Directed evolution of a β‐1,3‐1,4‐glucanase from Bacillus subtilis MA139 for improving thermal stability and other characteristics

Abstract

In order to improve some characteristics of a β-1,3-1,4-glucanase from Bacillus subtilis MA139, directed evolution was conducted in this study. After error-prone PCR, the β-1,3-1,4-glucanase gene, glu-opt, was cloned into the vector pBGP1 and transformed into Pichia pastoris X-33 to construct a mutant library. Three variants named as 7-32, 7-87, and 7-115 were screened from 8000 colonies. Amino-acid sequence analysis showed that these mutants had one or two amino-acid substitutions (7-32: T113S, 7-87: M44V/N53H, and 7-115: N157D). The variants were over-expressed in P. pastoris by methanol induction. After purification of the enzyme proteins, the characteristics of the variants were analyzed in detail. It indicated that these mutant enzymes had broader ranges of pH value and better pH stability than the wild-type enzyme. The mutant enzyme 7-87 had the best ability to tolerate an acid environment (pH 2.0), while the wild-type enzyme had no activity under this condition. Moreover, all these mutants demonstrated improved thermal stability. In particular, the mutant enzyme 7-32 had residual enzymatic activity of 60% and 40% after being incubated at 80 °C and 90 °C for 10 min. While, the wild-type enzyme had no residual enzymatic activity after being incubated at 80 °C for 4 min. In addition, the mutant enzymes had better tolerance to some chemicals than the wild-type enzyme. The improved stability could enhance the prospects for this enzyme to have use in the feed industry to reduce the effects of the anti-nutritional factor β-glucan.

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

“…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 ).…”

“…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. Urea slightly inhibited the enzyme activity, similar result was reported by Wang et al ( 2007 ).…”

“…The K m value of the purified enzyme (0.62 mg β-1,3–1,4-glucan/mL) was similar to that reported for fungal β-1,3–1,4-glucanases from Neocallimastix patriciarum (0.67 mg/mL) and Malbranchea cinnamomea (0.69 mg/mL) (Hung et al 2012 ; Yang et al 2014 ) and lower than the k m value of β-1,3–1,4-glucanases from Thermotoga maritima MSB8 (0.78 mg/mL) (Khan et al 2007 ), Bacillus subtilis MA139 (0.91 mg/mL) (Pei et al 2015 ), Bacillus pumilus US570 (2.2 mg/mL) (Elgharbi et al 2017 ), and the recombinant β-1,3–1,4-glucanase cloned from Clostridium Thermocellum (2.7 mg/mL) (Luo et al 2014 ). The V max value (7936 U/mg) was higher than that reported for β-1,3–1,4-glucanase enzyme from truncated Fibrobacter succinogenes 7833 U/mg (Chen et al 2010 ) and for the recombinant β-1,3–1,4-glucanase enzyme from Bacillus altitudinis YC-9 (7692 U/mg) (Mao et al 2013 ), and Bacillus amyloliquefaciens and Bacillus tequilensis CGX5-1 (7500 U/mg) (Sun et al 2012 ; Wang et al 2014 ).…”

“…X4 and Bacillus sp. SJ-10 cultures (Baek et al 2017 ; Tak et al 2019 ), and for both wild type and mutant β-1,3–1,4-glucanase purified from Bacillus subtilis MA139 (Pei et al 2015 ). Also, it was higher than that reported for recombinant β-1,3–1,4-glucanase purified from Saccharophagus degradans (30 °C) (Lafond et al 2016 ), and Bacillus velezensis ZJ20 (35 °C) (Xu et al 2016 ).…”