Separation of levan-formation and sucrose-hydrolysis catalyzed by levansucrase of Zymomonas mobilis using in vitro mutagenesis

Abstract

A levansucrase (SacB) of Zymomonas mobilis capable of sucrose hydrolysis but not levan formation was isolated through invitro mutagenesis of cloned sacB gene. When the sacB mutant gene was expressed in Escherichiacoli strains, only 50% of the sucrose-hydrolysing activity (2.0 U/mg) was produced, compared to the wild type levansucrase (4.0 U/mg). Sequencing of the sacB mutant gene revealed changes of two amino acid residues (Phe-102 to Leu and Trp-261 to Lys in the levansucrase). The absence of mutation at the site of Cys of SacB is contradictory to the inhibition kinetics that demonstrated the involvement of Cys in conferring the levan-forming activity to the SacB. The present finding is useful in understanding the mechanism of selective modulation of levan-forming (polymerase) activity of levansucrase.<br>Um levansucrase (SacB) do Zymomonas mobilis capaz de hidrolisar a sucrose mas não de produzir levan foi isolado completamente in vitro, do gene clonado de sacB. Quando o gene do mutante sacB foi expressado em cadeias de Escherichia coli, somente 50% da atividade sucrose-hydrolysing (2,0 U/mg) foi produzida, comparado ao levansucrase tipo selvagem 4,0 U/mg). Sequência do sacB (gene mutante) revelou mudanças de dois resíduos do amino-ácido (Phe-102 ao Leu e Trp-261 ao Lys no levansucrase). A ausência de mutação no local do Cys de SacB é contraditória à inibição do cinética que demonstrou a participação da Cys em conferir a atividade de formação do levan ao SacB. A presenta descoberta é útil ao entendimento do mecanismo da modulação seletiva da formação do levan (polymerase) atividade do levansucrase

Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

“…These results suggested that the expression of sacB in either Z. mobilis or E. coli was much lower than the expression of sacC gene, probably due to inefficiency of sacB promoter. However, it is observed that extracellular sucrase (SacC) was unstable than extracellular levansucrase (SacB) Sangiliyandi et al, 1999a;Sangiliyandi et al, 1999b). A high relative abundance of sacC specific transcripts might compensate for the instability of the Z. mobilis extracellular sucrase.…”

“…The optimum temperature for polymerase activity was at 30°C and hydrolase activity at 50°C. However, the polymerase activity was selectively abolished by para-chloromercuribenzoate (Sangiliyandi et al, 1999a) and also by in-vitro mutagenesis (Sangiliyandi et al,1999b). Recently, a levansucrase mutant was isolated by in-vitro mutagenesis using hydroxylamine.…”