Directed evolution of a bacterial alpha-amylase: toward enhanced pH-performance and higher specific activity

alpha-Amylases, in particular, microbial alpha-amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for alpha-amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the alpha-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild-type BAA and the mutants BAA S201N and BAA N297D were subjected to error-prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5-fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4- and 1.5-fold, respectively. Another mutant (BAA 29) possesses a wild-type-like pH profile but possesses a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial alpha-amylases.     

Structure‐based replacement of methionine residues at the catalytic domains with serine significantly improves the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica

The alkaline amylase requires high resistance towards chemical oxidation for use in the detergent and textile industries. This work aims to improve the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica by site‐directed mutagenesis based on the enzyme structure model. Five mutants were created by individually replacing methionine at positions 145, 214, 229, 247, and 317 in the amino acid sequence of alkaline amylase with oxidative‐resistant serine. The pH stability of the mutant enzymes was almost the same as that of the wild‐type (WT) enzyme (pH 7.0–11.0). The stable temperature range of the mutant enzymes M145S and M247S decreased from <50°C of the WT to <40°C, while the thermal stability of the other three mutant enzymes (M214S, M229S, and M317S) was almost the same as that of the WT enzyme. The catalytic efficiency (k_cat/K_m) of all the mutant enzymes decreased when compared to WT enzyme. The mutant enzymes showed increased activity in the presence of surfactants Tween‐60 and sodium dodecyl sulfate. When incubated with 500 mM H₂O₂ at 35°C for 5 h, the WT enzyme retained only 13.3% of its original activity, while the mutant enzymes M145S, M214S, M229S, M247S, and M317S retained 55.6, 70.2, 54.2, 62.5, and 46.4% of the original activities, respectively. The results indicated that the substitution of methionine residues at the catalytic domains with oxidative‐resistant serine can significantly improve the oxidative stability of alkaline amylase. This work provides an effective strategy to improve the oxidative stability of amylase, and the high oxidation resistance of the mutant enzymes shows their potential applications in the detergent and textile industries. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Investigating the potential of Bacillus subtilis α‐amylase as a pressure‐temperature‐time indicator for high hydrostatic pressure pasteurization processes

The potential of Bacillus subtilis α‐amylase (BSA) as a pressure‐temperature‐time indicator (pTTI) for high pressure pasteurization processing (400–600 MPa; T_i 10–40°C; 1–15 min) was investigated. A stepwise approach was followed for the development of an enzyme‐based, extrinsic, isolated pTTI. First, based on literature data on the pressure stability, BSA was selected as a candidate indicator. Next to the accuracy and ease of the measurement of the indicator's response (residual activity) to the pressure treatment, the storage and handling stability of BSA at atmospheric pressure was verified. Second, the stability of BSA at a constant temperature (T) and time in function of pressure (p) was investigated. Solvent engineering was used to shift the inactivation window of BSA in the processing range of interest. Third, the enzyme (1 g/L BSA—MES 0.05 M pH 5.0) was kinetically calibrated under isobaric‐isothermal conditions. Time dependent changes in activity could be modeled best by a first‐order model. Except for low pressures and high temperatures, a synergistic effect between pressure and temperature could be observed. Based on the model selected to describe the combined p,T‐dependency of the inactivation rate constant, an elliptically shaped isorate contour plot could be constructed, illustrating the processing range where BSA can be used to demonstrate temperature gradients. Fourth, the validity of the kinetic model was tested successfully under dynamic conditions similar to those used in food industry. Finally, the indicator was found suitable to demonstrate nonuniformity in two‐sectional planes of a vertical, single vessel system. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

Effects of the biocontrol agent Bacillus amyloliquefaciens SN16‐1 on the rhizosphere bacterial community and growth of tomato

Fusarium oxysporum is a common soil‐borne pathogen that causes serious economic losses in tomato crops worldwide. The purpose of this study was to evaluate the influence of the bio‐control agents Bacillus amyloliquefaciens SN16‐1 and Pseudomonas fluorescens SN15‐2 and the pathogen Fusarium oxysporum f.sp. lycopersici (FOL) inoculation on tomato rhizosphere bacterial communities and growth, as measured by terminal restriction fragment length polymorphism (T‐RFLP). Treatment with SN16‐1 and SN15‐2 had a transient influence on indigenous bacterial communities, withSN16‐1 showing great potential for controlling FOL. The corresponding genera of terminal restriction fragments (T‐RFs) that were significantly altered after 10 days were obtained using Ribosomal Database Project (RDP) database comparison. Genera that produce antibiotics and promote plant growth were activated by SN16‐1 and FOL treatments, indicating that SN16‐1 responds quickly to FOL invasion. Moreover, the bioremediation activity characteristic of certain genera and the levels of enzymes that degrade pathogen cell walls were decreased while bacterial nutrient cycling and plant growth promotion were enhanced with FOL treatment. In conclusion, we found that SN16‐1 possesses the capacity to control tomato wilt, acts synergistically with soil microbes and does not have a persistent effect on the rhizosphere bacterial communities of tomato.     

Structure,activity, and stability of metagenome‐derived glycoside hydrolase family 9 endoglucanase with an N‐terminal Ig‐like domain

A metagenome‐derived glycoside hydrolase family 9 enzyme with an N‐terminal immunoglobulin‐like (Ig‐like) domain, leaf‐branch compost (LC)‐CelG, was characterized and its crystal structure was determined. LC‐CelG did not hydrolyze p‐nitrophenyl cellobioside but hydrolyzed CM‐cellulose, indicating that it is endoglucanase. LC‐CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5–9. Its denaturation temperature was 81.4°C, indicating that LC‐CelG is a thermostable enzyme. The structure of LC‐CelG resembles those of CelD from Clostridium thermocellum (CtCelD), Cel9A from Alicyclobacillus acidocaldarius (AaCel9A), and cellobiohydrolase CbhA from C. thermocellum (CtCbhA), which show relatively low (29–31%) amino acid sequence identities to LC‐CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC‐CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC‐CelG. Removal of the Ig‐like domain reduced the activity and stability of LC‐CelG by 100‐fold and 6.3°C, respectively. Removal of the Gln40‐ and Asp99‐mediated interactions between the Ig‐like and catalytic domains destabilized LC‐CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig‐like domain contributes to the stabilization of LC‐CelG mainly due to the Gln40‐ and Asp99‐mediated interactions. Because the LC‐CelG derivative lacking the Ig‐like domain accumulated in Escherichia coli cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig‐like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain.     

Antidiabetic potential: in vitro inhibition effects of some natural phenolic compounds on α‐glycosidase and α‐amylase enzymes

α‐Glycosidase is a catalytic enzyme and it destroys the complex carbohydrates into simple absorbable sugar units. The natural phenolic compounds were tested for their antidiabetic properties as α‐glycosidase and α‐amylase inhibitors. The phenolic compounds investigated in this study have been used as antidiabetic common medicines. This paper aimed to consider their capability to inhibit α‐amylase and α‐glycosidase, two significant enzymes defined in serum glucose adjustment. These examination recorded impressive inhibition profiles with IC₅₀ values in the range of 137.36–737.23 nM against α‐amylase and 29.01–157.96 nM against α‐glycosidase.     

Deletion of the carboxyl‐terminal residue disrupts the amino‐terminal folding,self‐association,and thermal stability of an amphipathic antimicrobial peptide

Understanding the complex relationship between amino acid sequence and protein behaviors, such as folding and self‐association, is a major goal of protein research. In the present work, we examined the effects of deleting a C‐terminal residue on the intrinsic properties of an amphapathic α‐helix of mastoparan‐B (MP‐B), an antimicrobial peptide with the sequence LKLKSIVSWAKKVL‐NH2. We used circular dichroism and nuclear magnetic resonance to demonstrate that the peptide MP‐B^[1‐13] displayed significant unwinding at the N‐terminal helix compared with the parent peptide of MP‐B, as the temperature increased when the residue at position 14 was deleted. Pulsed‐field gradient nuclear magnetic resonance data revealed that MP‐B forms a larger diffusion unit than MP‐B^[1‐13] at all experimental temperatures and continuously dissociates as the temperature increases. In contrast, the size of the diffusion unit of MP‐B^[1‐13] is almost independent of temperature. These findings suggest that deleting the flexible, hydrophobic amino acid from the C‐terminus of MP‐B is sufficient to change the intrinsic helical thermal stability and self‐association. This effect is most likely because of the modulation of enthalpic interactions and conformational freedom that are specified by this residue. Our results implicate terminal residues in the biological function of an antimicrobial peptide. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

The effect of a beta‐lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study

Co‐administration of beta‐lactam antibiotics and beta‐lactamase inhibitors has been a favored treatment strategy against beta‐lactamase‐mediated bacterial antibiotic resistance, but the emergence of beta‐lactamases resistant to current inhibitors necessitates the discovery of novel non‐beta‐lactam inhibitors. Peptides derived from the Ala46–Tyr51 region of the beta‐lactamase inhibitor protein are considered as potent inhibitors of beta‐lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell‐penetrating peptides could guide the design of beta‐lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta‐lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell‐penetrating peptide pVEC, our approach involved the addition of the N‐terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta‐lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta‐lactam antibiotic ampicillin, and the beta‐lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N‐terminus of the beta‐lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N‐terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Influence of phosphate anions on the stability of immobilized enzymes. Effect of enzyme nature,immobilization protocol and inactivation conditions

A destabilizing effect at pH 7 of sodium phosphate on several lipases immobilized via interfacial activation is shown in this work. This paper investigates if this destabilizing effect is extended to other inactivation conditions, immobilization protocols or even other immobilized enzymes (ficin, trypsin, β-galactosidase, β-glucosidase, laccase, glucose oxidase and catalase). As lipases, those from Candida antarctica (A and B), Candida rugosa and Rhizomucor miehei have been used. Results confirm the very negative effect of 100 mM sodium phosphate at pH 7.0 for the stability of all studied lipases immobilized on octyl agarose, while using glutaraldehyde-support the effect is smaller (still very significant using CALA) and in some cases the effect disappeared (e.g., using CALB). The change of the pH to 5.0 or 9.0, or the addition of 1 M NaCl reduced the negative effect of the phosphate in some instances (e.g., at pH 5.0, this negative effect is only relevant for CALB). Regarding the other enzymes, only the monomeric β-galactosidase from Aspergillus oryzae is strongly destabilized by the phosphate buffer. This way, the immobilization protocol and the inactivation conditions strongly modulate the negative effect of sodium phosphate on the stability of immobilized lipases, and this effect is not extended to other enzymes.     

A comparative study of activity and apparent inhibition of fungal β‐glucosidases

β‐Glucosidases (BGs) from Aspergillus fumigates, Aspergillus niger, Aspergillus oryzae, Chaetomium globosum, Emericella nidulans, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and analyzed by isothermal titration calorimetry with respect to their hydrolytic activity and its sensitivity to glucose (product) using cellobiose as substrate. Global non‐linear regression of several reactions, with or without added glucose, to a product inhibition equation enabled the concurrent derivation of the kinetic parameters k_cat, K_m, and the apparent product inhibition constant _appK_i for each of the enzymes. A more simple fit is not advisable to use as the determined _appK_i are in the same range as their K_m for some of the tested BGs and produced glucose would in these cases interfere. The highest value for k_cat was determined for A. fumigatus (768 s^?1) and the lowest was a factor 9 less. K_m varied by a factor of 3 with the lowest value determined for C. globosum (0.95 mM). The measured _appK_i varied a factor of 15; the hydrolytic activity of N. crassa being the most resistant to glucose with an apparent product inhibition constant of 10.1 mM. Determination of _appK_i using cellobiose as substrate is important as it reflects to what extent the different BGs are hydrolytically active under industrial conditions where natural substrates are hydrolyzed and the final glucose concentrations are high. Biotechnol. Bioeng. 2010;107: 943–952. © 2010 Wiley Periodicals, Inc.     

On the pH‐optimum of activity and stability of proteins

Biological macromolecules evolved to perform their function in specific cellular environment (subcellular compartments or tissues); therefore, they should be adapted to the biophysical characteristics of the corresponding environment, one of them being the characteristic pH. Many macromolecular properties are pH dependent, such as activity and stability. However, only activity is biologically important, while stability may not be crucial for the corresponding reaction. Here, we show that the pH‐optimum of activity (the pH of maximal activity) is correlated with the pH‐optimum of stability (the pH of maximal stability) on a set of 310 proteins with available experimental data. We speculate that such a correlation is needed to allow the corresponding macromolecules to tolerate small pH fluctuations that are inevitable with cellular function. Our findings rationalize the efforts of correlating the pH of maximal stability and the characteristic pH of subcellular compartments, as only pH of activity is subject of evolutionary pressure. In addition, our analysis confirmed the previous observation that pH‐optimum of activity and stability are not correlated with the isoelectric point, pI, or with the optimal temperature. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Cationic oligopeptides with the repeating sequence L‐lysyl‐L‐alanyl‐L‐alanine: conformational and thermal stability study using optical spectroscopic methods

The infrared (IR), vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) spectra of short cationic sequential peptides (L ‐Lys‐L ‐Ala‐L ‐Ala)_n (n = 1, 2, and 3) were measured over a range of temperatures (20–90 °C) in aqueous solution at near‐neutral pH values in order to investigate their solution conformations and thermally induced conformational changes. VCD spectra of all three oligopeptides measured in the amide I′ region indicate the presence of extended helical polyproline II (PPII)‐like conformation at room temperature. UV‐ECD spectra confirmed this conclusion. Thus, the oligopeptides adopt a PPII‐like conformation, independent of the length of the peptide chain. However, the optimized dihedral angles ? and ψ are within the range ?82 to ?107° and 143–154°, respectively, and differ from the canonical PPII values. At elevated temperatures, the observed intensity and bandshape variations in the VCD and ECD spectra show that the PPII‐like conformation of the Lys‐Ala‐Ala sequence is still preferred, being in equilibrium with an unordered conformer at near‐neutral pH values within the range of temperatures from 20 to 90 °C. This finding was obtained from analysis of the temperature‐dependent spectra using the singular value decomposition method. The study presents KAA‐containing oligopeptides as conformationally stable models of biologically important cationic peptides and proteins. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of acidic pH on the stability of α‐synuclein dimers

Environmental factors, such as acidic pH, facilitate the assembly of α‐synuclein (α‐Syn) in aggregates, but the impact of pH on the very first step of α‐Syn aggregation remains elusive. Recently, we developed a single‐molecule approach that enabled us to measure directly the stability of α‐Syn dimers. Unlabeled α‐Syn monomers were immobilized on a substrate, and fluorophore‐labeled monomers were added to the solution to allow them to form dimers with immobilized α‐Syn monomers. The dimer lifetimes were measured directly from the fluorescence bursts on the time trajectories. Herein, we applied the single‐molecule tethered approach for probing of intermolecular interaction to characterize the effect of acidic pH on the lifetimes of α‐Syn dimers. The experiments were performed at pH 5 and 7 for wild‐type α?Syn and for two mutants containing familial type mutations E46K and A53T. We demonstrate that a decrease of pH resulted in more than threefold increase in the α‐Syn dimers lifetimes with some variability between the α‐Syn species. We hypothesize that the stabilization effect is explained by neutralization of residues 96–140 of α‐Syn and this electrostatic effect facilitates the association of the two monomers. Given that dimerization is the first step of α‐Syn aggregation, we posit that the electrostatic effect thereby contributes to accelerating α‐Syn aggregation at acidic pH. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 715–724, 2016.     

Influence of the H‐site residue 108 on human glutathione transferase P1‐1 ligand binding: Structure‐thermodynamic relationships and thermal stability

The effect of the Y108V mutation of human glutathione S‐transferase P1‐1 (hGST P1‐1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 → Val resulted in a 3D‐structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H‐site) and glutathione binding site (G‐site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H‐site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (K_d ～ 0.5 μM) when compared with those of the parent compounds, K ～ 13 μM, K ～ 25 μM. The EA moiety of the conjugate binds in the H‐site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the ΔC_p values of binding can also be correlated with the potential stacking interactions between ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information.     

Molecular cloning,expression profile,odorant affinity,and stability of two odorant‐binding proteins in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

The polyembryonic endoparasitoid wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is deployed successfully as a biocontrol agent for corn pest insects from the Lepidopteran genus Ostrinia in Europe and throughout Asia, including Japan, Korea, and China. The odorants are recognized, bound, and solubilized by odorant‐binding protein (OBP) in the initial biochemical recognition steps in olfaction that transport them across the sensillum lymph to initiate behavioral response. In the present study, we examine the odorant‐binding effects on thermal stability of McinOBP2, McinOBP3, and their mutant form that lacks the third disulfide bonds. Real‐time PCR experiments indicate that these two are expressed mainly in adult antennae, with expression levels differing by sex. Odorant‐binding affinities of aldehydes, terpenoids, and aliphatic alcohols were measured with circular dichroism spectroscopy based on changes in the thermal stability of the proteins upon their affinities to odorants. The obtained results reveal higher affinity of trans‐caryophelle, farnesene, and cis‐3‐Hexen‐1‐ol exhibits to both wild and mutant McinOBP2 and McinOBP3. Although conformational flexibility of the mutants and shape of binding cavity make differences in odorant affinity between the wild‐type and mutant, it suggested that lacking the third disulfide bond in mutant proteins may have chance to incorrect folded structures that reduced the affinity to these odorants. In addition, CD spectra clearly indicate proteins enriched with α‐helical content.     

Effect of compatible and noncompatible osmolytes on the enzymatic activity and thermal stability of bovine liver catalase

Catalase is an important antioxidant enzyme that catalyzes the disproportionation of H₂O₂ into harmless water and molecular oxygen. Due to various applications of the enzyme in different sectors of industry as well as medicine, the enhancement of stability of the enzyme is important. Effect of various classes of compatible as well as noncompatible osmolytes on the enzymatic activity, disaggregation, and thermal stability of bovine liver catalase have been investigated. Compatible osmolytes, proline, xylitol, and valine destabilize the denatured form of the enzyme and, therefore, increase its disaggregation and thermal stability. The increase in the thermal stability is accompanied with a slight increase of activity in comparison to the native enzyme at 25?°C. On the other hand, histidine, a noncompatible osmolyte stabilizes the denatured form of the protein and hence causes an overall decrease in the thermal stability and enzymatic activity of the enzyme. Chemometric results have confirmed the experimental results and have provided insight into the distribution and number of mole fraction components for the intermediates. The increase in melting temperature (T_m) and enzymatic rate could be further amplified by the intrinsic effect of temperature enhancement on the enzymatic activity for the industrial purposes.     

Unexpected impact of esterification on the antioxidant activity and (photo)stability of a eumelanin from 5,6‐dihydroxyindole‐2‐carboxylic acid

To inquire into the role of the carboxyl group as determinant of the properties of 5,6‐dihydroxyindole melanins, melanins from aerial oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) and its DHICA methyl ester (MeDHICA) were comparatively tested for their antioxidant activity. MALDI MS spectrometry analysis of MeDHICA melanin provided evidence for a collection of intact oligomers. EPR analysis showed g‐values almost identical and signal amplitudes (ΔB) comparable to those of DHICA melanin, but spin density was one order of magnitude higher, with a different response to pH changes. Antioxidant assays were performed, and a model of lipid peroxidation was used to compare the protective effects of the melanins. In all cases, MeDHICA melanin performed better than DHICA melanin. This capacity was substantially maintained following exposure to air in aqueous buffer over 1 week or to solar simulator over 3 hr. Different from DHICA melanin, MeDHICA melanin was proved to be fairly soluble in different water‐miscible organic solvents, suggesting its use in dermocosmetic applications.