Loss Of Thermostability Research Articles

Engineering an α-L-rhamnosidase from Aspergillus niger for efficient conversion of rutin substrate

α-L-Rhamnosidase has great potential in the food and pharmaceutical industries, but its use in industrial applications is limited by its low catalytic efficiency. In this study, an α-L-rhamnosidase (AnRha) from Aspergillus niger CCTCC M 2018240 was engineered to enhance its catalytic efficiency towards rutin. Based on molecular docking and structural analysis, alanine scanning, saturation and combinatorial mutagenesis were performed on 16 residues involved in the substrate binding pocket. Four mutants, L234A, L234C, F345S and L234A/S339V/F345S had more than 2-fold higher catalytic efficiency than wild-type (WT) enzyme, and one mutant, S339V had a markedly improved half-life at 65 °C. F345S had 2.5 times the catalytic efficiency of WT, without loss of thermostability. Molecular docking revealed that F345 is the critical substrate entrance tunnel residue in AnRha. Molecular dynamics simulation indicated that the structure of L234A is more flexible, but less stable, whereas S339V is more rigid. Crude enzyme was produced by the F345S mutant and used to hydrolyze an aqueous suspension (130 g/L) of low water-solubility rutin, with a conversion of 95.89 %, nearly 30 % higher than WT (67.18 %). F345S is by far the most effective biocatalyst reported for conversion of rutin. This study also provides useful reference data for engineering α-L-rhamnosidase.

Improvement of the Catalytic Activity of Chitosanase BsCsn46A from Bacillus subtilis by Site-Saturation Mutagenesis of Proline121.

BsCsn46A, a GH46 family chitosanase from Bacillus subtilis, has great potential for industrial chitooligosaccharide production due to its high activity and stability. In this study, a special amino acid Pro121 was identified not fit in the helix structure, which was located in the opposite side of the active center in BsCsn46A, by the PoPMuSiC algorithm. Then, saturation mutagenesis was performed to explore the role of the site amino acid 121. Compared with the wild type, the specific activity of P121N, P121C, and P121V was increased by 1.69-, 1.97-, and 2.15-fold, respectively. In particular, the specific activity of P121N was increased without loss of thermostability, indicating that replacing the structural stiffness of proline in the helical structure could significantly improve the chitosanase activity. The Km values of P121N, P121C, and P121V decreased significantly, indicating that the affinity between the enzyme-substrate complex was enhanced. Through molecular docking, it was found that the increase of hydrogen bonds and van der Waals force between the enzyme-substrate complex and the removal of unfavorable bonds might be the main reason for the change of enzyme properties. In addition, the optimal temperature of the three mutants changed from 60 to 55 °C. These results indicate that the site 121 plays a critical role in the catalytic activity and enzymatic properties of chitosanase. To our knowledge, the results provide novel data on chitosanase activity and identify an excellent candidate of industrial chitosanase.

Engineering Bacillus pumilus alkaline serine protease to increase its low-temperature proteolytic activity by directed evolution

BackgroundMesophilic alkaline serine proteases from various bacteria have been commercially applied in a range of industries owing to their high catalytic efficiency and wide substrate specificity. However, these proteases have an optimal catalytic temperature of approximately 50 °C, and their activity decreases significantly at low temperature. Therefore, to enhance their cold activity, it is necessary to improve the catalytic performance of these proteases at low temperature. The alkaline serine protease (DHAP) from Bacillus pumilus BA06 is a typical mesophilic enzyme, which has demonstrated great potential in various industrial applications. Here we attempted to improve the cold activity of DHAP via directed evolution.ResultsSeven variants (P9S, A1G/K27Q, A38V, A116T, T162I, S182R, and T243S) of DHAP from B. pumilus were obtained via directed evolution. The results showed that all of the variants had increased proteolytic activity at 15 °C towards both the casein and synthetic peptide substrates. With the exception of variant T243S, the thermostability of these variants did not decrease in comparison with the wild-type enzyme. Kinetic analysis indicated that the increase in catalytic efficiency was largely attributed to the increase in turnover number (kcat). Furthermore, the combined variants generated by site-directed mutagenesis showed a further increase in specific caseinolytic activity and the kcat value for hydrolysis of the synthetic peptide. The combined variants of P9S/K27Q and P9S/T162I exhibited an approximate 5-fold increase in caseinolytic activity at 15 °C and almost no loss of thermostability. Finally, the possible mechanism responsible for the change in catalytic properties for these variants was interpreted based on structural modeling.ConclusionsDirected evolution and site-directed mutagenesis were combined to engineer variants of the DHAP from B. pumilus. All of the variants exhibited an increase in hydrolytic efficiency at low temperature towards both of the substrates, casein and synthetic peptide, without any loss of thermostability compared with the wild-type. These data suggest that engineering low-temperature activity for a bacterial protease is not always associated with the loss of thermostability. Furthermore, our findings demonstrate that enhanced cold activity and thermostability could be integrated into a single variant.

The fragmentation behavior and variation of the pristane-phytane ratios of pure phytol in conditions of clay catalysis

ABSTRACTThe thermal evolution of phytol in thecatalytic environmentsof reduction, montmorillonite catalysis and illite catalysis has been investigated. The results show the following: (1)Montmorillonite and illite facilitate the defunctionalization and further cracking of phytol. (2) Pristane and phytane can no longer serve as effect biomarkers at the high thermal evolution stage due to loss of thermostability. (3) Ratios of pristane/phytane greater than 1derive from reduction and montmorillonite catalysis environments. However, the ratio of pristane/phytane was less than 1 under the condition of illite catalysis, which has not been shown before.

Enhancing the α-Cyclodextrin Specificity of Cyclodextrin Glycosyltransferase from Paenibacillus macerans by Mutagenesis Masking Subsite -7.

Cyclodextrin glycosyltransferases (CGTases) (EC 2.4.1.19) catalyze the conversion of starch or starch derivates into mixtures of α-, β-, and γ-cyclodextrins. Because time-consuming and expensive purification procedures hinder the widespread application of single-ingredient cyclodextrins, enzymes with enhanced specificity are needed. In this study, we tested the hypothesis that the α-cyclodextrin selectivity of Paenibacillus macerans α-CGTase could be augmented by masking subsite -7 of the active site, blocking the formation of larger cyclodextrins, particularly β-cyclodextrin. Five single mutants and three double mutants designed to remove hydrogen-bonding interactions between the enzyme and substrate at subsite -7 were constructed and characterized in detail. Although the rates of α-cyclodextrin formation varied only modestly, the rate of β-cyclodextrin formation decreased dramatically in these mutants. The increase in α-cyclodextrin selectivity was directly proportional to the increase in the ratio of their kcat values for α- and β-cyclodextrin formation. The R146A/D147P and R146P/D147A double mutants exhibited ratios of α-cyclodextrin to total cyclodextrin production of 75.1% and 76.1%, approximately one-fifth greater than that of the wild-type enzyme (63.2%), without loss of thermostability. Thus, these double mutants may be more suitable for the industrial production of α-cyclodextrin than the wild-type enzyme. The production of β-cyclodextrin by these mutants was almost identical to their production of γ-cyclodextrin, which was unaffected by the mutations in subsite -7, suggesting that subsite -7 was effectively blocked by these mutations. Further increases in α-cyclodextrin selectivity will require identification of the mechanism or mechanisms by which these small quantities of larger cyclodextrins are formed.

Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps*

Pentameric ligand-gated ion channels (LGICs) play an important role in fast synaptic signal transduction. Binding of agonists to the β-sheet-structured extracellular domain opens an ion channel in the transmembrane α-helical region of the LGIC. How the structurally distinct and distant domains are functionally coupled for such central transmembrane signaling processes remains an open question. To obtain detailed information about the stability of and the coupling between these different functional domains, we analyzed the thermal unfolding of a homopentameric LGIC, the 5-hydroxytryptamine receptor (ligand binding, secondary structure, accessibility of Trp and Cys residues, and aggregation), in plasma membranes as well as during detergent extraction, purification, and reconstitution into artificial lipid bilayers. We found a large loss in thermostability correlating with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of the 5-hydroxytryptamine receptor occurred in consecutive steps at distinct protein locations. A loss of ligand binding was detected first, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally lead to the formation receptor aggregates. Structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Our results are important because they quantify the stability of LGICs during detergent extraction and purification and can be used to create stabilized receptor proteins for structural and functional studies.

Replacing 32 Proline Residues by a Noncanonical Amino Acid Results in a Highly Active DNA Polymerase

Protein engineering may be achieved by rational design, directed evolution-based methods, or computational protein design. Mostly these methods make recourse to the restricted pool of the 20 natural amino acids. With the ability to introduce different new kinds of functionalities into proteins, the use of noncanonical amino acids became a promising new method in protein engineering. Here, we report on the generation of a multifluorinated DNA polymerase. DNA polymerases are highly dynamic enzymes that catalyze DNA synthesis in a template-dependent manner, thereby passing several conformational states during the catalytic cycle. Here, we globally replaced 32 proline residues by the noncanonical imino acid (4R)-fluoroproline in a DNA polymerase of 540 amino acids (KlenTaq DNA polymerase). Interestingly, the substitution level of the proline residues was very efficient (92%). Nonetheless, the introduction of (4R)-fluoroproline into the DNA polymerase resulted in a highly active fluorinated enzyme, which was investigated in primer extension and PCR assays to analyze activity, selectivity, and stability in comparison to the parental enzyme. The DNA polymerase retained fidelity, activity, and sensitivity as the parental wild-type enzyme accompanied by some loss in thermostability. These results demonstrate that a noncanonical amino acid can be used for substitutions of natural counterparts in a highly dynamic enzyme with high molecular weight without effecting crucial enzyme properties. Furthermore, the employed DNA polymerase represents a promising starting point for directed DNA polymerase evolution with noncanonical amino acids.

Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: Improved thermostability by a single-isoleucine mutant

A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.

Improvement of the enzymatic activity of the hyperthermophilic cellulase from Pyrococcus horikoshii

A hyperthermophilic beta-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type EGPh toward both CMC and crystalline cellulose (Avicel).

Thermostability profile of Newcastle disease virus (strain I–2) following serial passages without heat selection

I-2 is an avirulent strain of Newcastle disease virus. During establishment of the I-2 strain master vaccine seed, a series of selection procedures was carried out at 56 degrees C in order to enhance heat resistance. This master seed is used to produce a working seed, which is then employed to produce the vaccine. These two passages are done without further heat selection; however, it is not known how rapidly and to what extent thermostable variants would be lost during further passage. The study was therefore conducted to determine the effect of passage on thermostability of strain I-2. The virus was serially passaged and at various passage levels samples were subjected to heat treatment at 56 degrees C for 120 min. The inactivation rates for infectivity and haemagglutinin (HA) titres were assayed by use of chicken embryonated eggs and HA test, respectively. Thermostability of HA and infectivity of I-2 virus were reduced after 10 and 5 passages, respectively, without heat selection at 56 degrees C. These results suggest that 5 more passages could be carried out between the working seed and vaccine levels without excessive loss of thermostability. This would result in increased vaccine production from a single batch of a working seed.

The temperature dependence of activity and structure for the most prevalent mutant aldolase B associated with hereditary fructose intolerance

Hereditary fructose intolerance (HFI) is an autosomal recessive disorder in humans which is caused by mutations in the aldolase B gene. The most common HFI allele encodes an enzyme with an A149P substitution (AP-aldolase). A lysis method suitable for aggregation-prone proteins overexpressed in bacteria was developed. The enzyme’s structure and function is investigated as a function of temperature. Near-UV CD shows a qualitative difference in tertiary structure, whereas far-UV CD shows no difference in overall secondary structure, although both show increased temperature sensitivity for AP-aldolase compared to that seen with wild-type aldolase B. AP-aldolase exists as a dimer at all temperatures tested, unlike the tetrameric wild-type enzyme, thus providing a possible explanation for the loss in thermostability. AP-aldolase has sixfold lower activity than wild type at 10 °C, which decreases substantially at higher temperature. In addition to disruptions at the catalytic center, the kinetic constants toward different substrates suggest that there is a disruption at the C1-phosphate-binding site, which is not sensitive to temperature. The implications of these structural alterations are discussed with regard to the HFI disease.