PMC:3395577 / 2164-6234 JSONTXT

{"project":"MicrobeTaxon","denotations":[{"id":"T4","span":{"begin":1098,"end":1103},"obj":"3193"},{"id":"T5","span":{"begin":2858,"end":2901},"obj":"1517"},{"id":"T6","span":{"begin":3112,"end":3144},"obj":"1517"},{"id":"T7","span":{"begin":3335,"end":3367},"obj":"1517"},{"id":"T8","span":{"begin":3528,"end":3560},"obj":"1517"},{"id":"T9","span":{"begin":1782,"end":1805},"obj":"5068"},{"id":"T10","span":{"begin":1827,"end":1835},"obj":"5061"},{"id":"T11","span":{"begin":1818,"end":1827},"obj":"5062"},{"id":"T12","span":{"begin":1848,"end":1859},"obj":"63131"},{"id":"T13","span":{"begin":2689,"end":2694},"obj":"4751"},{"id":"T14","span":{"begin":2699,"end":2707},"obj":"2"},{"id":"T15","span":{"begin":2838,"end":2846},"obj":"2"},{"id":"T16","span":{"begin":2848,"end":2857},"obj":"2"},{"id":"T17","span":{"begin":3922,"end":3954},"obj":"1517"},{"id":"T66","span":{"begin":3112,"end":3144},"obj":"1517"}],"namespaces":[{"prefix":"_base","uri":"http://purl.bioontology.org/ontology/NCBITAXON/"}],"text":"Introduction\nCellulosic biomass is the most abundant renewable resource on earth, whose natural degradation represents an important part of the carbon cycle within the biosphere [1]. β-Glucosidase (EC 3.2.1.21) is a glucosidase enzyme that acts upon β 1–4 bonds linking two glucose or glucose-substituted molecules. It is an important component of the cellulase enzyme system. The limiting step in the enzymatic saccharification of cellulosic material is the conversion of short-chain oligosaccharides and cellobiose, which was resulted from the synergistic action of endogucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91), to glucose, a reaction catalyzed by β-glucosidases [2]. It is well established that cellobiose inhibits the activities of most cellobiohydrolases and endoglucanses [3]. β-glucosidases reduce cellobiose inhibition by hydrolyzing this disaccharide to glucose, thus allowing the cellulolytic enzymes to function more efficiently [4,5]. Furthermore, β-glucosidase is used as a flavor enzyme to enhance the flavor of wine, tea and fruit juice [6,7]. In fruits and other plant tissues many secondary metabolites, including flavor compounds, are accumulated in their glucosylated form [8,9]. Because β-glucosides constitute the majority of the known glycoconjugated flavor compounds, β-glucosidases play an important role in flavor liberation from these precursors. Therefore, producing high-activity and glucose-tolerant β-glucosidase has become important.\nRecently, the search for β-glucosidases insensitive to glucose has increased significantly, for these enzymes would improve the process of saccharification of lignocellulosic materials. A few microbial β-glucosidases have been reported to tolerate glucose [10-14]. For example, β-glucosidases from Aspergillus tubingensis CBS 643.92, A. oryzaeA. niger CCRC 31494, A. foetidus, and marine microbial metagenome displayed high inhibition constant by glucose (Ki) of 600 mM, 1390 mM, 543 mM, 520 mM, and 1000 mM, respectively. But these β-glucosidases have considerably lower specific activity for cellobiose than for p-nitrophenyl-β-D-glucopyranoside. Therefore, over-expression of thermostable β-glucosidase with high glucose tolerance and specific activity for cellobiose abilities will help shed light on degradation of cellulosic biomass.\nThermostable enzymes have several generic advantages, allowing a decreased amount of enzyme needed because of higher specific activity and elongated hydrolysis time due to higher stability. In addition, thermostable enzymes are generally more tolerant and allow more flexibility in process configurations [15,16]. Although some glucose-tolerant β-glucosidases from fungi and bacteria have been reported [10-14], the glucose-tolerant β-glucosidases genes have not been expressed and characterized from thermophilic bacteria. Bacterium Thermoanaerobacterium thermosaccharolyticum is a strict anaerobe that grows on wide range of hexose and pentose at temperature from 37°C to 75°C, which have attracted considerable interests to hydrogen production and thermostable enzyme production [17]. T. thermosaccharolyticum DSM 571 could utilize cellobiose, but the gene for β-glucosidase, the key enzyme in degradation cellobiose, was not reported in the Genbank (NC_014410.1). Because the optimal growth temperature for T. thermosaccharolyticum DSM 571 was at 60°C, the thermostable β-glucosidase could have a considerable potential for industrial applications. Owing to the inherent difficulty of cultivation of T. thermosaccharolyticum DSM 571, it is difficult to obtain a sufficient amount of cells for large-scale enzyme production. For the production of the recombinant protein, genetic engineering is the first choice because it is easy, fast, and cheap.\nIn this paper, we report the phylogenesis analysis, cloning, over-expression, and detailed biochemical characterization of the β-glucosidase from T. thermosaccharolyticum DSM 571. The favorable properties make the β-glucosidase a good candidate for utilization in biotechnological applications."}

{"project":"2_test","denotations":[{"id":"22571470-14538100-7770684","span":{"begin":179,"end":180},"obj":"14538100"},{"id":"22571470-20006926-7770685","span":{"begin":685,"end":686},"obj":"20006926"},{"id":"22571470-11272024-7770686","span":{"begin":798,"end":799},"obj":"11272024"},{"id":"22571470-20181208-7770687","span":{"begin":962,"end":963},"obj":"20181208"},{"id":"22571470-21294581-7770688","span":{"begin":1212,"end":1213},"obj":"21294581"},{"id":"22571470-11330708-7770689","span":{"begin":1741,"end":1743},"obj":"11330708"},{"id":"22571470-20890102-7770689","span":{"begin":1741,"end":1743},"obj":"20890102"},{"id":"22571470-11052758-7770689","span":{"begin":1741,"end":1743},"obj":"11052758"},{"id":"22571470-17589813-7770690","span":{"begin":2630,"end":2632},"obj":"17589813"},{"id":"22571470-21592333-7770691","span":{"begin":2633,"end":2635},"obj":"21592333"},{"id":"22571470-11330708-7770692","span":{"begin":2728,"end":2730},"obj":"11330708"},{"id":"22571470-20890102-7770692","span":{"begin":2728,"end":2730},"obj":"20890102"},{"id":"22571470-11052758-7770692","span":{"begin":2728,"end":2730},"obj":"11052758"},{"id":"22571470-21464600-7770693","span":{"begin":3107,"end":3109},"obj":"21464600"}],"text":"Introduction\nCellulosic biomass is the most abundant renewable resource on earth, whose natural degradation represents an important part of the carbon cycle within the biosphere [1]. β-Glucosidase (EC 3.2.1.21) is a glucosidase enzyme that acts upon β 1–4 bonds linking two glucose or glucose-substituted molecules. It is an important component of the cellulase enzyme system. The limiting step in the enzymatic saccharification of cellulosic material is the conversion of short-chain oligosaccharides and cellobiose, which was resulted from the synergistic action of endogucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91), to glucose, a reaction catalyzed by β-glucosidases [2]. It is well established that cellobiose inhibits the activities of most cellobiohydrolases and endoglucanses [3]. β-glucosidases reduce cellobiose inhibition by hydrolyzing this disaccharide to glucose, thus allowing the cellulolytic enzymes to function more efficiently [4,5]. Furthermore, β-glucosidase is used as a flavor enzyme to enhance the flavor of wine, tea and fruit juice [6,7]. In fruits and other plant tissues many secondary metabolites, including flavor compounds, are accumulated in their glucosylated form [8,9]. Because β-glucosides constitute the majority of the known glycoconjugated flavor compounds, β-glucosidases play an important role in flavor liberation from these precursors. Therefore, producing high-activity and glucose-tolerant β-glucosidase has become important.\nRecently, the search for β-glucosidases insensitive to glucose has increased significantly, for these enzymes would improve the process of saccharification of lignocellulosic materials. A few microbial β-glucosidases have been reported to tolerate glucose [10-14]. For example, β-glucosidases from Aspergillus tubingensis CBS 643.92, A. oryzaeA. niger CCRC 31494, A. foetidus, and marine microbial metagenome displayed high inhibition constant by glucose (Ki) of 600 mM, 1390 mM, 543 mM, 520 mM, and 1000 mM, respectively. But these β-glucosidases have considerably lower specific activity for cellobiose than for p-nitrophenyl-β-D-glucopyranoside. Therefore, over-expression of thermostable β-glucosidase with high glucose tolerance and specific activity for cellobiose abilities will help shed light on degradation of cellulosic biomass.\nThermostable enzymes have several generic advantages, allowing a decreased amount of enzyme needed because of higher specific activity and elongated hydrolysis time due to higher stability. In addition, thermostable enzymes are generally more tolerant and allow more flexibility in process configurations [15,16]. Although some glucose-tolerant β-glucosidases from fungi and bacteria have been reported [10-14], the glucose-tolerant β-glucosidases genes have not been expressed and characterized from thermophilic bacteria. Bacterium Thermoanaerobacterium thermosaccharolyticum is a strict anaerobe that grows on wide range of hexose and pentose at temperature from 37°C to 75°C, which have attracted considerable interests to hydrogen production and thermostable enzyme production [17]. T. thermosaccharolyticum DSM 571 could utilize cellobiose, but the gene for β-glucosidase, the key enzyme in degradation cellobiose, was not reported in the Genbank (NC_014410.1). Because the optimal growth temperature for T. thermosaccharolyticum DSM 571 was at 60°C, the thermostable β-glucosidase could have a considerable potential for industrial applications. Owing to the inherent difficulty of cultivation of T. thermosaccharolyticum DSM 571, it is difficult to obtain a sufficient amount of cells for large-scale enzyme production. For the production of the recombinant protein, genetic engineering is the first choice because it is easy, fast, and cheap.\nIn this paper, we report the phylogenesis analysis, cloning, over-expression, and detailed biochemical characterization of the β-glucosidase from T. thermosaccharolyticum DSM 571. The favorable properties make the β-glucosidase a good candidate for utilization in biotechnological applications."}