Immobilization of the recombinant xylanase B (XynB) from the hyperthermophilic Thermotoga maritima on metal-chelate Eupergit C 250L

Esterases, lipases, and serine proteases have been applied as versatile biocatalysts for preparing a variety of chiral compounds in industry via the kinetic resolution of their racemates. In order to meet this requirement, three approaches of enzyme engineering, medium engineering, and substrate engineering are exploited to improve the enzyme activity and enantioselectivity. With the hydrolysis of (R,S)-mandelates in biphasic media consisting of isooctane and pH 6 buffer at 55 degrees C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from V(S)/(E(t)) = 1.62 mmol/h g and V(S)/V(R) = 43.6 of (R,S)-ethyl mandelate (1) for a Klebsiella oxytoca esterase (named as SNSM-87 from the producer) to 16.7 mmol/h g and 867 of (R,S)-2-methoxyethyl mandelate (4) for the enzyme immobilized on Eupergit C 250L. The analysis is then extended to other (R,S)-2-hydroxycarboxylic acid esters, giving improvements of the enzyme performance from V(S)/(E(t)) = 1.56 mmol/h g and V(S)/V(R) = 41.9 of (R,S)-ethyl 3-chloromandelate (9) for the free esterase to 39.4 mmol/h g and 401 of (R,S)-2-methoxyethyl 3-chloromandelate (16) for the immobilized enzyme, V(S)/(E(t)) = 5.46 mmol/h g and V(S)/V(R) = 8.27 of (R,S)-ethyl 4-chloromandelate (10) for free SNSM-87 to 33.5 mmol/h g and 123 of (R,S)-methyl 4-chloromandelate (14) for the immobilized enzyme, as well as V(S)/(E(t)) = 3.0 mmol/h g and V(S)/V(R) = 7.94 of (R,S)-ethyl 3-phenyllactate (11) for the free esterase to 40.7 mmol/h g and 158 of (R,S)-2-methoxyethyl 3-phenyllactate (18) for the immobilized enzyme. The great enantioselectivty enhancement is rationalized from the alteration of ionization constants of imidazolium moiety of catalytic histidine for both enantiomers and conformation distortion of active site after the covalent immobilization, as well as the selection of leaving alcohol moiety via substrate engineering approach.

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“…Similar results were reported from the reduction of band absorption at 4,520 cm À1 in FT-NIR spectra ( Fig. 3 in Li et al, 2007).…”

Section: Enzyme Immobilizationsupporting

confidence: 92%

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization

Wang

Tsai

Chen

2008

Biotech & Bioengineering

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“…In this case, TMAI weight fraction decreased from the initial value of about 50% to the final one of 8.7%, thus proving the higher selectivity of Eu-Cu support toward TMAI. These results are coherent with the ones observed in the immobilization of the (His) 6 -tagged xylanase B from T. maritima (Li et al, 2007) on Nickel-chelate Eupergit ® C250L. Furthermore, after 48 h of enzyme-support interaction, most proteins remained bound to either support, not being released after washing steps with either water or a solution with imidazole (comparison of lines C3 and C4, respectively, with line H), indicating covalent immobilization.…”

Section: Purification and Characterization Of Tmaisupporting

confidence: 92%

“…This suggests that adsorption of TMAI through His tags resulted in fewer conformational changes which could lower the enzyme activity of the adsorbed protein. Similar results were found by (Li et al, 2007) who found Y T equal to 14.8% with Eu and a value of 65% with Nickel-chelate Eupergit ® C250L.…”

Section: Purification and Characterization Of Tmaisupporting

confidence: 92%

Immobilization of the recombinant (His)6-tagged l-arabinose isomerase from Thermotoga maritima on epoxy and cupper-chelate epoxy supports

Bortone

Fidaleo

2015

Food and Bioproducts Processing

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“…For example, the increase in laccase load from 22 to 440 mg/g resulted in the decrease of activity recovery: from 36.1 to 21.1% for Eupergit C and more so, from 88.4 to 43.8%, in the case of Eupergit C 250L. It is generally acknowledged that the immobilisation efficiency of enzymes on solid supports decreases when enzyme loading exceeds a certain value (Knezevic et al 2006;Li et al 2007). In fact, it was observed that Eupergit carriers presented a saturation value of laccase of 440 mg protein/g support.…”

Section: Immobilisation Of Laccasementioning

confidence: 99%

Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed reactor

et al. 2011

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Laccase from Myceliophthora thermophila was covalently immobilised on Eupergit C and Eupergit C 250L yielding specific activities of up to 17 and 80 U/g, respectively. Due to its superior activity, Eupergit C 250L was chosen for further research. The somewhat lower catalytic efficiency (based on the ratio between the turnover number and the Michaelis constant, k cat /K M ) of the immobilised enzyme in comparison with that of the free enzyme was balanced by its increased stability and broader operational window related to temperature and pH. The feasibility of the immobilised laccase was tested by using a packed bed reactor (PBR) operating in consecutive cycles for the removal of Acid Green 27 dye as model substrate. High degrees of elimination were achieved (88, 79, 69 and 57% in 4 consecutive cycles), while the levels of adsorption on the support varied from 18 to 6%, proving that dye removal took place mainly due to the action of the enzyme. Finally, a continuous PBR with the solid biocatalyst was applied for the treatment of a solution containing the following endocrine disrupting chemicals: estrone (E1), 17b-estradiol (E2) and 17a-ethinylestradiol (EE2). At steady-state operation, E1 was degraded by 65% and E2 and EE2 were removed up to 80% and only limited adsorption of these compounds on the support, between 12 and 22%, was detected. In addition, a 79% decrease in estrogenic activity was detected in the effluent of the enzymatic reactor while only 14% was attained by inactivated laccase.

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Immobilization of the recombinant xylanase B (XynB) from the hyperthermophilic Thermotoga maritima on metal-chelate Eupergit C 250L

Cited by 18 publications

References 35 publications

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization

Immobilization of the recombinant (His)6-tagged l-arabinose isomerase from Thermotoga maritima on epoxy and cupper-chelate epoxy supports

Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed reactor

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