Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.     

Microplate assay for aptamer-based thrombin detection using a DNA-enzyme conjugate based on histidine-tag chemistry

We report a method to prepare a DNA–enzyme conjugate using histidine-tag (His-tag) chemistry. A DNA oligonucleotide was modified with nitrilotriacetate (NTA), whose K_d was approximately 10^?6 (M^?1) toward a His-tag present on a recombinant protein via the complexation of Ni²⁺. His-tagged alkaline phosphatase (His-AP) was used as the model enzyme. Enzyme immobilization on the microplate revealed the conjugation of His-AP and the NTA-modified DNA via an Ni²⁺ complex. SPR measurements also proved the conjugation of His-AP with the NTA-modified DNA via an Ni²⁺ complex. The DNA–enzyme conjugate was then used for the detection of thrombin using a DNA aptamer. The DNA-AP conjugate successfully amplified the binding signal between the DNA aptamer and the thrombin, and the signal was measured as the fluorescent intensity derived from the AP-catalyzed reaction. The detection limit was 11 nM. Finally, we studied the effect of the release of the immobilized His-AP from the microplate on the AP activity, because the present strategy used a cleavable linker for the conjugation and the enzyme immobilization. The DNase-catalyzed release of the immobilized His-AP resulted in a 1.7-fold higher AP activity than observed when the His-AP was surface-immobilized.     

Immobilization of Bacillus subtilis α-amylase on zirconia-coated alkylamine glass with glutaraldehyde

Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The K_m value of the immobilized α-amylase was decreased by immobilization while V_max was unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.     

Rhus vernicifera Laccase Immobilization on Magnetic Nanoparticles to Improve Stability and Its Potential Application in Bisphenol A Degradation

In the present study, Rhus vernicifera laccase (RvLac) was immobilized through covalent methods on the magnetic nanoparticles. Fe₂O₃ and Fe₃O₄ nanoparticles activated by 3-aminopropyltriethoxysilane followed with glutaraldehyde showed maximum immobilization yields and relative activity up to 81.4 and 84.3% at optimum incubation and pH of 18 h and 5.8, respectively. The maximum RvLac loading of 156 mg/g of support was recorded on Fe₂O₃ nanoparticles. A higher optimum pH and temperature of 4.0 and 45 °C were noted for immobilized enzyme compared to values of 3.5 and 40 °C for free form, respectively. Immobilized RvLac exhibited better relative activity profiles at various pH and temperature ranges. The immobilized enzyme showed up to 16-fold improvement in the thermal stability, when incubated at 60 °C, and retained up to 82.9% of residual activity after ten cycles of reuses. Immobilized RvLac exhibited up to 1.9-fold higher bisphenol A degradation efficiency potential over free enzyme. Previous reports have demonstrated the immobilization of RvLac on non-magnetic supports. This study has demonstrated that immobilization of RvLac on magnetic nanoparticles is very efficient especially for achieving high loading, better pH and temperature profiles, and thermal- and solvents-stability, high reusability, and higher degradation of bisphenol A.     

Immobilization of β-galactosidase onto functionalized graphene nano-sheets using response surface methodology and its analytical applications

Background

β-Galactosidase is a vital enzyme with diverse application in molecular biology and industries. It was covalently attached onto functionalized graphene nano-sheets for various analytical applications based on lactose reduction.

Methodology/Principal Findings

Response surface methodology based on Box-Behnken design of experiment was used for determination of optimal immobilization conditions, which resulted in 84.2% immobilization efficiency. Native and immobilized functionalized graphene was characterized with the help of transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Functionalized graphene sheets decorated with islands of immobilized enzyme were evidently visualized under both transmission and scanning electron microscopy after immobilization. FTIR spectra provided insight on various chemical interactions and bonding, involved during and after immobilization. Optimum temperature and energy of activation (E_a) remains unchanged whereas optimum pH and K_m were changed after immobilization. Increased thermal stability of enzyme was observed after conjugating the enzyme with functionalized graphene.

Significance

Immobilized β-galactosidase showed excellent reusability with a retention of more than 92% enzymatic activity after 10 reuses and an ideal performance at broad ranges of industrial environment.     

Preparation of active corn peptides from zein through double enzymes immobilized with calcium alginate–chitosan beads

Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl₂ and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters V_max and apparent K_m were 32.3 mL/min and 456.62 g⁻¹, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications.     

Dual immobilization of biomolecule on the glass surface using cysteine as a bifunctional linker

A novel method for highly efficient enzyme immobilization on the glass surface, by incorporating cysteine as a linker has been demonstrated. The internal glass surface of test tube was pretreated with (3-mercaptopropyl) trimethoxysilane sol–gel and cysteine capped silver nanoparticles, to generate a cysteine layer. This, cysteine rich surface is then used to covalently immobilize alkaline phosphatase on both groups (amino and carboxyl) of cysteine through carbodiimide and glutaraldehyde treatment. The cysteine capped silver nanoparticles were synthesized with an average nanoparticle size of 61 nm as determined by particle size analyzer, while cysteine capping of nanoparticles was confirmed by Fourier transform infra-red spectroscopy. Enhanced enzymatic activity of about 73% was obtained using the dual immobilization technique, while 40% enzyme activity was recovered with carboxyl group and 51% with amino group only. The re-usability of the enzyme immobilized test tube was found to be 8 times and the enzyme retained 85% of its initial activity. With such high immobilization efficiency, cysteine provides a new approach for enhanced immobilization and its integration into different industrial processes and biosensor technology.     

Biochemical characterization and FAD-binding analysis of oleate hydratase from Macrococcus caseolyticus

A putative fatty acid hydratase gene from Macrococcus caseolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was a 68 kDa dimer with a molecular mass of 136 kDa. The enzymatic products formed from fatty acid substrates by the putative enzyme were isolated with high purity (>99%) by solvent fractional crystallization at low temperature. After the identification by GC–MS, the purified hydroxy fatty acids were used as standards to quantitatively determine specific activities and kinetic parameters for fatty acids as substrates. Among the fatty acids evaluated, specific activity and catalytic efficiency (k_cat/K_m) were highest for oleic acid, indicating that the putative fatty acid hydratase was an oleate hydratase. Hydration occurred only for cis-9-double and cis-12-double bonds of unsaturated fatty acids without any trans-configurations. The maximum activity for oleate hydration was observed at pH 6.5 and 25 °C with 2% (v/v) ethanol and 0.2 mM FAD. Without FAD, all catalytic activity was abolished. Thus, the oleate hydratase is an FAD-dependent enzyme. The residues G29, G31, S34, E50, and E56, which are conserved in the FAD-binding motif of fatty acid hydratases (GXGXXG_(A/S)X_(15–21)E_(D)), were selected by alignment, and the spectral properties and kinetic parameters of their alanine-substituted variants were analyzed. Among the five variants, G29A, G31A, and E56A showed no interaction with FAD and exhibited no activity. These results indicate that G29, G31, and E56 are essential for FAD-binding.     

Activity and stability of lipase in Aerosol-OT/isooctane reverse micelles

The stability of Candida rugosa lipase, which catalyzes the hydrolysis reaction of olive oil in AOT/isooctane reverse micelles, decreased with the increase of ₀ (defined as the molar ratio of water to surfactant) and Aerosol-OT concentration. The addition of a non-ionic cosurfactant, tetraethylene glycol dodecyl ether (C₁₂E₄), preserved enzymatic activity. The residual activity of the lipase was 53% after 24 h, while the enzyme completely lost its activity within 6 h in the absence of C₁₂E₄ addition. The stabilizing effect of C₁₂E₄ resulted in the increase of conversion. The enhancement of the activity and stability of lipase in reverse micelles by the addition of C₁₂E₄ may contribute to increase the rigidity of the micellar matrix stabilizing the enzyme structure.     

The Length of the A-M3 Linker Is a Crucial Determinant of the Rate of the Ca2+ Transport Cycle of Sarcoplasmic Reticulum Ca2+-ATPase

Ion translocation by the sarcoplasmic reticulum Ca²⁺-ATPase depends on large movements of the A-domain, but the driving forces have yet to be defined. The A-domain is connected to the ion-binding membranous part of the protein through linker regions. We have determined the functional consequences of changing the length of the linker between the A-domain and transmembrane helix M3 (“A-M3 linker”) by insertion and deletion mutagenesis at two sites. It was feasible to insert as many as 41 residues (polyglycine and glycine-proline loops) in the flexible region of the linker without loss of the ability to react with Ca²⁺ and ATP and to form the phosphorylated Ca₂E1P intermediate, but the rate of the energy-transducing conformational transition to E2P was reduced by >80%. Insertion of a smaller number of residues gave effects gradually increasing with the length of the insertion. Deletion of two residues at the same site, but not replacement with glycine, gave a similar reduction as the longest insertion. Insertion of one or three residues in another part of the A-M3 linker that forms an α-helix (“A3 helix”) in E2/E2P conformations had even more profound effects on the ability of the enzyme to form E2P. These results demonstrate the importance of the length of the A-M3 linker and of the position and integrity of the A3 helix for stabilization of E2P and suggest that, during the normal enzyme cycle, strain of the A-M3 linker could contribute to destabilize the Ca₂E1P state and thereby to drive the transition to E2P.The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)² is a membrane-bound ion pump that transports Ca²⁺ against a steep concentration gradient, utilizing the energy derived from ATP hydrolysis (1–3). It belongs to the family of P-type ATPases, in which the γ-phosphoryl group of ATP is transferred to a conserved aspartic acid residue during the reaction cycle. Both phospho and dephospho forms of the enzyme undergo transitions between so-called E1 and E2 conformations (Scheme 1). The E1 and E1P states display specificity for reaction with ATP and ADP, respectively (“kinase activity”), whereas E2P and E2 react with water and P_i instead of nucleotide (“phosphatase activity”). The E1 dephosphoenzyme of the Ca²⁺-ATPase binds two Ca²⁺ ions with high affinity from the cytoplasmic side, thereby triggering the phosphorylation from ATP. In E1P, the Ca²⁺ ions are occluded with no access to either side of the membrane, and Ca²⁺ is released to the luminal side after the conformational transition to E2P, likely in exchange for protons being countertransported. The structural organization and domain movements leading to Ca²⁺ translocation have recently been elucidated by crystallization of SERCA in various conformational states thought to represent intermediates in the pump cycle (4–7). SERCA is made up of 10 membrane-spanning mostly helical segments, M1–M10 (numbered from the N terminus), of which M4–M6 and M8 contribute liganding groups for Ca²⁺ binding, and a cytoplasmic headpiece separated into three distinct domains, named A (“actuator”), P (“phosphorylation”), and N (“nucleotide binding”). The A-domain appears to undergo considerable movement during the functional cycle. In the E1/E1P states, the highly conserved TGE¹⁸³S loop of the A-domain is at great distance from the catalytic center containing nucleotide-binding residues and the phosphorylated Asp³⁵¹ of the P-domain, but during the Ca₂E1P → E2P transition, the A-domain rotates ∼90° around an axis perpendicular to the membrane, thereby moving the TGE¹⁸³S loop into close contact with the catalytic site such that Glu¹⁸³ can catalyze dephosphorylation of E2P (8, 9). During the dephosphorylation, Glu¹⁸³ likely coordinates the water molecule attacking the aspartyl phosphoryl bond and withdraws a hydrogen. Hence, the movement of the A-domain during the Ca₂E1P → E2P transition is the event that changes the catalytic specificity from kinase activity to phosphatase activity. During the dephosphorylation of E2P → E2, there is only a slight change of the position of the A-domain, and a large back-rotation is needed to reach the E1 form from E2; thus, the A-domain rotation defines the difference between the E1/E1P class of conformations and the E2/E2P class. Because the A-domain is physically connected to transmembrane helices M1–M3 through the linker segments A-M1, A-M2, and A-M3, the A-domain movement occurring during the Ca₂E1P → E2P transition may be a key event in the opening of the Ca²⁺ sites toward the lumen, thus explaining the coupling of ATP hydrolysis to Ca²⁺ translocation. An important unanswered question is, however, how the movement of the A-domain is brought about. Which are the driving forces that destabilize Ca₂E1P and/or stabilize E2P such that the energy-transducing Ca₂E1P → E2P transition takes place? To answer this, it seems important to elucidate the exact roles of the linkers. Intriguing results have been obtained by Suzuki and co-workers, who demonstrated the importance of the A-M1 linker in connection with luminal release of Ca²⁺ from E2P (10). In this study, we have addressed the role of the A-M3 linker. An alignment of two crystal structures thought to resemble the Ca₂E1P and E2·P_i forms (5), respectively, is shown in Fig. 1. The A-domain rotation is associated with formation of a helix (“A3 helix”) in the N-terminal part of the A-M3 linker, and this helix seems to interact with a helix bundle consisting of the P5–P7 helices of the P-domain, a feature exhibited by all published crystal structures of the E2 type (cf. supplemental Fig. S1 and Ref. 11). Moreover, when structures of similar crystallographic resolution are compared (as in Fig. 1), the non-helical part of the A-M3 linker in E2-type structures has a higher relative temperature factor (“B-factor”) than the corresponding segment in Ca₂E1P (Fig. 1C, thick part colored orange-red for high temperature factor), thus suggesting a higher degree of freedom of movement relative to Ca₂E1P. Hence, the A-M3 linker appears more strained in Ca₂E1P compared with E2 forms, and the greater flexibility of the linker in E2 forms may promote the formation of the A3 helix.Open in a separate windowSCHEME 1.Ca²⁺-ATPase reaction cycle.Open in a separate windowFIGURE 1.A-M3 linker configuration in E1- and E2-type crystal structures. Crystal structures with Protein Data Bank codes 2zbd (Ca₂E1P analog) and 1wpg (E2·P_i analog) are shown aligned. A, overview of structure 2zbd in bluish colors with green A-M3 linker and structure 1wpg in reddish colors with wheat A-M3 linker. B, magnification of the A-M3 linker (corresponding to the red box in A) with arrows indicating site 1, between Glu²⁴³ and Gln²⁴⁴, and site 2, between Gly²³³ and Lys²³⁴, in both conformations. The green A-M3 linker to the right is structure 2zbd. The wheat A-M3 linker to the left is structure 1wpg. Note the kinked A3 helix forming part of the latter structure. C, same A-M3 linker structures as in B but with the magnitude of the temperature factor (B-factor) indicated in colors (red > orange > yellow > green > blue) and by tube diameter. Because the two crystal structures selected here as E1- and E2-type representatives have similar crystallographic resolution (2.40 and 2.30 Å, respectively), the differences in temperature factor in specific regions provide direct information about chain flexibility.Here, we have determined the functional consequences of changing the length (and thereby likely the strain) of the A-M3 linker. Polyglycine and glycine-proline loops of varying lengths were inserted at two different sites in the linker (Fig. 1), and deletions were also studied. Rather unexpectedly, we were able to insert as many as 41 residues in one of the sites without loss of expression or ability to react with Ca²⁺ and ATP, forming Ca₂E1P, but the Ca₂E1P → E2P transition was greatly affected.     

Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase

Improvement of enzyme function by engineering pH dependence of enzymatic activity is of importance for industrial application of Bacillus circulans xylanases. Target mutation sites were selected by structural alignment between B. circulans xylanase and other xylanases having different pH optima. We selected non-conserved mutant sites within 8 Å from the catalytic residues, to see whether these residues have some role in modulating pK_as of the catalytic residues. We hypothesized that the non-conserved residues which may not have any role in enzyme catalysis might perturb pK_as of the catalytic residues. Change in pK_a of a titratable group due to change in electrostatic potential of a mutation was calculated and the change in pH optimum was predicted from the change in pK_a of the catalytic residues. Our strategy is proved to be useful in selection of promising mutants to shift the pH optimum of the xylanases towards desired side.     

Effect of compressed fluids treatment on β-galactosidase activity and stability

This study aimed at assessing the influence of different pressurized fluids treatment on the enzymatic activity and stability of a lyophilized β-galactosidase. The effects of system pressure, exposure time and depressurization rate, using propane, n-butane, carbon dioxide and liquefied petroleum gas on the enzymatic activity were evaluated. The β-galactosidase activity changed significantly depending on the experimental conditions investigated, allowing the selection of the proper compressed fluid for advantageous application of this biocatalyst in enzymatic reactions. The residual activity ranged from 32.1 to 93.8?% after treatment. The storage stability of the enzyme after high-pressure pre-treatment was also monitored, and results showed that the biocatalyst activity presents strong dependence of the fluid used in the pretreatment. The activity gradually decreases over the time for the enzyme treated with LGP and propane, while the enzyme treated with n-butane maintained 96?% of its initial activity until 120?days. For CO₂, there was a reduction of around 40?% in the initial activity 90?days of storage. The enzyme treated with n-butane also showed a better thermostability in terms of enzymatic half-life.     

Molecular Engineering of Fungal GH5 and GH26 Beta-(1,4)-Mannanases toward Improvement of Enzyme Activity

Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in k_cat/K_M due to a significant decrease of K_M, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in k_cat/K_M compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (k_cat/K_M).     

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion: Analysis of N‐ and O‐linked glycans and identification and elimination of a xylose‐based O‐linked tetrasaccharide core in the linker region

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion (huLCAT‐Fc) is a chimeric protein produced by fusing human Fc to the C‐terminus of the human enzyme via a linker sequence. The huLCAT‐Fc homodimer contains five N‐linked glycosylation sites per monomer. The heterogeneity and site‐specific distribution of the various glycans were examined using enzymatic digestion and LC‐MS/MS, followed by automatic processing. Almost all of the N‐linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N‐linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N‐linked site exclusively contain typical asialobiantennary structures. HuLCAT‐Fc was also confirmed to have mucin‐type glycans attached at T₄₀₇ and S₄₀₉. When LCAT‐Fc fusions were constructed using a G‐S‐G‐G‐G‐G linker, an unexpected +632 Da xylose‐based glycosaminoglycan (GAG) tetrasaccharide core of Xyl‐Gal‐Gal‐GlcA was attached to S₄₁₈. Several minor intermediate species including Xyl, Xyl‐Gal, Xyl‐Gal‐Gal, and a phosphorylated GAG core were also present. The mucin‐type O‐linked glycans can be effectively released by sialidase and O‐glycanase; however, the GAG could only be removed and localized using chemical alkaline β‐elimination and targeted LC‐MS/MS. E₄₁₆ (the C‐terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O‐xylosyltransferase. HuLCAT‐Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPP E₄₁₆GS₄₁₈G G G GDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.     

Activity of myrosinase from Sinapis alba seeds immobilized into Ca-polygalacturonate as a simplified model of soil-root interface mucigel

Ca-polygalacturonate is a demethoxylated component of pectins which are constitutive of plant root mucigel. In order to define the role of root mucigel in myrosinase immobilization and activity at root level, a myrosinase enzyme which had been isolated from Sinapis alba seeds was immobilized into Ca-polygalacturonate. The activity profile for the immobilized and free enzyme was evaluated using the pH-Stat method as a function of time, temperature, and pH. The Michaelis-Menten kinetic parameters change between the immobilized (V _max ?=?127?±?13 U mg^?1 protein; K _M ?=?6.28?±?0.09?mM) and free (V _max ?=?17?±?1 U mg^?1 protein; K _M ?=?0.96?±?0.01?mM) forms of myrosinase, probably due to conformational changes involving the active site as a consequence of enzyme immobilization. Immobilized enzyme activity evaluated as a function of different substrates gave the highest value with nasturtin, the glucosinolate that is typical of several brassicaceae plant roots containing the glucosinolate-myrosinase defensive system. No feedback regulation mechanism was found in the presence of an excess of enzymatic reaction products (i.e. allyl isothiocyanate or sulphate). The high enzyme immobilization yield into Ca-polygalacturonate and its activity preservation under different conditions suggest that the enzyme released by plants at root level could be entrapped in root mucigel in order to preserve its activity.     

Immobilization of the α-amylase of Bacillus amyloliquifaciens TSWK1-1 for the improved biocatalytic properties and solvent tolerance

The α-amylase of Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033) was immobilized by various methods, including ionic binding with DEAE cellulose, covalent coupling with gelatin and entrapment in polyacrylamide and agar. The immobilization of the purified enzyme was most effective with the DEAE cellulose followed by gelatin, agar and polyacrylamide. The K _m increased, while V _max decreased upon immobilization on various supports. The temperature and pH profiles broadened, while thermostability and pH stability enhanced after immobilization. The immobilized enzyme exhibited greater activity in various non-ionic surfactants, such as Tween-20, Tween-80 and Triton X-100 and ionic surfactant, SDS. Similarly, the enhanced stability of the immobilized α-amylase in various organic solvents was among the attractive features of the study. The reusability of the immobilized enzyme in terms of operational stability was assessed. The DEAE cellulose immobilized α-amylase retained its initial activity even after 20 consequent cycles. The DEAE cellulose immobilized enzyme hydrolyzed starch with 27 % of efficiency. In summary, the immobilization of B. amyloliquifaciens TSWK1-1 α-amylase with DEAE cellulose appeared most suitable for the improved biocatalytic properties and stability.     

Promotion of multipoint covalent immobilization through different regions of genetically modified penicillin G acylase from E. coli

A novel approach is proposed to prepare a set of immobilized derivatives of a enzyme covalently rigidified through different regions of its surface. Six different variants of penicillin G acylase (PGA) from Escherichia coli (which lacks Cys) were prepared by introducing a unique Cys residue via site-directed mutagenesis in six different enzyme regions which were rich in Lys residues. All variants exhibited a similar activity and stability compared to those of the native enzyme. Each variant was immobilized on supports having a low concentration of reactive disulfide moieties and a high concentration of poorly reactive epoxy groups. After immobilization at pH 7.0 by site-directed thiol-disulfide intermolecular exchange, derivatives were further incubated at pH 10.0 for 48 h to promote an additional intramolecular reaction between Lys residues of enzyme and epoxy groups of the support. The establishment of at least three covalent attachments per PGA molecule was determined for all immobilized enzyme variants. The different derivatives exhibited diverse stability against several distorting agents and different selectivity in two interesting reactions. The derivative of the PGA variant obtained by replacement of GlnB380 by Cys was the most stable against heat and organic cosolvents: it preserved 90% of the initial activity and was 30-fold more stable than soluble PGA. This derivative also exhibited an improved enantioselectivity in the hydrolysis of chiral esters (E was improved from 8 to 16) and in kinetically controlled synthesis of amides (synthetic yields were increased from 31 to 49%).     

Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase

Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C₅₅-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C₅₅-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C₅₅-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C₅₅-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C₅₅-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C₅₅-PP molecules on the same (outer) side of the plasma membrane.     

Urea denaturation of the immobilized proteases of Streptomyces griseus (pronase)

The protease preparation (pronase, EC 3.4. group) from Streptomyces griseus has been covalently immobilized on porous succinamidopropyl glass using a carbodiimide carboxyl activation procedure. The separate activities of the individual proteases in this preparation were assayed using specific synthetic substrates. Stabilities of both soluble and immobilized preparations were determined and compared by assaying for each activity in urea solutions of various concentration. The loss of activity by the immobilized enzymes was shown to be reversible under most conditions. Analysis of the data in terms of a two-state transition showed that the urea concentration resulting in 50% loss of activity was increased for each enzyme as a result of immobilization. Also the m-value in the relation ΔG_D = ΔG^H₂O_D - m[urea] decreased for each enzyme upon immobilization. Thus, all of the enzymes were stabilized by immobilization and the apparent broadening of the transitions, as measured by the decreased m-value, was interpreted as the formation of a population of molecules with different stabilities. The degree of apparent stabilization upon immobilization varied with the magnitude decreasing as: aminopeptidases > carboxypeptidase ? trypsin > proteases A and B. Furthermore, it is suggested that stabilization may result from multipoint attachment since the magnitude was correlated with the number of potential enzyme reaction sites as reflected by their lysine contents.