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

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

G. Sangiliyandi; T.R. Kannan; K. Chandra Raj; P. Gunasekaran^*

Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai-625 021, India

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.

Key words: Levansucrase, polymerase, sucrase, in vitro mutagenesis, levan, selective modulation.

RESUMO

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.

INTRODUCTION

Zymomonas mobilis is an ethanologenic bacterium, which produces an extracellular levansucrase (SacB), an extracellular sucrase (SacC) and an intracellular sucrase (SacA) (O'Mullan et al., 1991). Among these,levansucrase (SacB) (sucrose: 2,6-ß-D-fructan-6-ß -D-fructosyl transferase; EC 2.4.1.10) forms levan as a byproduct during growth of Z. mobilis in sucrose medium, which reduces the efficacy of ethanol production (Viikari, 1984). Since an improvement in the ethanol yield depended on the loss or reduction of levan-formation in the fermentation medium, an understanding of the levansucrase catalysis is necessary (Kannan et al., 1993). The critical amino acids involved in the separate modulation of the levan-forming ability of SacB have been investigated by genetic or biochemical approaches. The sacB gene (Gunasekaran et al,. 1995), which allowed the easy purification of the enzyme and construction of its variants. The levansucrase expressed in E. coli has been purified and characterized encoding levansucrase has been cloned, sequenced and expressed in Escherichia coli (Sangiliyandi & Gunasekaran, 1998). The selective modulation of the levan-forming activity of the levansucrase by Cys has been demonstrated (Sangiliyandi et al., 1999). Among other sources, levansucrase from Bacillus subtilis has been well studied. So far, the mutation in sacB gene encoding levansucrase resulting in loss of levan-formation without affecting sucrose hydrolysis has been identified only from B. subtilis (Chambert & Petit-Glatron, 1991). We report here for the first time, the sequencing of mutant sacB gene of Z. mobilis encoding levansucrase catalyzing sucrose hydrolysis but not levan-formation and the present results will be helpful in revealing the mechanism of action of levansucrase.

MATERIALS AND METHODS

Bacterial strains and Plasmids:Escherichia coli strains BL21 (DE3) (F^-hsdS, gal), JM109 (recA1, endA1, gyrA96, thi, hsdR17, supE, relA1, (lac^-proAB) [F' traΔ 36, proAB, lacI^q, Δ M15] andXL1 Blue (recA1, lac^-, endA1, gyrA96, thi, hsdR17, supE44, recA1 [F’ proAB, lacI^q, lacΔ M15, Tn10]); plasmids-pBluescriptKS⁺ (Stratagene -USA) and pLS3(Sangiliyandi & Gunasekaran, 1998) were used.

In vitro mutagenesis:In vitro mutagenesis of plasmid DNA was carried out according to Busby et al., (1982) by treating plasmid DNA with 1M hydroxylamine in 50 mM Sodium pyrophosphate (pH 7.0), 100 mM Sodium chloride and 2 mM EDTA at 75°C (2 h). After transforming E. coli JM109 with mutagenised pLS3, levansucrase variants were isolated according to Kannan et al., (1993). Preparation of plasmid DNA and sequencing were carried out as described earlier (Gunasekaran et al., 1995).

Expression of levansucrase:E. coli strain JM109 or XL1Blue or BL21 was transformed with pLS3 or pLS3105 andgrown in M63 medium supplemented with glycerol (20g/l) (MY) or glucose (20g/l) (MG) or fructose (20g/l) (MF) and LB medium, supplemented with ampicillin (50 µg/ml). After 18 h, the cell lysate was assayed for sucrase or levan-forming activity. E. coli cultures were also grown in MY medium to OD₆₀₀ of 1.0, induced with the addition of 0.4 mM IPTG and after 4h, the cell lysate was assayed for enzyme activity (Studier & Moffat, 1986).

Enzyme assay: A reaction mixture containing enzyme extract (100 µl), 1M sucrose (200 µl) and sodium acetate buffer (pH 5.0, 700 µl) was incubated at 60°C for 30 min. and the reducing sugar released was estimated as previously described (Gunasekaran et al., 1995). The sucrase activity was expressed as the amount of enzyme required to release one µmol of reducing sugar in one minute. Levan formed was estimated by measuring the fructose residues after acid hydrolysis of levan (Kannan et al., 1993). Protein was measured by the method of Lowry using bovine serum albumin as a standard.

Zymogram analysis: The cellular proteins of E. coli BL21 (pLS3 or pLS3105) were separated on native PAGE,the gel was incubated at 30°C for 12 h in 5% (w/v) sucrose. Appearance of a slimy white band indicates levan-forming activity. Sucrase activity was identified according to O'Mullan et al., (1991).

RESULTS AND DISCUSSION

Screening of levansucrase mutant: The plasmid pLS3 containing sacB gene was treated with hydroxylamine and transferred in to E. coli JM109. The cell-lysate of the transformants was assayed for sucrase and levan-forming activities. Among 2000 transformants, one produced sucrose-hydrolysing activity but not levan-forming activity. The plasmid containing sacB mutant(encoding defective levansucrase) was isolated from this transformant and designated as pLS3105. In the zymogram analysis, the cellular extracts of E. coli (pLS3) or E. coli (pLS3105) showed the sucrase activity band corresponding to SacB, while a slimy white band of levan corresponding to SacB was showed by cellular extracts of E. coli (pLS3) only (data not shown). These results suggested the selective elimination of levan-forming activity of SacB by in vitro mutation. Similar type of mutants from B. subtilis have been reported but by in vivo mutation of the culture using ethylmethane-sulphonate (Lepasant et al., 1974).

Expression and induction of levansucrase: The expression of sucrose-hydrolysing and levan-forming activity of levansucrase in E. coli BL21 (pLS3) or E. coli BL21 (pLS3105) is shown in Fig. 1. It is obvious that the levan was not formed by the defective sacB expressed in E. coli BL21 (pLS3105), while a maximum of 2.0 U/mg of sucrose-hydrolysing activity was expressed at 21 h of growth. However, E. coli BL21 (pLS3), harboring the wild type sacB gene, expressed a maximum of 4.5 U/mg of sucrase activity and 1.8 U/mg of levan-forming activity.

The comparison of expression of sacB in E. coli (pLS3) and E. coli (pLS3105) was carried out by determining the sucrase activity, since the defective levanscurase did not exhibit the levan-forming activity. When pLS3105 or pLS3 was expressed in various E. coli strains, grown in LB or MY medium, a low level of sucrase activity (0.26-1.23 U/mg) was obtained in E. coli strain JM109 or XL1Blue compared to E. coli BL21 (0.95-4.0 U/mg) (Table 1).

The comparison of expression of sacB in E. coli (pLS3) and E. coli (pLS3105) was carried out by determining the sucrase activity, since the defective levanscurase did not exhibit the levan-forming activity. When pLS3105 or pLS3 was expressed in various E. coli strains, grown in LB or MY medium, a low level of sucrase activity (0.26-1.23 U/mg) was obtained in E. coli strain JM109 or XL1Blue compared to E. coli BL21 (0.95-4.0 U/mg) (Table 1).However, E. coli (pLS3105) expressed only 50% of sucrase compared to E. coli (pLS3), irrespective of growth medium or host strain. Levansucrase synthesis was not inducible with IPTG in E. coli strain JM109 or XL1Blue indicating the expression of levansucrase by its own promoter. But, in E. coli BL21-DE3, expression of levansucrase was induced by IPTG up to 16 fold (Table 1), suggesting the expression of levansucrase by T7 promoter in the plasmid pLS3 or pLS3105. Similarly, sacC gene encoding an extracellular sucrase of Z. mobilis was also overexpressed in E. coli BL21-DE3 up to 70 fold (Kannan et al., 1995).

Sequence analysis of the mutant of sacB

The nucleotide sequence of the sacB mutant gene is shown in Fig. 2. The comparison of this sequence with the already published sequence of sacB gene (Gunasekaran et al., 1995) revealed changes at positions 304 (TTT to CTT) and 781-782 (TGG to AAG) leading to two amino acid changes, Phe-102 to Leu and Trp-261 to Lys. Similarly, replacement of Arg-331 by Lys or Leu resulted in selective elimination of levan-forming activity of B. subtilis levansucrase (Chambert & Petit-Glatron,1991). But, in accordance with our earlier studies on chemical modification of levansucrase (Sangiliyandi et al., 1999), mutation at the site of Cys residues should have been observed for eliminating the levan-forming activity of SacB. Though Cys residues were not affected by in vitro mutation, the two mutations occurred in the vicinity of Cys residue (Fig. 2).

n conclusion, Levansucrase (SacB) of Z. mobilis transfers fructosyl residue from sucrose to water (hydrolysis), mono- (or) di-saccharides (or) fructo-oligosaccharides (levan-formation). The selective elimination of the levan-forming activity of SacB has been a subject of interest to us in understanding it's catalytic mechanism, since SacB reduces the yield of ethanol production by Z. mobilis. The earlier studies on inhibition kinetics indicated the role of Cys in separately modulating the levan-forming activity of SacB (Sangiliyandi et al., 1999). In SacB, three Cys residues are present at positions 121, 151 and 244 that the site of mutations (Phe-102 and Trp-261) were found in this vicinity (Cys-121 and Cys-244). This suggests the possibility of selective modulation of levan-forming activity by altering the amino acid sequence in these vicinities so that the conformation of protein is modified, which eliminates levan-formation. Accordingly, Chambert & Petit-Glatron, (1991) have also suggested by site directed mutation, the stringent steric-requirements for the levansucrase to exhibit the polymerase activity.

ACKNOWLEDGMENTS

This work was supported by grants (No. 38/0912/EMRII) from CSIR, New Delhi and (No. SP/SO/A52/96) from DST, New Delhi. KC Raj and GS thank CSIR and UGC New Delhi for the award of Research Associate and SRF respectively.

Received August 31, 1999;

Accepted September 28, 1999;

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*

Author for correspondence

Publication Dates

• Publication in this collection
  04 Mar 2011
• Date of issue
  1999

History

• Accepted
  28 Sept 1999
• Received
  31 Aug 1999