A Bifunctional Fusion Enzyme Of Bacillus-sourced Laccase And Endoglucanase Expressed In Escherichia Coli And Deinking Application

Fungal laccase and endoglucanase have been used in old newsprint deinking research andobtained certain achievements, however, most fungal enzymes had the maximum activity at lowpH,but,deinking process conditions usually were at neutral or alkaline pH. The activity of fungalenzymes would reduce increasingly thus effecting the quality of deinking.This paper we clonedBllac and Bleg from Bacillus licheniformis7172, on this basis, fusion genes of Bllac–Bleg wereconstructed by ligase. The characteristics of recombinant proteins as well as the quality ofdeinking were also studied.Bllac and Bleg from Bacillus licheniformis7172have been cloned in pET-28b(+) andexpressed in Escherichia coli BL21，respectively. SDS-PAGE analysis of purified recombinantBlLac and BlEG displayed protein bands of about60kDa and55kDa, respectively. The optimaltemperature of recombinant BlLac and BlEG were70℃and55℃respectively and the optimalpH value were4.5and6.5respectively. After incubation at40℃,50℃,60℃,70℃for45min,recombinant BlLac maintained more than half of its original activity and retained80%of theresidual enzyme activity over the pH range of4.0to6.0. Recombinant BlEG maintained morethan60%of its original activity after incubation at45℃,50℃and55℃for30min, while,completely lost all activity after45min at60℃,65℃,70℃, the enzyme activity decreasedrapidly after60℃. The recombinant BlLac maintained more than80%of its original activity overthe pH range of6.0to8.0. The specific activities of purified recombinant BlLac and BlEG were635.95±6.25U/μmol and297.08±15.58U/μmol, for ABTS and CMC, respectively.Fusion gene was successfully constructed and expressed in Escherichia coli BL21.SDS-PAGE analysis of purified recombinant fusion enzyme displayed protein bands located at120kDa, The optimal temperature of BlLac-BlEG were the same as BlLac and BlEG. Theoptimal pH value of recombinant BlLac-BlEG were5.0and6.5, respectively. After incubation at60℃for30min, laccase activities of BlLac-BlEG retained60%of the residual enzyme activity,while, incubation at60℃for45min, only retained40%of the residual enzyme activity; Thelaccase activities of BlLac-BlEG maintained more than80%of its original activity over the pHrange of4.0to4.5. After incubation at55℃for30min, endoglucanase activities of BlLac-BlEGretained half of its original activity, the endoglucanase activities of BlLac-BlEG maintained50%of the residual enzyme activity over the pH range of5.0to8.0. The specific activity of fusionenzyme were874.19±22.45U/μmol and385.99±10.72U/μmol, for ABTS and CMC,respectively.The optimal temperature of BlLac、BlEG and BlLac-BlEG for remove ink particles were60℃，45℃and55℃, respectively, while, the results showed little difference over the pH rangeof6.0to8.0. The optimal temperature of BlLac、BlEG and BlLac-BlEG for improve the pulpbrightness were55℃、45℃and55℃, respectively, while, the BlLac、BlEG and BlLac-BlEG improve the pulp brightness best were at pH6.0、7.0、7.0, respectively. The results showed thatlaccase improve the pulp brightness better than endoglucanase, while, endoglucanase remainedless ink particles.