Limits...
An efficient method for the immobilization of inulinase using new types of polymers containing epoxy groups.

Trytek M, Fiedurek J, Podkościelna B, Gawdzik B, Skowronek M - J. Ind. Microbiol. Biotechnol. (2015)

Bottom Line: A tenfold increase in the speed of flow resulted in an increase in the yield of oligosaccharides (DP2-DP6) up to ~41% in the overall hydrolysate, as analysed by HPLC-RID and LC-ESI/MS.These results indicate that two forms of inulinase, an exo- and an endo-acting enzyme, were immobilized on our carrier.There were no significant decreases in the efficiency of continuous hydrolysis during this time (about 17.4% in comparison to its initial value).

View Article: PubMed Central - PubMed

Affiliation: Department of Industrial Microbiology, Institute of Microbiology and Biotechnology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka St. 19, 20-033, Lublin, Poland, mtrytek1@tlen.pl.

ABSTRACT
New glycidyl methacrylate copolymers containing different numbers of epoxy groups were synthesized and used to develop effective procedures for inulinase immobilization. The beneficial characteristics of the carriers included a high degree of crosslinking, stability at ambient temperature, an appropriate surface, and the presence of reactive epoxy groups. Some factors affecting the efficiency of immobilization of crude inulinase, including the kind and amount of carrier, the number of epoxy groups, as well as buffer pH and buffer concentration were examined. The yield of immobilization of this enzyme on the investigated type of microspheres was higher than on the commercial carrier, Eupergit(®) C. After immobilization, the optimum temperature for inulinase activity shifted from 55 to 45 °C, whereas the optimum pH = 5 remained unchanged. The basic parameters of inulin hydrolysis were examined, and the possibility of applying the obtained biocatalyst in continuous conditions was tested. Inulin at a concentration of 0.5% (w/v) was almost completely hydrolyzed to fructose (in a yield of 98 %) at a flow rate of 0.1 mL/min. A tenfold increase in the speed of flow resulted in an increase in the yield of oligosaccharides (DP2-DP6) up to ~41% in the overall hydrolysate, as analysed by HPLC-RID and LC-ESI/MS. These results indicate that two forms of inulinase, an exo- and an endo-acting enzyme, were immobilized on our carrier. The enzyme showed good operational stability in a packed column over 28 days. There were no significant decreases in the efficiency of continuous hydrolysis during this time (about 17.4% in comparison to its initial value).

No MeSH data available.


Immobilization yield of crude inulinase on the newly synthesized polymer microspheres and the commercial carrier, Eupergit®. Conditions: 1 M phosphate buffer, pH = 7
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4464867&req=5

Fig4: Immobilization yield of crude inulinase on the newly synthesized polymer microspheres and the commercial carrier, Eupergit®. Conditions: 1 M phosphate buffer, pH = 7

Mentions: In a further study, carried out under optimal conditions for Eupergit® C (data not shown), we examined the ability of the four carriers to immobilize inulinase from crude post-culture medium (Fig. 4). A decision was made to use crude inulinase (as a concentrated medium obtained from a culture of A. niger) instead of the purified enzyme, because the latter, being an endoinulinase, did not hydrolyze sucrose. When the culture supernatant was used, fructose was produced from inulin, suggesting that apart from endoinulinase, an exo-acting enzyme was also present in the culture filtrate [38]. The highest efficiency of inulinase immobilization (15.4 %) was achieved for BES.DM-GMA (1:1) and the lowest for the carriers BES.DM-GMA (1:6) (3.29 %) and BES.DM-GMA-ST (1:1:3) (3.33 %). This result correlates with differences in the porosity of the polymers and may suggest that the yield of immobilization depends on their specific surface area. Ettalibi and Baratti [6] immobilized inulinase from Aspergillus ficuum on porous glassware activated with 3-aminopropyltriethoxysilane in toluene reaching an efficiency of inulinase immobilization varying from 29 % (3.000 Å) to 71 % (80 Å), depending on pore diameter. A high immobilization yield (82.6 % of inulinase specific activity) was also obtained by Paula et al. [28], in a gelatin–water support after treatment with glutaraldehyde as a cross-linking reagent. The authors did not report the performance of the obtained biocatalyst in semi-continuous conditions, though. In another study, partially purified exoinulinase was immobilized onto Amino-cellulofine using glutaraldehyde as the cross-linking agent, with an immobilization yield of 15 % based on the enzyme activity [13].Fig. 4


An efficient method for the immobilization of inulinase using new types of polymers containing epoxy groups.

Trytek M, Fiedurek J, Podkościelna B, Gawdzik B, Skowronek M - J. Ind. Microbiol. Biotechnol. (2015)

Immobilization yield of crude inulinase on the newly synthesized polymer microspheres and the commercial carrier, Eupergit®. Conditions: 1 M phosphate buffer, pH = 7
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4464867&req=5

Fig4: Immobilization yield of crude inulinase on the newly synthesized polymer microspheres and the commercial carrier, Eupergit®. Conditions: 1 M phosphate buffer, pH = 7
Mentions: In a further study, carried out under optimal conditions for Eupergit® C (data not shown), we examined the ability of the four carriers to immobilize inulinase from crude post-culture medium (Fig. 4). A decision was made to use crude inulinase (as a concentrated medium obtained from a culture of A. niger) instead of the purified enzyme, because the latter, being an endoinulinase, did not hydrolyze sucrose. When the culture supernatant was used, fructose was produced from inulin, suggesting that apart from endoinulinase, an exo-acting enzyme was also present in the culture filtrate [38]. The highest efficiency of inulinase immobilization (15.4 %) was achieved for BES.DM-GMA (1:1) and the lowest for the carriers BES.DM-GMA (1:6) (3.29 %) and BES.DM-GMA-ST (1:1:3) (3.33 %). This result correlates with differences in the porosity of the polymers and may suggest that the yield of immobilization depends on their specific surface area. Ettalibi and Baratti [6] immobilized inulinase from Aspergillus ficuum on porous glassware activated with 3-aminopropyltriethoxysilane in toluene reaching an efficiency of inulinase immobilization varying from 29 % (3.000 Å) to 71 % (80 Å), depending on pore diameter. A high immobilization yield (82.6 % of inulinase specific activity) was also obtained by Paula et al. [28], in a gelatin–water support after treatment with glutaraldehyde as a cross-linking reagent. The authors did not report the performance of the obtained biocatalyst in semi-continuous conditions, though. In another study, partially purified exoinulinase was immobilized onto Amino-cellulofine using glutaraldehyde as the cross-linking agent, with an immobilization yield of 15 % based on the enzyme activity [13].Fig. 4

Bottom Line: A tenfold increase in the speed of flow resulted in an increase in the yield of oligosaccharides (DP2-DP6) up to ~41% in the overall hydrolysate, as analysed by HPLC-RID and LC-ESI/MS.These results indicate that two forms of inulinase, an exo- and an endo-acting enzyme, were immobilized on our carrier.There were no significant decreases in the efficiency of continuous hydrolysis during this time (about 17.4% in comparison to its initial value).

View Article: PubMed Central - PubMed

Affiliation: Department of Industrial Microbiology, Institute of Microbiology and Biotechnology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka St. 19, 20-033, Lublin, Poland, mtrytek1@tlen.pl.

ABSTRACT
New glycidyl methacrylate copolymers containing different numbers of epoxy groups were synthesized and used to develop effective procedures for inulinase immobilization. The beneficial characteristics of the carriers included a high degree of crosslinking, stability at ambient temperature, an appropriate surface, and the presence of reactive epoxy groups. Some factors affecting the efficiency of immobilization of crude inulinase, including the kind and amount of carrier, the number of epoxy groups, as well as buffer pH and buffer concentration were examined. The yield of immobilization of this enzyme on the investigated type of microspheres was higher than on the commercial carrier, Eupergit(®) C. After immobilization, the optimum temperature for inulinase activity shifted from 55 to 45 °C, whereas the optimum pH = 5 remained unchanged. The basic parameters of inulin hydrolysis were examined, and the possibility of applying the obtained biocatalyst in continuous conditions was tested. Inulin at a concentration of 0.5% (w/v) was almost completely hydrolyzed to fructose (in a yield of 98 %) at a flow rate of 0.1 mL/min. A tenfold increase in the speed of flow resulted in an increase in the yield of oligosaccharides (DP2-DP6) up to ~41% in the overall hydrolysate, as analysed by HPLC-RID and LC-ESI/MS. These results indicate that two forms of inulinase, an exo- and an endo-acting enzyme, were immobilized on our carrier. The enzyme showed good operational stability in a packed column over 28 days. There were no significant decreases in the efficiency of continuous hydrolysis during this time (about 17.4% in comparison to its initial value).

No MeSH data available.