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      Proteolytic activities of bacteria, yeasts and filamentous fungi isolated from coffee fruit (Coffea arabica L.) Translated title: Atividade proteolítica de bactérias, leveduras e fungos filamentosos presentes em grãos de café (Coffea arabica L.)

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          Abstract

          One hundred forty-four microorganisms previously isolated from coffee fruit (Coffea arabica) were grown on casein agar to evaluate their proteolytic activities. Fifty percent of filamentous fungi, 52.5% of bacteria and 2.6% of yeasts were able to secrete proteases. Positive isolates were further examined in liquid culture for their protease activities by hydrolysis of casein at different pH values (5.0, 7.0 and 9.0) at 30 º C. Bacillus megaterium, B. subtilis, Enterobacter agglomerans, Kurthia sp, Pseudomonas paucimobilis and Tatumella ptyseos demonstrated the highest proteolytic activities at pH 9.0. One yeast isolate, Citeromyces matritensis, had a proteolytic activity of 2.40 U at pH 5.0. Aspergillus dimorphicus, A. ochraceus, Fusarium moniliforme, F. solani, Penicillium fellutanum and P. waksmanii showed the highest activities. Of the bacterial isolates, the highest enzyme activities were observed in B. subtilis 333 (27.1 U), Tatumella ptyseos (27.0 U) and B. megaterium 817 (26.2 U). Of the filamentous fungi, Aspergillus ochraceus (48.7 U), Fusarium moniliforme 221 (37.5 U) and F. solani 359 (37.4 U) had the highest activities at pH 9.0.

          Translated abstract

          Este trabalho teve por objetivos avaliar a capacidade de secreção de proteases extracelulares por 144 microrganismos, previamente isolados de grãos de café (Coffea arabica) durante fermentação por via seca, e determinar a atividade das enzimas produzidas. Os microrganismos foram cultivados em ágar-caseína para avaliação da produção de enzimas proteolíticas. Dos 40 isolados de bactéria presentes na amostra, 52,5% apresentaram resultado positivo para o teste. Considerando os 66 isolados de fungos filamentosos, 50% foram capazes de secretar proteases, enquanto que dos 38 isolados de leveduras, apenas 2,6% conseguiram promover a hidrólise da caseína do meio. Os isolados que apresentaram capacidade de secreção de proteases foram, posteriormente, cultivados em meio líquido para a determinação da atividade enzimática em diferentes valores de pH (5,0, 7,0 e 9,0) a 30 º C. Os isolados Aspergillus ochraceus (48,7 U), Fusarium moniliforme 221 (37,5 U) e F. solani 359 (37,4 U) apresentaram os melhores resultados de atividade enzimática, o que foi verificado em pH 9,0. Também neste pH os isolados Bacillus subtilis 333 (27,1 U), Tatumella ptyseos (27,0 U) e B. megaterium 817 (26,2 U) apresentaram maior valor de atividade. A levedura Citeromyces matritensis apresentou atividade em pH 5,0 (2.40 U).

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          Most cited references34

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          Developments in industrially important thermostable enzymes: a review.

          G Haki (2003)
          Cellular components of thermophilic organisms (enzymes, proteins and nucleic acids) are also thermostable. Apart from high temperature they are also known to withstand denaturants of extremely acidic and alkaline conditions. Thermostable enzymes are highly specific and thus have considerable potential for many industrial applications. The use of such enzymes in maximising reactions accomplished in the food and paper industry, detergents, drugs, toxic wastes removal and drilling for oil is being studied extensively. The enzymes can be produced from the thermophiles through either optimised fermentation of the microorganisms or cloning of fast-growing mesophiles by recombinant DNA technology. In this review, the source microorganisms and properties of thermostable starch hydrolysing amylases, xylanases, cellulases, chitinases, proteases, lipases and DNA polymerases are discussed. The industrial needs for such specific thermostable enzyme and improvements required to maximize their application in the future are also suggested.
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            Investigations on protease production by a wild-type Aspergillus terreus strain using diluted retentate of pre-filtered palm oil mill effluent (POME) as substrate

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              Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus.

              An investigation was carried out on the purification and characterization of an alkaline protease from Bacillus pumilus MK6-5. An alkalophilic Bacillus pumilus MK6-5 was grown in a laboratory fermenter containing 1% reverse osmosis concentrated cheese whey powder, 0.25% corn steep liquor, 1% glucose, 0.5% tryptone, 1% sodium citrate, 0.02% MgSO4.7H2O and 0.65% Na2CO3 at 35 degrees C and pH 9.6, agitation at 250 rev min(-1) and aeration of 1 vvm for 60 h. When the enzyme was purified using ammonium sulphate precipitation, ion exchange and gel filtration chromatographies, a 26.2% recovery of enzyme with 36.6-fold purification was recorded. The purified protease was found to be homogenous by SDS-PAGE with molecular mass estimate of 28 kDa. The enzyme was optimally active at pH 11.5 and temperature of 55-60 degrees C. The Km and kcat values observed with synthetic substrates at 37 degrees C and pH 8.0 were 1.1 mmol l(-1) and 624 s(-1) for Glu-Gly-Ala-Phe-pNA and 3.7 mmol l(-1) and 826 s(-1) for Glu-Ala-Ala-Ala-pNA, respectively. The kinetic data revealed that small aliphatic and aromatic residues were the preferred residues at the P1 position. Inhibition profile exhibited by PMSF suggested the B. pumilus protease to be an alkaline serine protease. Bacillus pumilus MK6-5 produced a calcium-dependent, thermostable alkaline serine protease. The thermostable alkaline protease from Bacillus pumilus MK6-5 will be extremely useful in ultrafiltration membrane cleaning due to its ability to work in broad pH and temperature ranges, and tolerance to detergents, unlike the mesophilic proteases which face these limitations.
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                Author and article information

                Contributors
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Journal
                asagr
                Acta Scientiarum. Agronomy
                Acta Sci., Agron.
                Editora da Universidade Estadual de Maringá - EDUEM (Maringá )
                1807-8621
                September 2011
                : 33
                : 3
                : 457-464
                Affiliations
                [1 ] Universidade Federal de Juiz de Fora Brazil
                [2 ] Universidade Federal de Lavras Brazil
                [3 ] Universidade Federal de Lavras Brazil
                Article
                S1807-86212011000300011
                10.4025/actasciagron.v33i3.6734
                9bf04257-9378-43f2-bcf9-09aff81070a4

                http://creativecommons.org/licenses/by/4.0/

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                SciELO Brazil

                Self URI (journal page): http://www.scielo.br/scielo.php?script=sci_serial&pid=1807-8621&lng=en
                Categories
                AGRONOMY

                Horticulture
                microbial enzyme,protease,coffee fermentation,enzimas microbianas,fermentação de café

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