Production of xylo-oligosaccharides by immobilized-stabilized derivatives of endo-xylanase from Streptomyces halstedii

An endoxylanase from Streptomyces halstedii was stabilized by multipoint covalent immobilization on glyoxyl-agarose supports. The immobilized enzyme derivatives preserved 65% of the catalytic activity corresponding to the one of soluble enzyme that had been immobilized. These immobilized derivatives were 200 times more stable 200 times more stable than the one-point covalently immobilized derivative in experiments involving thermal inactivation at 60 °C. The activity and stability of the immobilized enzyme was higher at pH 5.0 than at pH 7.0. The optimal temperature for xylan hydrolysis was 10 °C higher for the stabilized derivative than for the non-stabilized derivative. On the other hand, the highest loading capacity of activated 10% agarose gels was 75 mg of enzyme per mL of support. To prevent diffusional limitations, low loaded derivatives (containing 0.2 mg of enzyme per mL of support) were used to study the hydrolysis of xylan at high concentration (close to 1% (w/v)). 80% of the reducing sugars were released after 3 h at 55 °C. After 80% of enzymatic hydrolysis, a mixture of small xylo-oligosaccharides was obtained (from xylobiose to xylohexose) with a high percentage of xylobiose and minimal amounts of xylose. The immobilized-stabilized derivatives were used for 10 reaction cycles with no loss of catalytic activity.     

Immobilization and stabilization of a bimolecular aggregate of the lipase from Pseudomonas fluorescens by multipoint covalent attachment

The soluble lipase from Pseudomonas fluorescens (PFL) forms bimolecular aggregates in which the hydrophobic active centers of the enzyme monomers are in close contact. This bimolecular aggregate could be immobilized by multipoint covalent linkages on glyoxyl supports at pH 8.5. The monomer of PFL obtained by incubation of the soluble enzyme in the presence of detergent (0.5% TRITON X-100) could not be immobilized under these conditions. The bimolecular aggregate has two amino terminal residues in the same plane. A further incubation of the immobilized derivative under more alkaline conditions (e.g., pH 10.5) allows a further multipoint attachment of lysine (Lys) residues located in the same plane as the amino terminal residues. Monomeric PFL was immobilized at pH 10.5 in the presence of 0.5% TRITON X-100. The properties of both PFL derivatives were compared. In general, the bimolecular derivatives were more active, more selective and more stable both in water and in organic solvents than the monomolecular ones. The bimolecular derivative showed twice the activity and a much higher selectivity (100 versus 20) for the hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) in aqueous media at pH 5.0 compared to the monomeric derivative. In experiments measuring thermal inactivation at 75 °C, the bimolecular derivative was 5-fold more stable than the monomeric derivative (and 50-fold more stable than a one-point covalently immobilized PFL derivative), and it had a half-life greater than 4 h. In organic solvents (cyclohexane and tert-amyl alcohol), the bimolecular derivative was much more stable and more active than the monomeric derivative in catalyzing the transesterification of olive oil with benzyl alcohol.     

Site-directing an intense multipoint covalent attachment (MCA) of mutants of the Geobacillus thermocatenulatus lipase 2 (BTL2): Genetic and chemical amination plus immobilization on a tailor-made support

Immobilized enzymes are preferred over their soluble counterparts due to their robustness in harsh industrial processes; the most stable enzyme derivatives are often produced through multipoint covalent attachment (MCA). However, most enzymes are unable to establish optimal MCA to electrophile-type supports given the heterogeneous distribution and/or low content of primary amino groups on their surfaces; this restricts both the diversity of areas prone to react and the number of attachments to the support. To overcome this we propose combining site-directed immobilization and protein engineering to increase the number of bonds between a specific enzyme surface and a tailor-made support. We applied this novel strategy to engineered mutants of the lipase 2 from Geobacillus thermocatenulatus with one Cys exposed residue, that after genetic amination and/or chemical amination, were immobilized on glyoxyl-disulfide support using a site-directed MCA protocol. Two highly stabilized derivatives of chemically aminated lipase variants, in which site-directed MCA implied the surrounding surface of residues Cys344 or Cys40, were produced: the first one was 2.4-fold more productive than the reference derivative (648 g of hydrolyzed ester); the second derivative was 40% more selective (EPA/DHA molar ratio) and as active (1 μmol g catalyst⁻¹ min⁻¹) as the reference in the production of PUFAs.     

Efficient removal of toxic phthalate by immobilized serine-type aldehyde-tagged esterase G

Esterase G (EstG) from dibutyl phthalate (DBP)-degrading Sphingobium sp. SM42 was immobilized on amine-functionalized supports through aldehyde tag technology. Two different sulfatase motif tags, either LCTPSR (cysteine-type) or MSAPAR (serine-type), each of which is recognized by a specific formylglycine generating enzyme (FGE), were fused to the C-terminus of EstG. The cysteine-specific FGE was derived from Pseudomonas putida KT2440 while Klebsiella sp. SLS5 provided serine-specific FGE. The EstG with serine-type aldehyde tag showed a greater immobilization yield and higher specific activity by 4.8-fold and 1.8-fold, respectively. The immobilized EstG retained over 90% of its original activity after seven cycles of usage, and exhibited significantly improved thermostability by retaining 66% activity after 1 h incubation at 60 °C. Additionally, nearly 100% and over 30% of the DBP in 10 mM and 100 mM solutions, respectively, was degraded by the immobilized EstG within 18 h.     

Improved yield and stability of amylase by multipoint covalent binding on polyglutaraldehyde activated chitosan beads: Activation of denatured enzyme molecules by calcium ions

In this study raw starch digesting amylase (RSDA) from Aspergillus carbonarius (Bainier) Thom IMI 366159 was stabilized by covalent binding on polyglutaraldehyde (PG), glutaraldehyde (G) activated chitosan beads or post immobilization cross linking of enzyme adsorbed on chitosan. Presence of Ca²⁺ ions (0.5–1.5 mM) activated the PG and G derivatives but repressed the crosslinked enzyme. Optimum pH for cross linked derivative increased by 2 units but was unaltered for PG and G derivatives. Immobilized amylase exhibited improved thermal and storage stability. Immobilized derivatives had no loss of activity after 1 month storage and retained above 90% activity after 10 batch reactions of 60 min each. Immobilization successfully stabilized RSDA and immobilized enzyme from A. carbonarius can be applied in numerous industries for cheap, cost effective and environmentally friendly starch hydrolytic processes to simple sugars.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

Synthetic p-tetrasulphonatocalix[4]arene as novel excipient for lipase-complex

The present article describes formation of excipient-CRL complex from water soluble calix[4]arene derivative (3 as excipient) and Candida rugosa lipase (CRL), which is proposed as a reusable form of enzyme that is free from steric and diffusion limitations associated with those enzymes immobilized onto porous solid supports. The excipient-CRL could completely hydrolyze 50 mM p-nitrophenyl palmitate (p-NPP) in Tris–HCl buffer at a wide range of temperatures, i.e. 30–80 °C. It is stable under stirred conditions and could be reused multiple times without loss of enzyme activity. It was observed that excipient-CRL complex shows a significant effect on the enzyme activity with an enhancement in thermal stability, while pH and temperature affect the activity of excipient-CRL as well as free CRL. Consequently, the excipient-CRL was found more active than free CRL for the hydrolysis of p-NPP in respect of its reusability.     

Improvement of fungal arabinofuranosidase thermal stability by reversible immobilization

A gene encoding α-l-arabinofuranosidase (abfA) from Aspergillus niveus was identified, cloned, and successfully expressed in Aspergillus nidulans. Based on amino acid sequence comparison, the 88.6 kDa enzyme could be assigned to the GH family 51. The characterization of the purified recombinant AbfA revealed that the enzyme was active at a limited pH range (pH 4.0–5.0) and an optimum temperature of 70 °C. The AbfA was able to hydrolyze arabinoxylan, xylan from birchwood, debranched arabinan, and 4-nitrophenyl arabinofuranoside. Synergistic reactions using both AbfA and endoxylanase were also assessed. The highest degree of synergy was obtained after the sequential treatment of the substrate with endoxylanase, followed by AbfA, which was observed to release noticeably more reducing sugars than that of either enzyme acting individually. The immobilization of AbfA was performed via ionic adsorption onto various supports: agarose activated by polyethyleneimine polymers, cyanogen bromide activated Sepharose, DEAE-Sepharose, and Sepharose-Q. The Sepharose-Q derivative remained fully active at pH 5 after 360 min at 60 °C, whereas the free AbfA was inactivated after 60 min. A synergistic effect of arabinoxylan hydrolysis by AbfA immobilized in Sepharose-Q and endoxylanase immobilized in glyoxyl agarose was also observed. The stabilization of arabinofuranosidases using immobilization tools is a novel and interesting topic.     

A response surface methodological study on prediction of glucosylation yields of thiamin using immobilized β-glucosidase

Response surface methodology was used to predict the glucosylation yields of thiamin using immobilized β-glucosidase. A Central Composite Rotatable Design (CCRD) of 32 experiments with immobilized β-glucosidase, thiamin, incubation period, buffer concentration and pH, as five independent variables was employed at five levels. A second-order polynomial equation was developed, the regression coefficient values of which exhibited a R² value of 0.74. Contour plots explained the glucosylation behaviour of the enzyme through a reversal in glucosylation at a cross-over point corresponding to 60% (w/w d-glucose) immobilized β-glucosidase and 0.12 mM buffer concentration at pH 6. The highest conversion yield of 58% obtained experimentally compared well with the predicted yield of 52% under optimum conditions of 40% immobilized β-glucosidase, 0.55 mmol thiamin, 96 h incubation period and 0.16 mM buffer concentration at pH 7. Validation experiments carried out at certain random predictive conditions also showed good correspondence between experimental and predictive yields.     

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme–substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in V_max of immobilized enzyme from 8411.0 to 4919.0 U ml^?1 min^?1 whereas, K_m apparently increased from 1.71 to 3.17 mM ml^?1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.     

Characterization of functionally active immobilized carbonic anhydrase purified from sheep blood lysates

Carbonic anhydrase (CA) catalyzes the reversible reaction of hydration of CO₂ to bicarbonate and the dehydration of bicarbonate back to CO₂. Sequestration of CO₂ from industrial processes or breathing air may require a large amount of highly active and stable CA. Therefore, the objectives of the present study were to purify large amounts of CA from a cheap and easily accessible source of the enzyme and to characterize the enzymatic and kinetic properties of soluble and immobilized enzyme. We recovered 80% of pure enzyme with a specific activity of 4870 EU/mg protein in a single step using sheep blood lysates from slaughter house waste products and CA specific inhibitor affinity chromatography. Since affinity pure CA showed both anhydrase and esterase activities, we measured the esterase activities for enzymology. The Michaelis–Menten constant, K_M, pH optimum, activation energy, and thermal stability of soluble enzymes were 8 × 10^?2 M, 7.3 pH, 7.3 kcal/mol and 70 °C, respectively.The immobilization of the enzyme to Affigel-10 was very efficient and 83% of purified enzyme was immobilized. The immobilized enzyme showed a K_M of 5 × 10^?2 M and activation energy of 8.9 kcal/mol, suggesting a better preference of substrate for immobilized enzyme in comparison to soluble enzyme. In contrast to soluble enzyme, immobilized enzyme showed relatively higher activity at pH 6–8. From these results, we concluded that a shift in pH profile toward acidic pH is due to modification of lysine residues involved in the immobilization process. The immobilized enzyme was stable at higher temperatures and showed highest activity at 80 °C. The activity of immobilized enzyme in a flow reactor at 0.5–2.2 ml/min flow rate was unaffected. Collectively, results from the present study suggested the application of blood lysate waste from animal slaughterhouses for purification of homogeneous enzyme for CO₂ capture in a flow reactor.     

Optimization of the immobilization of sweet potato amylase using glutaraldehyde-agarose support. Characterization of the immobilized enzyme

A simplified procedure for the preparation of immobilized beta-amylase using non-purified extract from fresh sweet potato tubers is established in this paper, using differently activated agarose supports. Beta-amylase glutaraldehyde derivative was the preparation with best features, presenting improved temperature and pH stability and activity. The possibility of reusing the amylase was also shown, when this immobilized enzyme was fully active for five cycles of use. However, immobilization decreased enzyme activity to around 15%. This seems to be mainly due to diffusion limitations of the starch inside the pores of the biocatalyst particles. A fifteen-fold increase in the Km was noticed, while the decrease of Vmax was only 30% (10.1 U mg^?1 protein and 7.03 U mg^?1 protein for free and immobilized preparations, respectively).     

Enhanced activity of immobilized pepsin nanoparticles coated on solid substrates compared to free pepsin

In the present work nanoparticles (NPs) of pepsin were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on low-density polyethylene (PE) films, or on polycarbonate (PC) plates, or on microscope glass slides. The pepsin NPs coated on the solid surfaces have been characterized by HRSEM, TEM, FTIR, XPS and DLS. The amount of enzyme introduced on the substrates, the leaching properties, and the catalytic activity of the immobilized enzyme on the three surfaces are compared. Catalytic activities of pepsin deposited onto the three solid surfaces as well as free pepsin, without sonication, and free pepsin NPs were compared at various pH levels and temperatures using a hemoglobin assay. Compared to native pepsin, pepsin coated onto PE showed the best catalytic activity in all the examined parameters. Pepsin immobilized on glass exhibited better activity than the native enzyme, especially at high temperatures. Enzyme activity of pepsin immobilized on PC was no better than native enzyme activity at all temperatures at pH 2, and only over a narrow pH range at 37 °C was the activity improved over the native enzyme. A remarkable observation is that immobilized pepsin on all the surfaces was still active to some extent even at pH 7, while free pepsin was completely inactive. The kinetic parameters, K_m and V_max were also calculated and compared for all the samples. Relative to the free enzyme, pepsin coated PE showed the greatest improvement in kinetic parameters (K_m = 15 g/L, V_max = 719 U/mg versus K_m = 12.6 g/L and V_max = 787 U/mg, respectively), whereas pepsin coated on PC exhibited the most unfavorable kinetic parameters (K_m = 18 g/L, V_max = 685 U/mg). The values for the anchored enzyme-glass were K_m = 19 g/L, V_max = 763 U/mg.     

Immobilization of phytase on epoxy-activated Sepabead EC-EP for the hydrolysis of soymilk phytate

In this work, an active phytase concentrated extract from soybean sprout was immobilized on a polymethacrylate-based polymer Sepabead EC-EP which is activated with epoxy groups. The immobilized enzyme exhibited an activity of 0.1 U/g of carrier and activity yield of 64.7%. The optimum temperature and pH for the activity of both free and immobilized enzymes were found as 60 °C and pH 5.0, respectively. The immobilized enzyme was more stable than free enzyme in the range of pH 3.0–8.0 and more than 70% of the original activity was recovered. Both the enzymes completely retained nearly about 84% of their original activity at 65 °C. The K_m and V_max values were measured as 5 mM and 0.63 U/mg for free enzyme and 12.5 mM and 0.71 U/mg for immobilized enzyme, respectively. Free and immobilized soybean sprout phytase enzymes were also used in the biodegradation of soymilk phytate. The immobilized enzyme hydrolysed 92.5% of soymilk phytate in 7 h at 60 °C, as compared with 98% hydrolysis observed for the native enzyme over the same period of time. The immobilization procedure on Sepabead EC-EP is very cheap and also easy to carry out, and the features of the immobilized enzyme are very attractive that the potential for practical application is considerable.     

Immobilization of alkaline serine endopeptidase from Bacillus licheniformis on SBA-15 and MCF by surface covalent binding

An industrial enzyme, alkaline serine endopeptidase, was immobilized on surface modified SBA-15 and MCF materials by amide bond formation using carbodiimide as a coupling agent. The specific activities of free enzyme and enzyme immobilized on SBA-15 and MCF were studied using casein (soluble milk protein) as a substrate. The highest activity of free enzyme was obtained at pH 9.5 while this value shifted to pH 10 for SBA-15 and MCF immobilized enzyme. The highest activity of immobilized enzymes was obtained at higher temperature (60 °C) than that of the free enzyme (55 °C). Kinetic parameters, Michaelis–Menten constant (K_m) and maximum reaction velocity (V_max), were calculated as K_m = 13.375, 11.956, and 8.698 × 10^?4 mg/ml and V_max = 0.156, 0.163 and 0.17 × 10^?3 U/mg for the free enzyme and enzyme immobilized on SBA-15 and MCF, respectively. The reusability of immobilized enzyme showed 80% of the activity retained even after 15 cycles. Large pore sized MCF immobilized enzyme was found to be more promising than the SBA-15 immobilized enzyme due to the availability of larger pores of MCF, which offer facile diffusion of substrate and product molecules.     

Immobilization of the cross-linked para-nitrobenzyl esterase of Bacillus subtilis aggregates onto magnetic beads

The para-nitrobenzyl esterase (PNBE), which was encoded by pnbA gene from Bacillus subtilis, was immobilized on amino-functionalized magnetic supports as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE-CLEA immobilized on the magnetic beads using glutaraldehyde as a coupling agent was 31.4 mg/g of beads with a 78% activity recovery after the immobilization. The performance of immobilized PNBE-CLEA was evaluated under various conditions. As compared to its free form, the optimal pH and temperature of PNBE-CLEA were 1 unit (pH 8.0) and 5 °C higher (45 °C), respectively. Under different temperature settings, the residual enzyme activity was highest for the PNBE-CLEA, followed by covalently fixed PNBE without further cross-linking and the free PNBE. During 40 days of storage pried, the PNBE-CLEA maintained more than 90% of its initial activity while the free PNBE maintained about 60% under the same condition. PNBE-CLEA also retained more than 80% activity after 30 reuses with 30 min of each reaction time, indicating stable reusability under aqueous medium.     

Characterization of a thermophilic l-arabinose isomerase from Thermoanaerobacterium saccharolyticum NTOU1

l-Arabinose isomerase (EC 5.3.1.4, l-AI) mainly catalyzes the reversible aldose–ketose isomerization between l-arabinose and l-ribulose. l-AIs can also catalyze other reactions, such as the conversion of d-galactose to d-tagatose. In this study, the araA gene encoding l-AI was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. The recombinant l-AI was purified from the cell-free extract using nickel nitrilotriacetic acid metal-affinity chromatography. The purified enzyme showed an optimal activity at 70 °C and pH 7–7.5. The enzyme was stable at pHs ranging from 6.5 to 9.5 and the activity was fully retained after 2 h incubation at 55–65 °C. The low concentrations of divalent metal ions, either 0.1 mM Mn²⁺ or 0.05 mM Co²⁺, could improve both catalytic activity and thermostability at higher temperatures. The recombinant T. saccharolyticum NTOU1 l-AI has the lowest demand for metal ions among all characterized thermophilic l-AIs. This thermophilic l-AI shows a potential to be used in industry to produce d-tagatose from d-galactose.     

Highly efficient covalent immobilization of catalase on titanate nanotubes

In this study, titanate nanotubes (TNTs) with desirable biocompatibility and hydrophilicity have been synthesized by a facile and cost-effective alkaline hydrothermal method, and used to immobilize the enzyme. The characterization results reveal that the prepared TNTs have a regular tubular morphology with a length about 100–180 nm and an outer diameter about 10 nm, and a BET specific surface area of 305.4 m² g⁻¹. Catalase (CAT), as the model enzyme, was pre-modified by 3-(3,4-dihydroxyphenyl) propionic acid (3,4-diHPP) via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry, and then covalently immobilized on the TNTs surface by the chelation of catechol groups with Ti⁴⁺ ions. It is found that TNTs exhibits excellent performances as the immobilized supporter of enzyme: the enzyme loading is as high as 820 mg g of support⁻¹; the relative activity of immobilized enzyme is about 60% of that of free enzyme; the immobilized CAT demonstrates enhanced storage and recycling stability.     

Laccase mediator system for activation of agarose gel: Application for immobilization of proteins

Cross-linked Sepharose beads were treated with laccase–TEMPO system for oxidation of the primary alcohol groups on the sugar moieties. Optimal activation conditions using Trametes versicolor laccase were at pH 5 and 22 °C, giving an aldehyde content of 55 μmol g⁻¹ Sepharose with 28 units g⁻¹ of laccase and 12.5 mM TEMPO. The activated Sepharose was used for immobilization of trypsin as model protein. Highest degree of immobilization was obtained at pH 10.5 but the activity yield was only 31% of that loaded on the gel. The yield of gel bound trypsin activity was increased to 76% (corresponding to about 43 U g⁻¹ Sepharose) when the immobilization was performed in the presence of trypsin inhibitor, benzamidine. The immobilization yields were comparable to that obtained on the matrix activated using sodium periodate (containing 72 μmol aldehyde per g Sepharose). Recycling and storage of the immobilized trypsin preparations showed high stability of the enzyme bound to laccase–TEMPO activated gel.     

Immobilization of lipase on epoxy-activated Purolite® A109 and its post-immobilization stabilization

In this study, Purolite^® A109, polystyrenic macroporous resin, was used as immobilization support due to its good mechanical properties and high particle diameter (400 μm), which enables efficient application in enzyme reactors due to lower pressure drops. The surface of support had been modified with epichlorhydrine and was tested in lipase immobilization. Optimized procedure for support modification proved to be more efficient than conventional procedure for hydroxy groups (at 22 °C for 18 h), since duration of procedure was shortened to 40 min by performing modification at 52 °C resulting with almost doubled concentration of epoxy groups (563 μmol g⁻¹). Lipase immobilized on epoxy-modified support showed significantly improved thermal stability comparing to both, free form and commercial immobilized preparation (Novozym^® 435). The highest activity (47.5 IU g⁻¹) and thermal stability (2.5 times higher half-life than at low ionic strength) were obtained with lipase immobilized in high ionic strength. Thermal stability of immobilized lipase was further improved by blocking unreacted epoxy groups on supports surface with amino acids. The most efficient was treatment with phenylalanine, since in such a way blocked immobilized enzyme retained 65% of initial activity after 8 h incubation at 65 °C, while non-blocked derivative retained 12%.