Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques

Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi-subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross-linking of previously immobilized enzyme with tailor-made dextran-aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl-agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl-agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross-linked with dextran-aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity.     

Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase.

The five glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase were each purified from extracts of Zymomonas mobilis cells, by using dye-ligand chromatography as the principal step. Two procedures, producing three and two of the enzymes respectively, are described in detail. Z. mobilis glyceraldehyde-phosphate dehydrogenase was found to be similar in most respects to the enzyme from other sources, except for having a slightly larger subunit size. Phosphoglycerate kinase has properties typical for this enzyme; however, it did not show the sulphate activation effects characteristic of this enzyme from most other sources. Phosphoglycerate mutase is a dimer, partially independent of 2,3-bisphosphoglycerate, and has a high specific activity. Enolase was found to be octameric; otherwise its properties were very similar to those of the yeast enzyme. Pyruvate kinase is unusual in being dimeric, and not requiring K+ for activity. It is not allosterically activated by sugar phosphates, having a high activity in the absence of any effectors. Some quantitative differences in the relative amounts of these enzymes, compared with eukaryotic species, are ascribed to the fact that Z. mobilis utilizes the Entner-Doudoroff pathway rather than the more common Embden-Meyerhoff glycolytic route.     

小分子封闭提高固定化嗜热菌蛋白酶的热稳定性

为了提高固定化嗜热菌蛋白酶的热稳定性,在制备共价固定化嗜热菌蛋白酶的基础上,通过选择氨基酸和醇类小分子来封闭载体表面未反应的活化基团,并考察了固定化酶的催化活性及热稳定性。结果发现：L-Trp和L-Val封闭修饰固定化酶时,在80℃的水浴中加热150 min后其剩余活力仍为93.4%和98.6%,其效果约为未经小分子封闭的固定化嗜热菌蛋白酶的2倍。所筛选的几种小分子物质中,叔戊醇、L-Trp、L-Val及L-Ala不仅能提高固定化嗜热菌蛋白酶的热稳定性,而且也可以提高固定化酶的相对活力,从而更有利于其在工业生产中的应用。     

Preparation of an immobilized–stabilized catalase derivative from Aspergillus niger having its multimeric structure stabilized: The effect of Zn on enzyme stability

Multimeric catalase from Aspergillus niger was immobilized on CNBr activated agarose, increasing the enzyme stability. However, it was found that some enzyme subunits could be desorbed to the supernatant after boiling the enzyme preparation in the presence of SDS or during thermal inactivation. Moreover, a positive enzyme concentration-enzyme stability correlation was detected in the immobilized preparation. This suggested the existence of some dissociation mechanism as a first step in the enzyme inactivation. The treatment of the immobilized enzyme with aldehyde–dextran permitted to fully stabilize its multimeric structure, but even this preparation exhibited an enzyme concentration-stability correlation. The presence of EDTA reduced the enzyme stability, suggesting that some cation could be involved in enzyme stability. It was found that 10 mM Zn²⁺ increased the enzyme stability of this immobilized–stabilized preparation. Now, the dilution of the biocatalyst did not produce a reduction in the enzyme stability.Thus, we have prepared an immobilized enzyme that does not release any subunit to the medium even after inactivation, and found that Zn²⁺ has a very positive effect on the stability of this immobilized–stabilized enzyme.     

Stabilization of a highly active but unstable alcohol dehydrogenase from yeast using immobilization and post-immobilization techniques

The alcohol dehydrogenase (ADH) from Baker's yeast is very active but extremely unstable under several different conditions. Mild immobilization methods such as one-point attachment to agarose activated with cyanogen bromide groups or ionic adsorption to agarose activated with charged groups allow high activity recoveries (80–100%) but do not promote protein stabilization. In contrast, immobilization methods that force the enzyme to be covalently attached at multiple points on the support fully inactivate the enzyme. Herein, we propose an interesting solution to address the dichotomy between activity and stability. We have developed a protocol in which the enzyme is immobilized on agarose activated with glyoxyl groups in the presence of acetyl cysteine, which results in the recovery of 25% of the enzyme activity but increases the thermal stability of the soluble enzyme 50-fold. However, this immobilization technique does not stabilize the enzyme quaternary structure. Hence, a post-immobilization technique using functionalized polymers has been used to cross-link all enzyme subunits. In this method, polycationic polymers (polyethylenimine) cross-link the quaternary structure with a negligible effect on catalytic activity, which results in a derivative that is 5-fold more stable than non-cross-linked derivatives under very dilute and acidic conditions that highly favor subunit dissociation. Therefore, the stability was increased 500-fold for this optimal derivative compared to diluted soluble enzyme, although the relative expressed activity was low (25%). However, the low expressed activity may be overcome by designing immobilized biocatalysts with high volumetric activities.     

Muscle contraction: viscous-like frictional forces and the impulsive model

The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.     

Immobilization of pig muscle enolase. Studies on the activity of subunits

The native dimeric form of enolase from pig muscle was immobilized on Sepharose 4B activated with cyanogen bromide. The amount of matrix-bound enolase, its specific activity and kinetic properties depend on the extent of gel activation with CNBr. Only on the Sepharose activated with small quantities of CNBr the amount of protein which remained after treatment with Gdn.HCl was about 50% of the initially bound enolase, indicating that the enzyme was bound covalently to the matrix through a single subunit. The matrix-bound monomers obtained in this way were inactive and were unable to reassociate to dimers on addition of free subunits. The matrix-bound monomers obtained after KBr treatment were inactive but retained the ability to reassociate into active dimers after addition of free subunits. The results indicate that single matrix-bound subunits of pig muscle enolase are enzymatically inactive and dimeric structure is essential for catalytic activity.     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric Cα structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

Immobilized diol dehydrase and its use in studies of cobalamin binding and subunit interaction.

Coenzyme B12 dependent diol dehydrase from Aerobacter aerogenes was immobilized by covalent binding to CNBr-activated Sepharose 4B. The Sepharose-bound enzyme exhibited a markedly high catalytic activity, viz., 75-95% of the specific activity of the original free enzyme. The apoenzyme acquired much greater stability to heat by immobilization. No significant difference between the immobilized and free enzymes was observed in the following properties: the affinity for coenzyme B12; the sensitivity to a sulfhydryl-modifying agent; the absolute requirement for a certain monovalent cation, such as K+, for catalysis; the susceptibility toward oxygen upon incubation with coenzyme B12 in the absence of substrate. These results suggest that the structure and function of the enzyme are not significantly influenced by immobilization on Sepharose. The immobilized enzyme was found to provide a convenient method for a study of ligand interaction with the enzyme. The subunit interaction between two dissimilar subunits, components F and S, was investigated using the component S immobilized on CNBr-activited Sepharose and free component F, and it was demonstrated that the substrate (1,2-propanedoil) promotes the hybrid formation between component F and component S, but K+ alone rather retarded the subunit association to some extent. Na+ markedly weakens the forces which bind the subunits together. The relationship between cobalamin binding and subunit structure is also discussed.     

Immobilization of Aspergillus oryzae tannase and properties of the immobilized enzyme

Tannase enzyme from Aspergillus oryzae was immobilized on various carriers by different methods. The immobilized enzyme on chitosan with a bifunctional agent (glutaraldehyde) had the highest activity. The catalytic properties and stability of the immobilized tannase were compared with the corresponding free enzyme. The bound enzyme retained 20·3% of the original specific activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme. The optimum temperature of the reaction was determined to be 40 °C for the free enzyme and 55 °C for the immobilized form. The stability at low pH, as well as thermal stability, were significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilized enzyme retained about 85% of the initial catalytic activity, even after being used 17 times.     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric C structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

Immobilization of goat brain aminopeptidase B in calcium alginate beads

Aminopeptidase B, an arginyl aminopeptidase, was purified from goat brain with a purification factor of ～280 and a yield of 2.7%. It was entrapped in calcium alginate together with bovine serum albumin. The optimal conditions for immobilization for maximum activity yield were 1% CaCl₂ and 2.5% alginate. The immobilized enzyme retained ～62% of its initial activity and could be used for five successive batch reactions with retention of 30% of the initial activity. The pH and temperature optima of the free and immobilized enzyme were pH 7.4, 45°C and pH 7.8, 50°C respectively, while the pH and thermal stability as well as the stability of the enzyme in organic solvents were improved significantly after entrapment. The K_m value for the immobilized enzyme was about twofold higher than that of the soluble enzyme. Because of this increased stability, the immobilized enzyme may be useful in the meat processing industry.     

A smart bioconjugate of chymotrypsin

alpha-Chymotrypsin was immobilized on Eudragit S-100 via covalent coupling with 93% retention of proteolytic activity. The conjugate behaved as a smart biocatalyst and functioned as a pH-dependent reversibly soluble-insoluble biocatalyst. The pH optimum of chymotrypsin broadened on immobilization, and the immobilized preparation showed better stability at and above pH 6.5 as compared to the free enzyme. The immobilized enzyme showed a slight shift in the temperature optimum and enhanced thermal stability retaining 70% of its original activity after 1 h of exposure to 40 degrees C as compared to the 25% residual activity for the free enzyme under identical conditions. K(m) and V(max) values did not change on immobilization. Also, the immobilized preparation was quite stable to reuse, it retained almost 85% of its original activity even after a fifth precipitation cycle. UV spectroscopy and circular dichroism were used to probe structural changes in the enzyme upon immobilization.     

Structure and conformational stability of a tetrameric thermostable N‐succinylamino acid racemase

The N‐succinylamino acid racemases (NSAAR) belong to the enolase superfamily and they are large homooctameric/hexameric species that require a divalent metal ion for activity. We describe the structure and stability of NSAAR from Geobacillus kaustophilus (GkNSAAR) in the absence and in the presence of Co²⁺ by using hydrodynamic and spectroscopic techniques. The Co²⁺, among other assayed divalent ions, provides the maximal enzymatic activity at physiological pH. The protein seems to be a tetramer with a rather elongated shape, as shown by AU experiments; this is further supported by the modeled structure, which keeps intact the largest tetrameric oligomerization interfaces observed in other homooctameric members of the family, but it does not maintain the octameric oligomerization interfaces. The native functional structure is mainly formed by α‐helix, as suggested by FTIR and CD deconvoluted spectra, with similar percentages of structure to those observed in other protomers of the enolase superfamily. At low pH, the protein populates a molten‐globule‐like conformation. The GdmCl denaturation occurs through a monomeric intermediate, and thermal denaturation experiments indicate a high thermostability. The presence of the cofactor Co²⁺ did alter slightly the secondary structure, but it did not modify substantially the stability of the protein. Thus, GkNSAAR is one of the few members of the enolase family whose conformational propensities and stability have been extensively characterized. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 757–772, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Immobilization of glucooligosaccharide oxidase of Acremonium strictum for oligosaccharic acid production

Summary The glucooligosaccharide oxidase was covalently immobilized to chitosan with polyethyleneimine and glutaraldehyde. Immobilization improved thermal stability. When used for conversion of starch hydrolysate to oligosaccharic acids, the immobilized enzyme maintained 75% initial activity after 60 days of continuous operation. Strong substrate inhibition was seen at high concentrations of cellobiose and lactose for free enzyme but not for immobilized enzyme.     

The mechanism of cobalamin binding to hog intrinsic factor

Chicken brain Arylsulfatase A (E.C.3.1.6.1) was immobilized by interaction with Concanavalin A. The immobilized enzyme retained its catalytic activity and this enzyme can be reused without appreciable loss of activity. The storage stability of bound and soluble enzymes was comparable and binding of enzyme to Concanavalin A increases its thermal stability. Kinetic studies indicated that bound enzyme shows similar anomalous kinetics as that of free enzyme but slight change was observed in relation to pH optima, Km value and activation energy.     

Direct immobilization of tyrosinase enzyme from natural mushrooms (Agaricus bisporus) on D-sorbitol cinnamic ester

Mushroom tyrosinase was immobilized from an extract onto the totally cinnamoylated derivative of D-sorbitol by direct adsorption as a result of the intense hydrophobic interactions that took place. The immobilization pH value and mass of lyophilized mushrooms were important parameters that affected the immobilization efficiency, while the immobilization time and immobilization support concentration were not important in this respect. The extracted/immobilized enzyme could best be measured above pH 3.5 and the optimum measuring temperature was 55 degrees C. The apparent Michaelis constant using 4-tert-butylcatechol as substrate was 0.38+/-0.02 mM, which was lower than for the soluble enzyme from Sigma (1.41+/-0.20 mM). Immobilization stabilized the extracted enzyme against thermal inactivation and made it less susceptible to activity loss during storage. The operational stability was higher than in the case of the tyrosinase supplied by Sigma and immobilized on the same support. The results show that the use of p-nitrophenol as enzyme-inhibiting substrate during enzyme extraction and immobilization made the use of ascorbic acid unnecessary and is a suitable method for extracting and immobilizing the tyrosinase enzyme, providing good enzymatic activity and stability.     

Studies on the properties of immobilized urokinase: effects of pH and temperature

Human urokinase was immobilized on an ethylene vinyl acetate copolymer surface. Soluble urokinase showed its maximum activity at pH 8.5, while the immobilized enzyme was most active at pH 9.0. Apparently, the shift in optimal pH was due to the polyanionic nature of the carrier surface on which the enzyme was immobilized. Optimal temperatures of soluble urokinase and immobilized enzyme were identical, i.e., 37 degrees C. The stability of immobilized enzyme against thermal degradation was several times higher than that of the soluble enzyme. Its stability at higher temperatures is one of the main reasons for the clinical use of immobilized urokinase as an antithrombotic material.     

Immobilization of adenosine deaminase onto agarose and casein

In the present study adenosine deaminase (ADA) was immobilized onto two different polymeric materials, agarose and casein. The factors affecting the amount of enzyme attachment onto the polymeric supports such as incubation time were investigated. The maximum amount of enzyme immobilized onto different polymeric supports occurred at incubation pH value 7.5 and ADA concentration 42 units/g and the incubation time needed for the maximum amount of enzyme attachment to the polymeric supports was found to be 8 h. Some phsicochemical properties of the free and immobilized ADA such as operational stability, optimum temperature and thermal stability, pH optimum and stability, storage stability, and the effect of gamma-radiation were studied. The operational stability of the free and immobilized enzyme showed that the enzyme immobilized by a cross-linking technique using gultaric dialdehyde showed poor durability and the relative activity decreased sharply due to the leakage after repeated washing, while the enzymes immobilized by covalent bonds to the carriers showed a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4-6 months, showed that the free enzyme lost its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzyme to a dose of gamma radiation of 0.5-10 Mrad showed complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to gamma radiation up to a dose of 5 Mrad.