Immobilization of mutated xylanase from Neocallimastix patriciarum in E. coli and application for kraft pulp biobleaching

Abstract As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.

Resumo Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.

Immobilization of mutated xylanase from Neocallimastix patriciarum in E coli and application for kraft pulp biobleaching / Imobilização de xilanase mutada de Neocallimastix patriciarum em E coli e aplicação para polpa kraft

As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.

Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.

Immobilization of mutated xylanase from Neocallimastix patriciarum in E. coli and application for kraft pulp biobleaching / Imobilização de xilanase mutada de Neocallimastix patriciarum em E. coli e aplicação para polpa kraft

Abstract As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.

Resumo Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.

Immobilization of mutated xylanase from Neocallimastix patriciarum in E coli and application for kraft pulp biobleaching / Imobilização de xilanase mutada de Neocallimastix patriciarum em E coli e aplicação para polpa kraft

As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.(AU)

Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.(AU)

Isolation and characterization of a novel endo--1, 4-glucanase from a metagenomic library of the black-goat rumen

ABSTRACT The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50 °C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50 °C at a pH of 5-7.(AU)

High levels of B-xylosidase in Thermomyces lanuginosus: potential use for saccharification

A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its -xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of -xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high -xylosidases activities 1003 U/mL. According our known, this study is the first to show so high levels of -xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce -xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60 °C showing better thermostability at 55 °C. The saccharification ability of -xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50 °C. Our data strongly indicated that the -xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production.(AU)

Preliminary studies of new strains of Trametes sp. from Argentina for laccase production ability

Oxidative enzymes secreted by white rot fungi can be applied in several technological processes within the paper industry, biofuel production and bioremediation. The discovery of native strains from the biodiverse Misiones (Argentina) forest can provide useful enzymes for biotechnological purposes. In this work, we evaluated the laccase and manganese peroxidase secretion abilities of four newly discovered strains of Trametes sp. that are native to Misiones. In addition, the copper response and optimal pH and temperature for laccase activity in culture supernatants were determined.The selected strains produced variable amounts of laccase and MnP; when Cu2+ was added, both enzymes were significantly increased. Zymograms showed that two isoenzymes were increased in all strains in the presence of Cu2+. Strain B showed the greatest response to Cu2+ addition, whereas strain A was more stable at the optimal temperature and pH. Strain A showed interesting potential for future biotechnological approaches due to the superior thermo-stability of its secreted enzymes.(AU)

Laccase produced by a thermotolerant strain of Trametes trogii LK13

Thermophilic and thermotolerant micro-organisms strains have served as the natural source of industrially relevant and thermostable enzymes. Although some strains of the Trametes genus are thermotolerant, few Trametes strains were studied at the temperature above 30 °C until now. In this paper, the laccase activity and the mycelial growth rate for Trametes trogii LK13 are superior at 37 °C. Thermostability and organic cosolvent tolerance assays of the laccase produced at 37 °C indicated that the enzyme possessed fair thermostability with 50% of its initial activity at 80 °C for 5 min, and could remain 50% enzyme activity treated with organic cosolvent at the concentration range of 25%–50% (v/v). Furthermore, the test on production of laccase and lignocellulolytic enzymes showed the crude enzymes possessed high laccase level (1000 U g−1) along with low cellulose (2 U g−1) and xylanase (140 U g−1) activity. Thus, T. trogii LK13 is a potential strain to be applied in many biotechnological processes.(AU)

TERMITEIROS ARBORÍCOLAS (Microcerotermes indistinctus e Constrictotermes cyphergaster) COMO SUBSTRATO DE NIDIFICAÇÃO PARA A ABELHA SEM FERRÃO Partamona seridoensis NA CAATINGA

A diagnose morfológica da espécie de abelha sem ferrão, endêmica da Caatinga, Partamona seridoensis é amplamente descrita e consolidada, porém, os aspectos arquitetônicos, estruturais e termodinâmicos dos cupinzeiros hospedeiros desta abelha termitófila obrigatória são pouco conhecidos. Diante disso, este trabalho buscou realizar um estudo base que teve por objetivos: (1) indicar qual melhor metodologia de dimensionamento de volume e área superficial de cupinzeiros arborícolas, oferecendo um modelo matemático de ajuste; (2) realizar uma descrição comparativa entre as estruturas arquitetônicas de ninhos de P. seridoensis localizados em termiteiros das espécies Microcerotermes indistinctus e Constrictotermes cyphergaster; (3) estudar as características térmicas de ninhos de abelhas e dos substratos circundantes; (4) apresentar um modelo detalhado de uma colmeia racional voltada exclusivamente para espécies termitófilas. O período experimental compreendeu os meses de abril a novembro de 2018, em que foram utilizados vinte cupinzeiros para os estudos, sendo dez de cada espécie, onde metade era habitado por abelhas e a outra metade inativa. (1) Para o primeiro objetivo utilizou-se a modelagem 3D e equações aritméticas para os cálculos de volume e área superficiais dos termiteiros. Os resultados encontrados para os dimensionamentos dos termiteiros mostraram que a compartimentalização é a metodologia que apresentou volumetria mais próxima à realidade. Porém devido à sua complexidade, é inviável de ser utilizada em campo, tornando o modelo elipsoidal mais vantajoso, uma vez que as medidas necessárias à sua utilização são apenas a largura, a altura e o comprimento dos cupinzeiros. (2) Para a descrição das características gerais dos termiteiros e a morfologia dos ninhos de P. seridoensis seguiu-se metodologias estabelecidas pela literatura. Verificou-se que os aspectos gerais e arquitetônicos dos ninhos de P. seridoensis, inseridos em cupinzeiros com substratos e dimensionamentos distintos, apresentaram características estruturais semelhantes e típicas das abelhas termitófilas do gênero Partamona. (3) Para a análise das características térmicas, sensores de temperatura foram inseridos em locais específicos (substrato, invólucro, áreas de cria e com potes de alimento) para cupinzeiros que possuíam ninhos de abelhas e, em termiteiros inativos, nas profundidades de 5, 10, 15, e 20 cm. Os dados ambientais foram obtidos de estação meteorológica automática instalada no local do experimento. A escolha do cupinzeiro mais adequado por P. seridoensis para nidificar pode ser por conta do tipo de substrato termoisolante, mas outros fatores podem influenciar, como a agressividade do hospedeiro, a cavidade pré-existente ou mesmo o grau de antropização do ambiente. Contudo, quando se avaliou apenas a questão térmica, os cupinzeiros de M. indistinctus foram melhores para a P. seridoensis, uma vez que exigiram menos da temperatura ambiental e possuíram maior isolamento térmico quando comparados aos termiteiros de C. cyphergaster, mesmo que estes também tivessem características desejáveis como a termoestabilidade. (4) Por fim, para a construção da colmeia racional foram utilizados os resultados provenientes deste estudo, unindo as características arquitetônicas mais usuais das colmeias já utilizadas, em que as observações preliminares em P. seridoensis indicaram que estas abelhas se habituaram rapidamente ao modelo de colmeia desenvolvido.

Morphological characteristics of the stingless bee Partamona seridoensis (Apidae, Meliponini), endemic to the Caatinga, are well known. However, the architectural, structural and thermodynamic aspects of the nests of this obligatory thermophilic bee within arboreal termite nests are poorly known. The present thesis aimed at increasing the knowledge about these facts, fundamental for future ecological studies on this meliponine species. The main goals were to: (1) indicate the best methodology for estimating the volume and surface area of arboreal termite nests and provide a mathematical adjustment model; (2) compare the architectural structures of P. seridoensis nests within the nests of the termite species Microcerotermes indistinctus and Constrictotermes cyphergaster; (3) study the thermal characteristics of both bee and surrounding termite nests; (4) elaborate a model of a new rational hive for thermophilic stingless bee species. Between April and November 2018, twenty termite nests, ten of each species, were used for the investigations. Half of the termite nests had been colonized by bees whereas the other half was inactive. (1) 3D-modelling and geometric shapes were used to estimate the volume and surface area of the arboreal termite nests. A combination of superimposed cylinders with spherical caps presented values closer to reality (3D-model) than ellipsoids. However, due to its complexity, it is not feasible for use in the field, rendering the ellipsoidal model, which requires only 3 linear measurements, more advantageous. (2) Methodologies established in literature were adopted to describe the characteristics of both termite nests and the nests of P. seridoensis. Despite differences in nest characteristics between the two termite species, the architecture of P. seridoensis nests showed no significant differences between the surrounding substrates. (3) For the analysis of the thermal characteristics of the nests, temperature sensors were inserted in specific locations in and around the bee nests (substrate, involucrum, brood area, and food area). As control, sensors were placed at depths of 5, 10, 15, and 20 cm in inactive termite nests. Environmental temperature was monitored through a weather station at the site of the experiment. The bees'choice which termite species' nest to colonize may be based on the thermal insulation characteristics of substrate. Yet, other factors such as the hosts' aggressiveness, the pre- existence of a inhabitable cavity, or even the anthropization of the environment may have an impact on the bees' choice. Concerning the thermal characteristics, the nests of M. indistinctus showed better insulation properties, given that temperatures inside the nests were more independent from ambient temperature the compared to the nests of C. cyphergaster. However, the nests of latter species presented an elevated thermostability, which is a characteristic in favour of the colonization by bees. (4) The results from this study were used in combination with the most common architectural characteristics of rational hives for the construction of rational hives for P. seridoensis. Preliminary observations on the success of the new hive model indicate that the bees quickly get used to the rational hive developed.

Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106 s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.(AU)

PRODUÇÃO E PURIFICAÇÃO DE PROTEASE OBTIDA DO FUNGO FILAMENTOSO Aspergillus sydowii POR FERMENTAÇÃO EM ESTADO SÓLIDO UTILIZANDO RESÍDUO DE CAFÉ COMO SUBSTRATO

As proteases são um grupo de enzimas hidrolíticas, que possuem a capacidade de hidrolisar ligações peptídicas extremamente importantes para indústria, sendo seu uso correspondente a 60% das enzimas comercializadas. Devido essa importância, faz-se necessária a utilização de novas fontes de obtenção ou de novas formas de baratear a produção. Pensando nisso a utilização de resíduos agroindustrial em associação com processos fermentativos pode ser uma forma de obtenção de novas enzimas alem de ajudar a combater os sérios danos ambientais causados pela eliminação desses resíduos na natureza. A Fermentação em Estado Sólido (FES) vem como tentativa de reaproveitamento desse material que pode ser utilizado como um substrato de alta qualidade por um baixo preço para produção de proteases com potencial biotecnológico. O presente trabalho teve como objetivo utilizar Aspergillus sydowii para a produção de protease através da FES, além de purificar a enzima alvo através de processos de cromatográficos, bem como a determinação das características bioquímicas da protease produzida e isolada. O micro-organismo utilizado foi isolado diretamente do café e identificado. Em seguida, foi realizada uma verificação do seu potencial de produção para protease. Para a produção foram avaliados o tempo de fermentação, a umidade e a quantidade de substrato utilizada. Para o processo de purificação foram utilizadas técnicas de precipitação com acetona, colunas cromatográficas utilizando como resina a DEAE-Sephadex e gel filtração Superdex 75. Foram analisados os extratos brutos e purificados. Os extratos foram submetidas à caracterização quanto a sua termoestabilidade, temperatura e pH ótimos, efeitos de inibidores e íons metálicos. O extrato bruto apresentou atividade proteolítica de 412 U/mL, tendo uma inibição de pelo Fenilmetilsulfonilflúor (85%) e o íon Fe+3 aumentou a atividade em 69%. A atividade ótima foi encontrada em pH 8 a 45°C, com estabilidade termica do extrato entre 25°C e 45°C, mantendo 85% de sua atividade inicial. A protease purificada apresentou um tamanho de 48 KDa, a técnica utilizada apresentou um alto rendimento de 5,9 vezes, a recuperação de 53% e a atividade proteolítica de 256 U/mL. A enzima teve uma inibição pelo Fluoreto de Fenilmetilsulfonila (60%) e o íon Cu+2 (56,5%). A atividade ótima foi encontrada em pH 8,0 a 45°C, com estabilidade da enzima entre 35°C e 50°C, mantendo 70% de sua atividade inicial. A linhagem de A. sydowii isolada foi eficaz para a produção de protease com potencial biotecnologico atraves da FES utilizando o resíduo de café, além de uma possivel solução para o dano ambiental provocado pelo borra do café.

Proteases are hydrolytic enzymes able to hydrolyze peptide bonds and thereby presenting several applications for industrial purposes (corresponding to 60% of the commercial enzymes). Wasting of agro-industrial raw material generates environmental problems at the same time that represents an economic hindrance. Solid State Fermentation emerges as an alternative to reuse this raw material by providing high quality substrate at a low price for the production of proteases with biotechnological potential. The present work aimed to select Aspergillus sydowii strains to perform Solid State Fermentation and then to purify a protease through chromatography process, as well as the determination of its biochemical characteristics. In this study the microorganism cultivated directly in the coffee substrate was isolated and identified regarding to its production potential, the fermentation time, the humidity and the amount of the substrate. For the purification process a number of techniques were used, acetone precipitation, gravitational column using DEAE-Sephadex resin and Superdex 75 gel filtration using the AKTA avant system. Solid state fermentation is a promising technology largely used in a biotechnology process and is a suitable strategy for producing low cost enzymatic products. At the present study, an enzyme obtained through solid state fermentation (SSF) by Aspergillus sydowii was herein purified, characterized and tested for its antimicrobial activity. The fermentations used coffee ground residues as substrate and the crude enzyme was submitted through further purification steps of: cetonic precipitation, chromatography through DEAE-Sephadex column and FPLC system (gel filtration in na Äkta avant system, Superdex 75 collumn). Both crude and purified enzymes were submitted to characterization of their thermostability, optimal temperature and pH, effects of inhibitors and metal ions. A purified protease (48 KDa) was obtained with high yield (5.9 fold) and recovery (53%) with proteolytic activity of 256 U/mL. The enzyme was highly inhibited by phenylmethanesulfonyl fluoride (60%) and the ion Cu+2 (56.5%). The optimal activity was found at pH 8, at 45 °C of temperature, with the enzyme stability between 35° C and 50° C (maintaining 70% of its highest activity). It was possible to determine appropriate conditions to the obtainment of thermostable proteases with biotechnological interest associated with a method that concomitantly shows excellent production levels and recovery waste raw material in a very profitable process.

Thermostability of xylanolytic enzymes produced by Lentinula edodes UFV70

Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65ºC for 21 hours, losing only 15-25% activity. Fungus incubation at 50ºC for 12 hours and at 65ºC for 24 hours increased the amount of xylose produced.

Comparative study of two purified inulinases from thermophile Thielavia Terrestris NRRL 8126 and mesophile Aspergillus Foetidus NRRL 337 grown on Cichorium Intybus l

Thirty fungal species grown on Cichorium intybus L. root extract as a sole carbon source, were screened for the production of exo-inulinase activities. The thermophile Thielavia terrestris NRRL 8126 and mesophile Aspergillus foetidus NRRL 337 gave the highest production levels of inulinases I & II at 50 and 24 ºC respectively. Yeast extract and peptone were the best nitrogen sources for highest production of inulinases I & II at five and seven days of incubation respectively. The two inulinases I & II were purified to homogeneity by gel-filtration and ion-exchange chromatography with 66.0 and 42.0 fold of purification respectively. The optimum temperatures of purified inulinases I & II were 75 and 50 ºC respectively. Inulinase I was more thermostable than the other one. The optimum pH for activity was found to be 4.5 and 5.5 for inulinases I & II respectively. A comparatively lower Michaelis-Menten constant (2.15 mg/ml) and higher maximum initial velocity (115 µmol/min/mg of protein) for inulinase I on inulin demonstrated the exoinulinase's greater affinity for inulin substrate. These findings are significant for its potential industrial application. The molecular mass of the inulinases I & II were estimated to be 72 & 78 kDa respectively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i) motile ii) rod shaped iii) non spore forming iv) catalase and amylase positive v) able to grow in presence of 10 % NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7) exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98% sequence similarity, 1201 bp). The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 % activity at 80 0C after 4 h and more than 70 % activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 % active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

Produção, purificação, caracterização e aplicação de tanase de Aspergillus melleus URM 5827 produzida por fermentação em estado sólido utilizando sementes de achachairú (Garcinia humilis (Vahl) C. D. Adam).

Tanino acil hidrolase (TAH) conhecida como tanase (E.C:3.1.1.20) é uma enzima que hidrolisa ésteres e ligações laterais de taninos hidrolisáveis. O ácido tânico é um típico tanino hidrolisável, que pode ser hidrolisado por tanase em glicose e ácido gálico. A tanase pode ser obtida a partir de fontes vegetais, animais e microbianas, sendo o meio microbiológico a fonte mais importante de obtenção desta enzima. O chá verde apresenta várias substâncias, dentre elas, as catequinas que são uma importante fonte de antioxidante, que ajudam na manutenção do organismo.A atividade e a purificação de tanase produzida por Aspergillus melleus URM 5827 foi avaliada por fermentação em estado sólido utilizando como substrato sementes de achachairú (Garcinia humilis (Vahl) C. D. Adam). Foram utilizadas 42 culturas de fungos do gênero Aspergillus para seleção qualitativa das culturas com potencial para produção da tanase. Com a cultura selecionada foi realizada uma fermentação em estado sólido utilizando sementes de achachairú como substrato. Um planejamento fatorial (23) foi utilizado para analisar a influência das variáveis de produção: quantidade de substrato, umidade inicial e quantidade de ácido tânico sobre a atividade da tanase. A purificação foi avaliada por cromatografia de troca iônica em DEAE- Sephadex. A máxima atividade foi produzida por Aspergillus melleus URM 5827 com 452,55 unidades por grama de substrato na base seca (U/gss) utilizando 5,0 g de substrato, com umidade inicial de 60% e 2,0% de ácido tânico em 48 horas de fermentação. A enzima purificada apresentou peso molecular de 69,52 kDa em Superdex G-75, enquanto que em eletroforese SDS-PAGE apresentou 66,5 kDa. Quanto a caracterização, apresentou pH e temperatura ótima de 5,5 e 40ºC respectivamente, obtendo termoestabilidade a 30ºC. A atividade enzimática na presença de íons, surfactantes e inibidores de protease, foi inibida na presença dos íons ZnCl2, ZnSO4 e dos surfactantes triton X-100, SDS, reduzida com os íons CaCl2, KCl, NaCl, MgSO4, CuSO4, dos surfactantes Tween 20, Tween 80 e dos inibidores de protease EDTA e -mercaptoetanol. A tanase aumentou a atividade antioxidante do chá verde significativamente. Os resultados obtidos no presente estudo, mostram o potencial promissor da tanase produzida por Aspergillus melleus URM 5827 e na sua utilização no aumento do potencial antioxidante do chá verde.

Tannin acylhydrolase (TAH) known as tannase (E.C:3.1.1.20) is an enzyme which hydrolizes esters and lateral bonds of hydrolizable tannins. The tannic acid is a typical hydrolizable tannin, which can be hydrolized by tannase along with glucose and gallic acid. Tannase can be obtained from vegetables, animal and microbial sources. From those, the last is the most important source to obtain the enzyme. Green tea has several substances, among which catechins are a major source of antioxidant, which help in maintaining the organism.The activity and purification of tannase produced by Aspergillus melleus URM 5827 was evaluated by semi solid fermentation using seeds of mangosteen (Garcinia humilis (Vahl) C. D. Adam) as substract. Forty two cultures fungical cultures of Aspergillus were used for qualitative selection purposes in order to verify potential for tannase production. After selecting cultures, it was performed a semi solid fermentation using seeds of mangosteen as substrate. A factorial planning (2³) was used to verify the influence of production variables such as: quantity of substrate; initial moisture and amount of tannic acid over tannase activity. The purification was evaluated by ionic change chromatography at DEAE-Sephadex. Maximum activity was produced by Aspergillus melleus URM 5827 with 452.55 units per gram of dry-based substract (U/gss) using 5.0 grams of substrate, with initial moisture of 60% and 2% of tannic acid through 48 hours fermentation. The purified enzyme has a molecular weight of 69.52 kDa on Superdex G-75, while on SDS-PAGE electrophoresis showed 66.5 kDa. As for the characterization, the optimum pH and temperature was 5.5 and 40ºC, respectively, achieving thermostability at 30ºC. Coughing increases the antioxidant activity of green tea significantly. The results obtained in this study show the promising potential of tannase produced by Aspergillus melleus URM 5827 and its use in improving the antioxidant potential of green tea.

Application of microbial -amylase in industry - A review

Amylases are one of the main enzymes used in industry. Such enzymes hydrolyze the starch molecules into polymers composed of glucose units. Amylases have potential application in a wide number of industrial processes such as food, fermentation and pharmaceutical industries. -Amylases can be obtained from plants, animals and microorganisms. However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors. The production of -amylase is essential for conversion of starches into oligosaccharides. Starch is an important constituent of the human diet and is a major storage product of many economically important crops such as wheat, rice, maize, tapioca, and potato. Starch-converting enzymes are used in the production of maltodextrin, modified starches, or glucose and fructose syrups. A large number of microbial -amylases has applications in different industrial sectors such as food, textile, paper and detergent industries. The production of -amylases has generally been carried out using submerged fermentation, but solid state fermentation systems appear as a promising technology. The properties of each -amylase such as thermostability, pH profile, pH stability, and Ca-independency are important in the development of fermentation process. This review focuses on the production of bacterial and fungal -amylases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

Hyperexpression of two Aspergillus niger xylanase genes in Escherichia coli and characterization of the gene products

The analysis of individual gene product should enable to clarify the role of a particular enzyme in a complex xylanase system of A. niger. The two genes encoding precursors of co-produced endo-1,4--D-xylanases, xynA1 and xynB, were isolated from Aspergillus niger SCTCC 400264 (SCTCC, China) by using RT-PCR technique and then successfully expressed in Escherichia coli BL21. The nucleotide sequences of the xynA1 and xynB genes revealed that they were only 52.5% homology to each other. Characterization of the recombinant enzymes revealed the different properties: the specific activity of recombinant XYNA1 was 16.58 U/mg compared to 1201.7 U/mg for recombinant XYNB; The optimum temperature and pH of the recombinant XYNA1 were 35 ºC and 3.0, respectively, whereas the corresponding values for the recombinant XYNB were 55 ºC and 5.0, respectively; The recombinant XYNB showed much more thermostability than recombinant XYNA1; The recombinant XYNB showed 94% of maximal activity after incubating in water for 60 min at 60 ºC compared to no activity for recombinant XYNA1. Various metal ions had different effects on activity between the two recombinant xylanases.

PRODUÇÃO, PURIFICAÇÃO E CARACTERIZAÇÃO PARCIAL DA FITASE EXTRACELULAR PRODUZIDA POR ASPERGILLUS NIGER VAR. PHOENICIS URM 4924 E SUA ATIVIDADE HIDROLÍTICA E ANTI-PROTEOLÍTICA IN VITRO EM RAÇÕES COMERCIAIS DE AVES E SUÍNOS

As enzimas exógenas têm a capacidade de auxiliar na degradação de ingredientes específicos presentes em alimentos. Dentre os principais aditivos enzimáticos utilizados na alimentação de animais aves e suínos pode-se destacar as lipases, xilanases, glucanases, proteases e fitases. O fitato é um composto considerado como fator anti-nutricional, pois é responsável por perdas nutricionais significativas. Fitases formam um grupo de enzimas, denominadas genericamente de mio-inositol-hexafosfato fosfohidrolase, que hidrolizam o fitato, liberando sais de inositol e fosfato inorgânico. O objetivo deste trabalho foi avaliar o efeito das variáveis (pH e temperatura) que influenciam à produção de fitase por A. niger var. phoenicis URM 4924, pré-purificação da enzima através da fermentação extrativa em SDFA PEG/citrato, caracterização bioquímica do pré e pós-purificado, atividade anti-proteolítica frente à pepsina gástrica suína e à tripsina pancreática, bem como o perfil hidrolítico in vitro da fitase em rações comerciais de aves e suínos. Temperatura e pH mostraram ser importantes parâmetros na produção da fitase e o conteúdo de ergosterol mostrou-se como um bom indicador para estimar a produção da biomassa. A fitase em estudo apresentou temperatura ótima a 30 °C e pH ótimo a 4,0. Após a extração e purificação da fitase em sistemas de duas fases aquosas por fermentação extrativa obteve-se máxima recuperação em atividade (150,38%) utilizando MPEG (8000 g/mol), CPEG, (20,0% m/m), CCIT (20,0% m/m), pH (6,0) e agitação (100 rpm). Através dos resultados obtidos verifica-se que após o processo de extração e purificação utilizando fermentação extrativa por SDFA PEG/citrato, a termoestabilidade da fitase diminuiu consideravelmente (38,4% da atividade residual em 90°C por 120 minutos para 50,0% da atividade residual em 70°C por 25 minutos). Observa-se que a enzima pré-purificada foi inibida em concentrações de fósforo de 6 moles de PO4-2, entretanto a fitase pós-purificada em SDFA. PEG/citrato foi inibida em concentração de fósforo menor do que a pré-purificada. Quanto a resistência quanto à proteólise, a fitase manteve 60,0% da sua atividade residual por 40 minutos. No entanto, quando exposta a atividade proteolítica da tripsina entérica, a fitase manteve aproximadamente 20,0% da atividade residual. A fitase mostrou-se com bom desempenho na resistência proteolítica, bem como na hidrólise do fitato em rações comerciais de aves e suínos, características bioquímicas essenciais para uma enzima com potencial aplicação industrial. Os resultados deste presente trabalho demostram o potencial do fungo Aspergillus niger var. phoenicis URM 4924 para produzir fitase sob fermentação submersa para aplicação na alimentação de animais não-ruminantes.

Exogenous enzymes have ability to assist on degradation of specific compounds in foods. Among some enzyme additives fed to poultry and pigs can be lipases, xylanases, glucanases, proteases and phytases. Phytate is a compound considered an anti-nutritional factor since it is responsible for significant nutritional losses. Phytases are a group of enzymes, generically known as myo-inositol hexaphosphate fosfohidrolase that hydrolyze phytate, releasing inositol and inorganic phosphate salts. This aim of this study was to evaluate the effect of variables (pH and temperature) that influence the production of phytase by A. niger var. phoenicis URM 4924, pre-purification of the enzyme by extractive fermentation in ATPS PEG/citrate, biochemical characterization of pre- and post-purified, anti- proteolytic activity against pig gastric pepsin and pancreatic trypsin, and the profile hydrolytic in vitro phytase in commercial diets of poultry and pigs. Temperature and pH proved to be important parameters in the production of phytase. Ergosterol content proved to be a good indicator to estimate biomass production. Phytase studied showed optimum temperature at 30°C and optimum pH 4.0. After extraction and purification of phytase in aqueous two-phase systems by extractive obtained maximum recovery in activity (150.38 %) using MPEG (8000 g/mol), CPEG (20.0 % m/m), CCIT (20.0% w/w), pH (6.0) and agitation (100 rpm). From the results obtained it can be seen that after the extraction process using extractive fermentation and purification in ATPS PEG/citrate, thermostability of phytase decreased significantly (38.4% residual activity at 90° C for 120 minutes to 50.0 % of residual activity at 70° C for 25 minutes). It is observed that the pre- purified enzyme was inhibited by concentrations of phosphorus of 6 mols of PO4-2, however post-purified phytase in ATPS PEG/citrate inhibited by low phosphorus concentration of the pre-purified. For resistance to proteolysis as the phytase kept 60.0 % of residual activity. after 40 minutes. However, when exposed to the proteolytic activity of trypsin enteric the phytase kept approximately 20.0 % of residual activity. Phytase proved diserable performance on proteolytic resistance, as well as phytate hydrolysis in commercial diets of poultry and pig, essential biochemical characteritics for an enzyme with potential industrial application. The results of this study demonstrate the potential of Aspergillus niger var. phoenicis URM 4924 to produce phytase in submerged fermentation for use in animal feed.

Highly thermostable and alkaline -amylase from a halotolerant-alkaliphilic Bacillus sp. AB68

An alkaliphilic and highly thermostable -amylase producing Bacillus sp. was isolated from Van soda lake. Enzyme synthesis occurred at temperatures between 25ºC and 40ºC. Analysis of the enzyme by SDS-PAGE revealed a single band which was estimated to be 66 kDa. The enzyme was active in a broad temperature range, between 20ºC and 90ºC, with an optimum at 50ºC; and maximum activity was at pH 10.5. The enzyme was almost completely stable up to 80ºC with a remaining activity over 90% after 30 min pre-incubation. Thermostability was not increased in the presence of Ca2+. An average of 75% and 60ºC of remaining activity was observed when the enzyme was incubated between pH 5 and 9 for 1 h and for 2 h, respectively. The activity of the enzyme was inhibited by SDS and EDTA by 38% and 34%, respectively.

Bacillus sp AB68 alcalifílico produtor de -amilase alcalina termoestável foi isolado do lago Van soda. A síntese da enzima ocorreu entre 25ºC e 40ºC. A análise da enzima por SDS-PAGE revelou uma única banda estimada em 66 kDa. A enzima foi ativa em uma ampla faixa de temperatura, entre 20ºC e 90ºC, com um ótimo a 50ºC. A atividade máxima foi em pH 10,5. A enzima foi estável até 80ºC, mantendo 90% de atividade após 30 min de pré-incubação. A termoestabilidade não aumentou na presença de Ca2+. Quando incubada em pH entre 5 e 9 por 1h e por 2h, a enzima manteve 75% e 60% de atividade, respectivamente. SDS e EDTA causaram redução de 38% e 34% na atividade da enzima, respectivamente.

Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources

Glucoamylase is widely used in the food industry to produce high glucose syrup, and also in fermentation processes for production beer and ethanol. In this work the productivity of the glucoamylase of Aspergillus awamori expressed by the yeast Saccharomyces cerevisiae, produced in submerged fermentation using different starches, was evaluated and characterized physico-chemically. The enzyme presented high specific activity, 13.8 U/mgprotein or 2.9 U/mgbiomass, after 48 h of fermentation using soluble starch as substrate. Glucoamylase presented optimum activity at temperature of 55ºC, and, in the substratum absence, the thermostability was for 1h at 50ºC. The optimum pH of activity was pH 3.5 - 4.0 and the pH stability between 5.0 and 7.0. The half life at 65ºC was at 30.2 min, and the thermal energy of denaturation was 234.3 KJ mol-1. The hydrolysis of different substrate showed the enzyme's preference for the substrate with a larger polymerization degree. The gelatinized corn starch was the substratum most susceptible to the enzymatic action.

A glucoamilase é amplamente utilizada na indústria de alimentos no processamento do amido para a produção de xarope com alto teor de glicose e também muito empregada nos processos de fermentação para produção de cerveja e etanol. Neste trabalho a glucoamilase de Aspergillus awamori expressa em Saccharomyces cerevisiae produzida sob fermentação líquida foi avaliada quanto à produtividade em diferentes amidos e caracterizada físico-quimicamente. A enzima apresentou alta atividade específica de 13,8 U/mg proteína e de 2,9 U/mg biomassa ao final de 48 h de fermentação em meio contendo amido solúvel. A glucoamilase apresentou temperatura ótima de atividade a 55ºC, e temperatura de desnaturação térmica na ausência de substrato por 1h a 50ºC. O pH ótimo de atividade foi na faixa de 3,5 - 4,0 e a estabilidade ao pH entre os valores 5,0 e 7,0. A meia vida a 65ºC foi 30,2 min., e a energia de desnaturação foi de 234.3 KJ mol-1. A hidrólise em diferentes substratos mostrou a preferência da enzima pelos substratos com maior grau de polimerização, sendo o amido de milho gelatinizado o substrato preferencial à ação enzimática.