Production of polysaccharides and oligosaccharides from immobilized dextransucrase produced from honey bacterial isolates

Abstract

The present investigation deals with the study of microbial dextransucrase. It included studies aiming to searching for a potent microorganism producing dextransucrase isolated from honey. The studies also included optimization of fermentation parameters for maximal enzyme production and partial purification of the potent enzyme. Moreover, studies on enzyme stabilization by immobilization on different carriers were also included. The comparative study between the properties of the partially purified and immobilized enzyme was also carried out. Finally, The immobilized enzyme used for the production of dextran with different molecular weights and production of oligosaccharides. The biological activities of the products were tested. The first part of this work was undertaken to investigate the productivity of dextransucrase and levansucrase by different bacterial honey isolates. Of twelve tested microorganisms, the bacteria (isolate 2) was found to be the most potent for dextransucrase production (45.19 U/ml) with no levansucrase production after 24 h. So, it was used throughout the study. The results are summarized as follow: • The selected dextransucrase producing strain was identified according to its morphological criteria and phylogenetic analysis (16s rRNA) that showed a high level of similarity (99%) with Enterococcus faecalis CGLBL115.

• The produced dextran was precipitated by ethanol and then acid hydrolysis was undertaken for the precipitate and the products of hydrolysis were illustrated chromatographically showing glucose only which evidence for presence of dextran only.

• The safety of selected bacteria were undertaken by measuring the antibiotic sensitivity test and found that the bacteria are sensitive to all antibiotics with different concentrations and also the blood haemolysis test (ability to break down the blood) was tested and found that it had no ability to breakdown red blood cells which indicates safe using of bacteria for human products.

• Different cultivation parameters were investigated for the optimization of dextransucrase by Enterococcus faecalis. The maximum activity of enzyme (45.19 U/ml) was obtained after 24 hours incubation.

• The shaking and static techniques of fermentation were studied and found that shaking technique was the most potent technique for dextransucrase production after 24 hours incubation.

• Different concentrations of wheat were added to sucrose in the production medium resulted in 1.5 g/ 50 ml is the most potent concentration for dextransucrase production (45.63 U/ ml).

• The effect of different organic (yeast extract, peptone, corn steep liquor, urea, baker’s yeast and soya bean) and inorganic nitrogen sources (sodium nitrate) in the production of dextransucrase by Enterococcus faecalis was also studied. Among all the used nitrogen sources yeast extract achieved the highest dextransucrase activity (45.20 U/ml). Also corn steep liquor concentrations were studied for the production of maximal dextransucrase but still yeast extract is the most potent nitrogen source for dextransucrase production.

• Also, Maximal dextransucrase production was observed at 0.3% K2HPO4(46.37 U/ml). • When the effect of some additives on the production of dextransucrase was studied, maximum enzyme activity was obtained in culture medium consisted of in (g/L): Sucrose, 100; K2HPO4, 3; MgSO4.7H2O, 0.2; yeast extract, 1and wheat, 20.

• The study was extend to explore the optimization of Enterococcus faecalisdextransucrase by using statistical based experimental designs:

1- In the first namely Plackett-Burman design, seven independent variables were screened in organized 11 combinations. The activity of dextransucrase is increased to 63.22 U/ml with 1.36 fold when compared to the basal medium.

2- The second namely, Central Composite Design, The coded and levels of the three independent variables investigated at five different levels. The activity of dextransucrase increased to 70.67 U/ml with 1.52 fold when compared to the basal medium.

• The second part of this study was extended to partial purification and characterization of the produced dextransucrase from Enterococcus faecalis.

• Partial purification of dextransucrase enzyme was achieved by fractional precipitation of crude enzyme by ethanol, acetone and ammonium sulfate. A weak recovery of enzyme from the total enzyme activity was obtained which results to subjecting the enzyme to ultrafiltration and lyophilization.

• The third part of this study was the stabilization of the partially purified enzyme by immobilization by using two methods. In this part Enterococcus faecalis dextransucrase was immobilized on water insoluble carriers by different immobilization techniques, this include: physical adsorption, covalent binding. Of all the tested immobilization techniques, the highest immobilization yield (94.35 %)and activity (90.23 U/g carrier) were observed by covalent binding to Alg.CMC (2:3) FeCl3 (PEI/GA), respectively. Thus, Alg.CMC (2:3) FeCl3 (PEI/GA)was chosen as an ideal carrier through this study.