Purification and characterization of a novel lichenase from Bacillus licheniformis GZ‐2

Abstract

A novel lichenase from Bacillus licheniformis GZ-2 was purified to homogeneity by two steps ion-exchange chromatography with a specific activity of 8231.3 U/mg. The purified enzyme showed as a single protein band with a molecular mass of 25 kDa. The optimum pH and temperature for the enzyme activity were 6.5 and 60 °C, respectively. The enzyme exhibited strict specificity for β-1,3-1,4-d-glucans. The kinetic parameters Km and Vmax were 5.11 mg/mL and 2097 µmol/Min/mg for lichenan and 7.42 mg/mL and 1440 µmol/Min/mg for barley β-glucan. Compared to most of the reported β-1,3-1,4-glucanases (lichenase), the activity of the purified enzyme for lichenan was much higher than that for barley β-glucan. The main products of β-glucan hydrolyzed by the lichenase were cellubiosyltriose (DP3) and cellutriosyltraose (DP4). The lichenase gene from B. licheniformis GZ-2 was cloned and sequenced. The open reading frame of gene gz-2 contained 642 bp coding for a 214 amino acid mature protein. The gene was cloned into an expression vector pET 28a and expressed in Escherichia coli BL21. The activity in cell lysate supernatant was 137.9 U/mg.

Purification, characterization, immobilization and applications of an enzybiotic β-1,3–1,4-glucanase produced from halotolerant marine Halomonas meridiana ES021

“…This result was dissimilar to the activating effect of Mg ++ ions recorded by Jung et al ( 2010 ), Cheng et al ( 2014 ), Zhang et al ( 2017 ) and Schröder et al ( 2018 ). Partial inhibition of the enzyme occurred by high concentration of Ca ++ , while no change in activity observed with low level, similarly to these data slight inhibition with Ca ++ ions was reported by Apiraksakorn et al ( 2008 ), Elgharbi et al ( 2013 ) and Wang et al ( 2014 ) and dissimilarly Ca ++ ions made activation for other β-1,3–1,4-glucanases (Gao 2016 ; Elgharbi et al 2017 ; Bernardi et al 2018 ). Complete inhibition of Halomonas meridiana ES021 β-1,3–1,4-glucanase was also observed with Cu ++ ions, similarly to Yang et al ( 2008 ), Elgharbi et al ( 2013 ) and Pei et al ( 2015 ), this was disagreed with the activating effect reported by Celestino et al ( 2006 ), Wang et al ( 2007 ), Yang et al ( 2014 ) and Niu et al ( 2016b ) for the same metal ion.…”

“…In contrast with these results some investigators reported an inhibitory effect of Co ++ ions on β-1,3–1,4-glucanase (Chaari et al 2012 ; Elgharbi et al 2013 ; Chaari et al 2014 ; Wang et al 2014 ; Elgharbi et al 2017 ; Ali et al 2019 ). The enzyme was also slightly activated in presence of Mn ++ , this result agreed with the data reported by (Teng et al 2007 ; Kim et al 2014 ; Yan et al 2018 ), while disagreed with (Wang et al 2007 ; Apiraksakorn et al 2008 ; Hong et al 2009 ; Luo et al 2014 ; Pei et al 2015 ; Gao 2016 ) who reported partial inhibition effect of Mn ++ ions while complete inhibition of the enzyme was reported by (Yang et al 2008 ; Elgharbi et al 2013 ; Chaari et al 2014 ; Kim et al 2014 ; Elgharbi et al 2017 ).…”

“…Co ++ was the best activating metal ion with a relative activity about 157.38% which was higher than those reported for bacterial β-1,3–1,4-glucanases from Bacillus altitudinis YC-9 (124.78%) (Mao et al 2013 ), Bacillus licheniformis GZ-2 (116.6%) (Gao 2016 ), and fungal β-1,3–1,4-glucanases from Paecilomyces thermophila (117%) (Yang et al 2008 ), Laetiporus sulphureus var. miniatus (109%) (Hong et al 2009 ), albranchea cinnamomea (147.2%) (Yang et al 2014 ), while it is slightly lower the relative activities of fungal β-1,3–1,4-glucanases from Aspergillus fumigatus (161.1%) (Bernardi et al 2018 ), and Thermoascus aurantiacus (159.8%) (Yan et al 2018 ).…”

“…On the other hand, Halomonas meridiana ES021 β-1,3–1,4-glucanase was partially inhibited by low concentration of Ni ++ , while the high concentration completely inhibited it. The inhibitory effect of Ni ++ was also reported by Yang et al ( 2008 ) in contrast with Hong et al ( 2009 ), Yang et al ( 2014 ), Gao ( 2016 ) and Bernardi et al ( 2018 ) as they reported activation of β-1,3–1,4-glucanase with Ni ++ ions. Mg ++ ions inhibited β-1,3–1,4-glucanase activity, similar effect was reported by Celestino et al ( 2006 ), Wang et al ( 2007 ), Apiraksakorn et al ( 2008 ), Yang et al ( 2008 ), Hong et al ( 2009 ), Chaari et al ( 2014 ), Yang et al ( 2014 ) and Elgharbi et al ( 2017 ).…”

Small RNA mediated repression of subtilisin production in Bacillus licheniformis

“…(II) Since the organisms live on organic matter they are evolved for high concentrations of nutrient supply which enables the them to reach high cell densities in fermentations 7 . Finally, (III) they secrete high amounts of many efficient bio-polymer hydrolases 8 like lichenases 9 , lipases 10 , thermostable alpha-amylases 11 , and many more 12–20 . Exoproteases of the subtilisin Carlsberg family are an industrial fermentation product of B .…”