Thermostable α-galactosidase fromBacillus stearothermophilusNUB3621: cloning, sequencing and characterization

Abstract

An alpha-galactosidase gene from the thermophilic bacterium Bacillus stearothermophilus NUB3621 was cloned, sequenced, expressed in Escherichia coli and the recombinant protein was purified. The Bacillus enzyme, designated AgaN, is similar to alpha-galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme was estimated to be a tetramer with a molecular mass of subunits 80.3 kDa. The purified AgaN is thermostable and has a temperature optimum of activity at 75 degrees C and a half-life of inactivation of 19 h at 70 degrees C. AgaN displays high affinity for oligomeric substrates such as melibiose and raffinose and is able to hydrolyze raffinose in the presence of 60% sucrose with high efficiency.

Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering

“…Our genome sequence data indicated that GsNUB3621 possesses two genes that encode alpha-galactosidases. One matched the previously published GsNUB3621 agaN sequence (Fridjonsson et al 1999), while the other was more distantly related. The former homologue was PCR-amplified from GsNUB3621 and cloned downstream of surT -P surP , thereby completing our surT -P surP - agaN -pNW33N expression vector.…”

Genomic analysis of six new Geobacillus strains reveals highly conserved carbohydrate degradation architectures and strategies

“…The intracellular α-galactosidases of MC52 and MC61 share 100% identity with each other and 98% identity with the G. stearothermophilus α-galactosidase identified as AgaA (Merceron et al, 2012 ). The intracellular α-galactosidase of YS93 shares only 81–82% identity with G. stearothermophilus AgaA and theα-galactosidases of MC52 and MC61, but shares 93% identity with the G. stearothermophilus α-galactosidase identified as AgaN (Fridjonsson et al, 1999 ).…”

“…Other Geobacillus species have been identified as sources of thermostable xylanase (Gerasimova and Kuisiene, 2012 ; Liu et al, 2012 ; Verma and Satyanarayana, 2012 ; Anand et al, 2013 ; Bhalla et al, 2014 ), with all the enzymes showing properties similar to those of the G. stearothermophilus enzyme. A range of other enzymes with potential industrial applications have been identified in Geobacillus species including α-galactosidases (Fridjonsson et al, 1999 ; Merceron et al, 2012 ) for use in soy processing, β-galactosidases (Goodman and Pederson, 1976 ; Hirata et al, 1984 , 1986 ; Solomon et al, 2013 ) for use in milk processing, lipases (Jeong et al, 2001 ; Sinchaikul et al, 2002 ; Abdul Rahman et al, 2009 ; Ebrahimpour et al, 2011 ; Balan et al, 2012 ) and proteases (Nishiya and Imanaka, 1990 ; Jang et al, 1992 ; Hawumba et al, 2002 ; Chen et al, 2004 ; Itoi et al, 2006 ) for use in detergents, and amylases (Sen and Oriel, 1989 ; Brumm et al, 1991 ; Narang and Satyanarayana, 2001 ; Kamasaka et al, 2002 ; Ferner-Ortner-Bleckmann et al, 2009 ; Mok et al, 2013 ; Nasrollahi et al, 2013 ) for use in corn wet milling, baking and ethanol production.…”

Some (bacilli) like it hot: genomics of Geobacillus species

“…Industrially important enzymes originating from Geobacillus spp. include lipases (Schmidt-Dannert et al ., 1998 ), glycoside hydrolases (Fridjonsson et al ., 1999 ; Bartosiak-Jentys et al ., 2013 ; Suzuki et al ., 2013 ), N-acylhomoserine lactonase (Seo et al ., 2011 ) and DNA polymerase I (Sandalli et al ., 2009 ) and protease (Chen et al ., 2004 ) among others. The advantages of using thermophilic bacteria as whole-cell biocatalysts were recently discussed in this journal (Taylor et al ., 2011 ) and include reduced risk of contamination, acceleration of biochemical processes and easier maintenance of anaerobic conditions.…”