Gene Cloning,Expression And Biochemical Characteristics Of Novel Feruloyl Esterases From Bacillus Amyloliquefaciens H47

Feruloyl esterases?FAEs,EC 3.1.1.73? are a subclass of the carboxylic esterases that hydrolyze the ester bonds between ferulate acid and polysaccharides in plant cell walls.Feruloyl esterases have wide application prospects in food,feed processing,paper making,medicine,cosmetics,biological energy and other fields.To date,few of feruloyl esterases from bacteria have been reported.Moreover,biochemical characteristics of feruloyl esterases from bacillus spp.have not been reported.In this study,the feruloyl esterases from bacillus were expressed and enzymology properties were researched firstly.The main research results are showed as follows:1)A strain with high activity of FAEs,Bacillus amyloliquefaciens H47 was screened from 122 Bacillus spp.preserved in our laboratory.2)The whole genome sequences of B amyloliquefaciens H47 were analyzed,and ten potential feruloyl esterases were predicted.The ten sequences are as follows:Ba Fae01?906bp?,Ba Fae02?780 bp?,Ba Fae03?774 bp?,Ba Fae04?768 bp?,Ba Fae05?732 bp?,Ba Fae06?723 bp?,Ba Fae07?744 bp?,Ba Fae08?696 bp?,Ba Fae09?741 bp?,Ba Fae10?822 bp?.The10 genes were cloned and expressed,and three feruloyl esterases?Ba Fae04,Ba Fae06,and Ba Fae09?were identified according to enzyme activity.The apparent molecular masses of Ba Fae04,Ba Fae06,and Ba Fae09 were approximately 30,29,and 31 k Da,respectively.Comparing with other bacterial FAEs,these novel feruloyl esterases exhibited low sequence identities?less than 30%?.3)Enzymology properties of the three overexpressed proteins were researched.Ba Fae04,Ba Fae06,and Ba Fae09 showed maximal activity with an optimal p H of 8.0 at 30°C,35°C and 40°C,respectively.Ba Fae04 showed high stability in the temperature range of 25–60°C for 1 h and retained 59%of its activity at 60°C.The profiles of 52 esterase substrates showed that the three feruloyl esterases had a broad substrate spectrum and could effectively hydrolyze several common feruloyl esterases substrates.Furthermore,the three feruloyl esterases also can release ferulic acid from destarched wheat bran.The kinetic constants for methyl ferulate,ethyl ferulate,methyl p-coumarate,and chlorogenic acid were as follows:for Ba Fae04,the K_m values were 1.14,2.15,0.78 and 6.08 mmol·L^-1,respectively,and the k_cat/K_m values were 10.57,11.72,11.40 and 45.31 L·mmol^-1·s^-1,respectively;for Ba Fae06,the K_m values were 1.99,0.58,1.42 and 1.51 mmol·L^-1,and the k_cat/K_m values were 4.32,5.48,30.90and 4.32 L·mmol^-1·s^-1,respectively;for Ba Fae09,the K_m values were 0.49,0.86,1.81 and4.79 mmol·L^-1,and the k_cat/K_m values were 0.69,1.23,1.21 and 2.52 L·mmol^-1·s^-1,respectively.The present study displays some useful characteristics of FAEs for potential industrial application and contributes to our understanding of FAEs.