dias de armazenamento, os frutos maduros apresentaram reduzida incidência de lanosidade, adequada suculência e firmeza da polpa. Os tratamentos AC1 e Controle não apresentaram condições de comercialização após 14 dias de frigoconservação.

This study was carried out with one of the most important cultivar grown in the State of Sao Paulo, Brazil, which has gained the preference of consumers, due to its sweet taste, intense skin color and large size; however, these fruits are susceptible to chilling injury when cold stored for long periods. The use of controlled atmosphere (CA) with elevated CO2 and reduced O2 concentrations prevent the onset of the chilling symptom. Thus, the effect of three different conditions of controlled atmosphere (CA1, CA2, CA3 and Control) was evaluated in order to extend the storage life of 'Douradão' peaches. After 14, 21 and 28 days, samples were withdrawn from CA and kept in fresh air at 25 ± 1 °C and 90 ± 5% RH to complete ripening. On the day of removal and after 4 days, were the peaches quality characteristics were evaluated. The results showed that the use of CA during cold storage reduced weight loss and prevented postharvest decay. CA2 and CA3 treatments were effective in keeping good quality of 'Douradão' peaches during 28 days of cold storage, the ripe fruits showed reduced incidence of woolliness, adequate juiciness and flesh firmness. CA1 and Control treatments did not present marketable conditions after 14 days of cold storage.

O presente trabalho visou a aplicação da β-1,3 glucanase lítica, obtida do microrganismo Cellulosimicrobium cellulans 191, na produção de protoplastos e na lise da parede celular de leveduras. A preparação bruta da enzima foi capaz de lisar as leveduras Kluyveromyces lodderi, Saccharomyces cerevisiae (Fleischmann e Itaiquara), S. cerevisiae KL-88, S. diastaticus NCYC 713, S. cerevisiae NCYC 1001, Candida glabrata NCYC 388, Kluyveromyces marxianus NCYC 587 e Hansenula mrakii NCYC 500. A β-1,3 glucanase purificada foi capaz de lisar as leveduras Saccharomyces cerevisiae KL-88, Saccharomyces capensis, Debaromyces vanriji, Pachysolen tannophillus, Kluyveromyces drosophilarum, Candida glabrata, Hansenula mrakii e Pichia membranaefaciens e formar protoplastos de Saccharomyces cerevisiae KL-88.

The aim of this work was the application of lytic β-1,3 glucanase obtained from Cellulosimicrobium cellulans strain 191 in the production of protoplasts and lysis of yeast cell walls. The crude extract demonstrated lysis activity against the yeasts Kluyveromyces lodderi, Saccharomyces cerevisiae (Fleischmann and Itaiquara), S. cerevisiae KL-88, S. diastaticus NCYC 713, S. cerevisiae NCYC 1001, Candida glabrata NCYC 388, Kluyveromyces marxianus NCYC 587, and Hansenula mrakii NCYC 500. The purified β-1,3 glucanase demonstrated lysis activity against the yeasts Saccharomyces cerevisiae KL-88, Saccharomyces capensis, Debaromyces vanriji, Pachysolen tannophillus, Kluyveromyces drosophilarum, Candida glabrata, Hansenula mrakii, and Pichia membranaefaciens, and it was able to produce Saccharomyces cerevisiae KL-88 protoplasts.

Yeast cell wall contains polymers glucan and mannan-protein that have received much attention with respect to their biological activities. Conventional isolation process involving treatments with hot alkali and acids cause degradation of these polymers. The aim of this paper was to study a low-degrading process for the isolation of glucan and mannan-protein from S. cerevisiae cell wall comprising physic and enzymatic treatments. Yeast cell glucan was obtained in a purity of 87.4% and a yield of 33.7%. The isolated mannan-protein presented antioxidant activity that was increased after thirty minutes of protease treatment. Antioxidant activity was determined by β-carotene/linoleate model system.

Objetivou-se, no presente trabalho, a aplicação de β-1,3 glucanases e quitinases da linhagem Cellulosimicrobium cellulans 191 na lise de leveduras e inibição de fungos, respectivamente. O delineamento experimental mostrou que as melhores condições para a lise de Saccharomyces cerevisiae KL-88 pela β-1,3 glucanase foi pH 6,5 e 35ºC. As células de leveduras incubadas por 10 h em frascos sem agitação mostraram-se mais susceptíveis à lise pela ação da enzima. Foi obtido maior lise da levedura quando a suspensão de células foi submetida ao tratamento com β-1,3 glucanase e cisteína 1mM. A enzima invertase intracelular ou ligada à célula de S. cerevisiae KL-88 e K. marxianus NCYC 587 foi extraída após tratamento da suspensão celular com β-1,3 glucanase, sendo que o tratamento prévio das leveduras com a enzima aumentou a susceptibilidade das células à lise com ultra-som. A preparação de quitinase foi capaz de formar halos de inibição de alguns fungos.

The aim of this work was the application of β-1,3 glucanases and chitinases by Cellulosimicrobium cellulans 191 strain on yeast cell lysis and fungi inhibition, respectively. The experimental design study showed that the best conditions to Saccharomyces cerevisiae KL-88 lysis by β-1,3 glucanase extract were pH 6,5 and 35ºC. This study also demonstrated that the yeast cells were more susceptible to lysis after 10 h of cultivation in flasks without agitation. Lysis activity was increased when S. cerevisiae KL-88 cell suspension was treated with β-1,3 glucanase and cystein 1mM. The enzyme invertase of S. cerevisiae KL-88 and Kluyveromyces marxianus NCYC 587 was extracted after treatment of cell suspension with β-1,3 glucanase and the previous treatment of yeasts with the enzyme, increased the susceptibility to lysis when ultrasonic treatment was used. The chitinase presented growth inhibition halos for some of the fungi.

O presente trabalho visou o estudo do efeito de diferentes variáveis na produção de β-1,3 glucanases, proteases e de quitinases pela linhagem Cellulosimicrobium cellulans 191, em meios de cultivo A, B e C, respectivamente. Foram realizados planejamentos fatoriais 2³, e os fatores estudados foram: pH inicial, temperatura e agitação dos frascos. No planejamento experimental para a produção de β-1,3 glucanase em meio de cultivo A foi verificada maior produção da enzima (0,64 U.mL-1) com pH inicial de 8,5 após 24 horas de fermentação a 33 ºC e 200 rpm. Os parâmetros pH, agitação dos frascos e interação entre todos os parâmetros foram estatisticamente significativos. No planejamento experimental para a produção de protease em meio de cultivo B foi verificada maior produção da enzima (4,25 U.mL-1) com pH inicial de 6,5 após 30 horas de fermentação a 20 ºC e 200 rpm. O pH foi o único parâmetro estatisticamente significativo. No planejamento experimental para a produção de quitinase em meio de cultivo C foi verificada maior produção da enzima (7,06 U.mL-1) com pH inicial de 5,5 após 72 horas de fermentação a 25 ºC e 200 rpm. Todos os parâmetros estudados e suas interações foram estatisticamente significativos. Os coeficientes de determinação, as análises de variância e os teste F demonstraram que os modelos de primeira ordem obtidos foram considerados estatisticamente significativos adotando um nível de confiança de 95%.

The aim of this work was to study the effects of different parameters in the production of β-1,3 glucanases, proteases and chitinases by Cellulosimicrobium celulans 191, in culture media A, B and C, respectively. The experimental designs employed were 2³ factorial designs, and the factors studied were: initial pH, temperature and rotary shaker speed. The experimental results for the production of β-1,3 glucanase in culture medium A showed maximum activity (0.64 U.mL-1) with an initial pH of 8.5 after 24 hours of fermentation at 33 ºC and 200 rpm. The parameters pH, rotary shaker speed and the interaction of all the parameters presented statistical significance. The experimental results for protease production in culture medium B showed maximum activity (4.25 U.mL-1) with an initial pH of 6.5 after 30 hours of fermentation at 20 ºC and 200 rpm. The pH was the only parameter that presented statistical significance. The experimental results for chitinase production in culture medium C showed maximum activity (7.06 U.mL-1) at an initial pH of 5.5 after 72 hours of fermentation at 25 ºC and 200 rpm. All the parameters and their interactions presented statistical significance. The determination coefficients of the analysis of variance and F tests demonstrated that the linear regression models obtained were significant at a confidence level of 95%.

Isomaltulose, a functional isomer of sucrose, is a non-cariogenic reducing disaccharide; has a low glycemic index; selectively promotes growth of beneficial bifidobacteria in the human intestinal microflora; and has greater stability than sucrose in some foods and beverages. Isomaltulose is a nutritional sugar that is digested more slowly than sucrose, and has health advantages for diabetics and nondiabetics. Immobilization techniques, especially entrapment of the cells, are widely used for conversion of sucrose into isomaltulose. Immobilization offers advantages such as minimum downstream processing, continuous operation and reusability of cells. Isomaltulose is currently considered to be a promising sugar substitute.

Finding a new microbial source of transglutaminase (MTGase) and the study of the medium composition for MTGase production were the goals of this work. A total of 200 actinomycete-like strains were isolated from Brazilian soil samples and two of them named T10b and P20 were selected based on their ability to produce 0.15 U.mL-1 and 0.25 U.mL-1 of MTGase, respectively. Strain P20 was chosen to continue the study and was identified as Streptomyces sp. In order to optimize the MTGase activity, modifications of the usual media composition described for enzyme production were tested. The strategy adopted was: (1) screening experiment for the best carbon and nitrogen sources; (2) fractional factorial design (FFD) to elucidate the key ingredients in the media (the results indicated that the soybean flour, peptone, KH2PO4 and MgSO4.7H2O had a significant effect on MTGase) production and (3) central composite design (CCD) to optimize the concentration of the key components. The experimental results were fitted to a second-order polynomial model at the 95% level of significance (P < 0.05). Under the proposed optimized conditions, the model predicted a MTGase activity of 1.37 U.mL-1, very closely matching the experimental activity of 1.4 U.mL-1.

Isomaltulose is a structural isomer of sucrose commercially used in food industries. Glucosyltransferase produced by Erwinia sp. D12 catalyses an intramolecular transglucosylation of sucrose giving isomaltulose. The Experimental design and response surface methodology were applied for the optimization of the nutrient concentration in the culture medium for the enzyme production in shaken flasks at 200 rpm and 30ºC. The three variables involved in this study were sugar cane molasses, bacteriological peptone and yeast extract Prodex Lac SD®. The statistical analysis of the results showed that, in the range studied, all the factors had a significant effect (p < 0.05) on glucosyltransferase production and the highest enzyme activity was observed in culture medium containing sugar cane molasses (160 g/L), bacteriological peptone (20 g/L) and yeast extract Prodex Lac SD® (15 g/L). Maximum glucosyltransferase activity of 29.88 U/mL was achieved in a 6.6-L fermenter using the optimized medium. Free Erwinia sp. D12 cells were used for isomaltulose production from sucrose during fifteen successive batches. The final isomaltulose concentration of 75.6% obtained in the first batch increased to 77.21% (mean value) in the other fourteen batches and the productivity of 1.1 g/L x hr was obtained in batch process.

Lytic enzymes such as beta-1,3 glucanases, proteases and chitinases are able to hydrolyse, respectively, beta-1,3 glucans, mannoproteins and chitin, as well as the cell walls of many yeast species. Lytic enzymes are useful in a great variety of applications including the preparation of protoplasts; the extraction of proteins, enzymes, pigments and functional carbohydrates; pre-treatment for the mechanical rupture of cells; degradation of residual yeast cell mass for the preparation of animal feed; analysis of the yeast cell wall structure and composition; study of the yeast cell wall synthesis and the control of pathogenic fungi. This review presents the most important aspects with respect to lytic enzymes, especially their production, purification, cloning and application.

A glicosiltransferase obtida pela linhagem Erwinia sp. é uma enzima intracelular que catalisa a conversão de sacarose em isomaltulose. A isomaltulose é um dissacarídeo redutor, não cariogênico e comercialmente utilizado em alimentos como substituto da sacarose. A metodologia de superfície de resposta e planejamento fatorial composto central-2³ foram utilizados para otimizar o meio de cultivo para a produção de glicosiltransferase de Erwinia sp. em frascos sob agitação a 200 rpm e 30ºC. As três variáveis independentes envolvidas no estudo foram o melaço de cana de açúcar, a água de maceração de milho e o extrato de levedura Prodex Lac SD. As análises estatísticas dos resultados mostraram que, dentro da faixa estudada das concentrações dos componentes de meio de cultivo, todas as variáveis apresentaram efeito significativo na produção de glicosiltransferase. O meio de cultivo otimizado foi composto de 100 gL-1 de melaço de cana de açúcar, 60 gL-1 de água de maceração de milho e 8 gL-1 de extrato de levedura Prodex Lac SD, apresentando atividade de glicosiltransferase de 6.65 U mL-1.

Glucosyltransferase produced by strain Erwinia sp. is an intracellular enzyme that catalyzes the formation of isomaltulose from sucrose. Isomaltulose is a non-cariogenic reducing dissacharide commercially used in foods. Response surface methodology and 2³-factorial central composite design were employed to optimize a fermentation medium for the production of glucosyltransferase by Erwinia sp. in shaken flasks at 200 rpm and 30ºC. The three variables involved in this study were sugar cane molasses (SCM), corn steep liquor (CSL) and yeast extract Prodex Lac SD (YEP). The statistical analysis of the results showed that, in the range studied, all the factors had a significant effect on glucosyltransferase production and the optimum medium composition for enzyme production was (in g l-1) SCM-100, CSL-60 and YEP-8, which lead to a glucosyltransferase activity of 6.65 U mL-1.