Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods

Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in T_m. The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability.     

Enhancing long-term thermal stability in mesophilic glutamate dehydrogenase from Clostridium symbiosum by eliminating cysteine residues

Glutamate dehydrogenase from Clostridium symbiosum has two cysteine residues, C144 and C320. The single mutant C320S and a double mutant with both cysteines replaced by serine have been compared with one another in terms of long-term stability and other properties. Specific activities and kinetic parameters were relatively little affected, but stability was improved—e.g. at 25 °C sterile, sealed samples of wild-type enzyme, C320S and the double mutant at 0.1 mg/ml in 0.1 M phosphate buffer, pH 7 lost 50%, 42% and 32% of activity over 60 days. For the first two proteins this loss was partly reversible with dithiothreitol. When wild-type enzyme was deliberately contaminated with 1 μM Cu²⁺ it became less stable and formed aggregates, whereas the double mutant was not affected. The double mutation thus removes a source of instability through –SH oxidation that would be accentuated by any heavy metal contamination of solutions.     

The state-of-the-art strategies of protein engineering for enzyme stabilization

Enzymes generated by natural recruitment and protein engineering have greatly contribute in various sets of applications. However, their insufficient stability is a bottleneck that limit the rapid development of biocatalysis. Novel approaches based on precise and global structural dissection, advanced gene manipulation, and combination with the multidisciplinary techniques open a new horizon to generate stable enzymes efficiently. Here, we comprehensively introduced emerging advances of protein engineering strategies for enzyme stabilization. Then, we highlighted practical cases to show importance of enzyme stabilization in pharmaceutical and industrial applications. Combining computational enzyme design with molecular evolution will hold considerable promise in this field.     

Structural investigation into the C‐terminal extension of the ene‐reductase from Ralstonia (Cupriavidus) metallidurans

Ene‐reductases (ERs), or Old Yellow Enzymes, catalyze the asymmetric reduction of various activated alkenes. This class of biocatalysts is considered an attractive alternative to current chemical technologies for hydrogenation due to their high selectivity and specificity. Here the X‐ray crystal structure of RmER, a “thermophilic”‐like ER from Ralstonia (Cupriavidus) metallidurans, is reported. Unlike other members of this class of ERs, RmER is monomeric in solution which we previously related to its atypical elongated C‐terminus. A typical dimer interface was however observed in our crystal structure, with the conserved Arg‐“finger” forming part of the adjacent monomer's active site and the elongated C‐terminus extending into the active site through contacting the “capping” domain. This dimerization also resulted in the loss of one FMN cofactor from each dimer pair. This potential transient dimerization and dissociation of FMN could conceivably explain the rapid rates previously observed when an FMN light‐driven cofactor regeneration system was used during catalysis with RmER.     

Changing the recognition site of a conjugative relaxase by rational design

TrwC is a relaxase protein, which starts and finishes DNA processing during bacterial conjugation in plasmid R388. TrwC recognizes a specific sequence of DNA (25 nucleotides) in the donor cell: the nic-site. As a model example, a single transversion C24G in nic avoids DNA processing by TrwC. Using this simple model, our objective was to obtain a proof of principle that TrwC specificity can be changed. Several structures of DNA–TrwC complexes were used as reference to design a focused saturation mutagenesis library (NNK) randomizing amino acid Lys262, since its side chain seems to sterically hinder the recognition of the C24G nic mutation by wild-type TrwC. Using bacterial conjugation as an in vivo selection system, several TrwC variants were found that show changes in substrate specificity. These variants were also tested in a competitive assay to evaluate their conjugation efficiency.     

Xenobiotic reductase A in the degradation of quinoline by Pseudomonas putida 86: physiological function, structure and mechanism of 8-hydroxycoumarin reduction

A continuous evolutionary pressure of the biotic and abiotic world has led to the development of a diversity of microbial pathways to degrade and biomineralize aromatic and heteroaromatic compounds. The heterogeneity of compounds metabolized by bacteria like Pseudomonas putida indicates the large variety of enzymes necessary to catalyse the required reactions. Quinoline, a N-heterocyclic aromatic compound, can be degraded by microbes along different pathways. For P. putida 86 quinoline degradation by the 8-hydroxycoumarin pathway has been described and several intermediates were identified. To select enzymes catalysing the later stages of the 8-hydroxycoumarin pathway P. putida 86 was grown with quinoline. The FMN-containing enzyme xenobiotic reductase A (XenA) was isolated and analysed for its reactivity with intermediates of the 8-hydroxycoumarin pathway. XenA catalyses the NADPH-dependent reduction of 8-hydroxycoumarin and coumarin to produce 8-hydroxy-3,4-dihydrocoumarin and 3,4-dihydrocoumarin, respectively. Crystallographic analysis of XenA alone and in complex with the two substrates revealed insights into the mechanism. XenA shows a dimeric arrangement of two (beta/alpha)(8) barrel domains each binding one FMN cofactor. High resolution crystal structures of complexes with 8-hydroxycoumarin and coumarin show different modes of binding for these molecules in the active site. While coumarin is ideally positioned for hydride transfer from N-5 of the isoalloxazine ring to C-4 of coumarin, 8-hydroxycoumarin forms a non-productive complex with oxidised XenA. Orientation of 8-hydroxycoumarin in the active site appears to be dependent on the electronic state of the flavin. We postulate that XenA catalyses the last step of the 8-hydroxycoumarin pathway before the heterocyclic ring is hydrolysed to yield 3-(2,3-dihydroxyphenyl)-propionic acid. As XenA is also found in P. putida strains unable to degrade quinoline, it appears to have more than one physiological function and is an example of how enzymes with low substrate specificity can help to explain the variety of degradation pathways possible.     

Engineering the thermostability of a TIM-barrel enzyme by rational family shuffling

A possible approach to generate enzymes with an engineered temperature optimum is to create chimeras of homologous enzymes with different temperature optima. We tested this approach using two family-10 xylanases from Thermotoga maritima: the thermophilic xylanase A catalytic domain (TmxAcat, T_opt = 68 °C), and the hyperthermophilic xylanase B (TmxB, T_opt = 102 °C). Twenty-one different chimeric constructs were created by mimicking family shuffling in a rational manner. The measured temperature optima of the 16 enzymatically active chimeras do not monotonically increase with the percentage of residues coming from TmxB. Only four chimeras had a higher temperature optimum than TmxAcat, the most stable variant (T_opt = 80 °C) being the one in which both terminal segments came from TmxB. Further analysis suggests that the interaction between the N- and C-terminal segments has a disproportionately high contribution to the overall thermostability. The results may be generalizable to other enzymes where the N- and C-termini are in contact.     

Recent advances in the improvement of enzyme thermostability by structure modification

Abstract

Thermostability is considered to be an important parameter to measure the feasibility of enzymes for industrial applications. Generally, higher thermostability makes an enzyme more competitive and desirable in industry. However, most natural enzymes show poor thermostability, which restricts their application. Protein structure modification is a desirable method to improve enzyme properties. In recent years, tremendous progress has been achieved in protein thermostability engineering. In this review, we provide a systemic overview on the approaches of protein structure modification for the improvement of enzyme thermostability during the last decade. Structure modification approaches, including the introduction of non-covalent interactions and covalent bonds, increase of proline and/or decrease in glycine, reinforcement of subunit–subunit interactions, introduction of glycosylation sites, truncation and cyclization have been highlighted.     

老黄酶OYE家族的蛋白质工程的研究进展 *

老黄酶OYE家族酶是一类广泛分布的能够催化烯烃化合物不对称还原的酶类,其能够用于多种手性化合物的制备.分析了OYE家族酶的系统分类及催化反应类型,针对目前该类酶在应用过程中出现的稳定性差,活性低及底物特异性强等问题,综述了蛋白质工程方法对该类酶进行改造的研究进展,为深入研究该家族酶的催化机制及进一步改造提供参考,同时为进一步拓展OYE酶的工业化应用奠定基础.     

The role of N-glycosylation sites in the activity,stability, and expression of the recombinant elastase expressed by Pichia pastoris

The Pseudomonas aeruginosa elastase (PAE), produced by Pseudomonas aeruginosa (P. aeruginosa), is a promising biocatalyst for peptide synthesis in organic solvents. As P. aeruginosa is an opportunistic pathogen, the enzyme has been heterologously over-expressed in the safe and efficient host, Pichia pastoris (P. pastoris) for its industrial application. The recombinant elastase (rPAE) contains three potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences), and is heterogeneously N-glycosylated. To investigate the role of N-glycosylation in the activity, stability, and expression of rPAE, these potential N-glycosylation sites (N43, N212, and N280) were mutated using site-directed mutagenesis. Specifically the asparagine (Asn, N) residues were converted to glutamine (Gln, Q). The enzymatic activity and stability of non-glycosylated and glycosylated rPAE were then compared. The results indicated that the influence of N-glycosylation on its activity was insignificant. The non- and glycosylated isoforms of rPAE displayed similar kinetic parameters for hydrolyzing casein in aqueous medium, and when catalyzing bipeptide synthesis in 50% (v/v) DMSO, they exhibited identical substrate specificity and activity, and produced similar yields. However, N-glycosylation improved rPAE stability both in aqueous medium and in 50% (v/v) organic solvents. The half-lives of the glycosylated and non-glycosylated forms of rPAE at 70 °C were 32.2 and 23.1 min, respectively. Mutation of any potential N-glycosylation site was detrimental to its expression in P. pastoris. There was a 23.9% decrease in expression of the N43Q mutant, 63.6% of the N212Q mutant, and 63.7% of the N280Q mutant compared with the wild type. Furthermore, combined mutation of these sites resulted in an additional decrease in the caseinolytic activities of the mutants. These results indicated that all of the N-glycosylation sites were necessary for high-level expression of rPAE.     

Biosynthesis of a rosavin natural product in Escherichia coli by glycosyltransferase rational design and artificial pathway construction

Phytochemicals are rich resources for pharmaceutical and nutraceutical agents. A key challenge of accessing these precious compounds can present significant bottlenecks for development. The cinnamyl alcohol disaccharides also known as rosavins are the major bioactive ingredients of the notable medicinal plant Rhodiola rosea L. Cinnamyl-(6′-O-β-xylopyranosyl)-O-β-glucopyranoside (rosavin E) is a natural rosavin analogue with the arabinopyranose unit being replaced by its diastereomer xylose, which was only isolated in minute quantity from R. rosea. Herein, we described the de novo production of rosavin E in Escherichia coli. The 1,6-glucosyltransferase CaUGT3 was engineered into a xylosyltransferase converting cinnamyl alcohol monoglucoside (rosin) into rosavin E by replacing the residue T145 with valine. The enzyme activity was further elevated 2.9 times by adding the mutation N375Q. The synthesis of rosavin E from glucose was achieved with a titer of 92.9 mg/L by combining the variant CaUGT3^T145V/N375Q, the UDP-xylose synthase from Sinorhizobium meliloti 1021 (SmUXS) and enzymes for rosin biosynthesis into a phenylalanine overproducing E. coli strain. The production of rosavin E was further elevated by co-overexpressing UDP-xylose synthase from Arabidopsis thaliana (AtUXS3) and SmUXS, and the titer in a 5 L bioreactor with fed-batch fermentation reached 782.0 mg/L. This work represents an excellent example of producing a natural product with a disaccharide chain by glycosyltransferase engineering and artificial pathway construction.     

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.     

Hydrophilization of immobilized model enzymes suggests a widely applicable method for enhancing protein stability in polar organic co-solvents

β-Galactosidases from Escherichia coli, Kluyveromyces lactis and Aspergillus oryzae were used to characterize the potential for enzyme stabilization of a two-step strategy: (i) immobilization on glutaraldehyde-agarose (Glut90), (ii) subsequent generation of a hydrophilic nano-environment by reaction with polyaldehyde-dextran polymer (Glut90-Pal), followed by polyamine-dextran polymer (Glut90-Pal-Pam). The derivatives were characterized by kinetics parameters, co-solvent (ethanol and acetone) and temperature stability. Hydrophilization achieved important co-solvent stabilization in all cases. One of the most remarkable results obtained was a 25-fold increase in the half-life of the A. oryzae Glut90-Pal-Pam derivative in 50% (v/v) acetone. Stabilization achieved in very drastic co-solvent concentrations is directly related to the hydrophilization of the nano-environment. The K_M values show that the hydrophilic shell appears to behave as an open structure and may create a “partition effect” that protects the enzymes from denaturation. These results show the potential of hydrophilization for building up additional stabilization of immobilized enzymes which would make possible the development of industrial applications.     

Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting

Sortases are a group of enzymes displayed on the cell-wall of Gram-positive bacteria. They are responsible for the attachment of virulence factors onto the peptidoglycan in a transpeptidation reaction through recognition of a pentapeptide substrate. Most housekeeping sortases recognize one specific pentapeptide motif; however, Streptococcus pyogenes sortase A (SpSrtA WT) recognizes LPETG, LPETA and LPKLG motifs. Here, we examined SpSrtA's flexible substrate specificity by investigating the role of the β7/β8 loop in determining substrate specificity. We exchanged the β7/β8 loop in SpSrtA with corresponding β7/β8 loops from Staphylococcus aureus (SaSrtA WT) and Bacillus anthracis (BaSrtA WT). While the BaSrtA-derived variant showed no enzymatic activity toward either LPETG or LPETA substrates, the activity of the SaSrtA-derived mutant toward the LPETA substrate was completely abolished. Instead, the mutant had an improved activity toward LPETG, the preferred substrate of SaSrtA WT.     

疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计提高其活性和温度稳定性

目的:通过对疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计,获得高酶活与耐高温的脂肪酶品种,为脂肪酶在饲料、油脂加工和生物柴油等领域的应用奠定基础.方法:对脂肪酶典型结构域lid和loop区域的系统发育分析,找到候选的位点,理性设计并通过实验验证,获得脂肪酶活性和耐高温特性显著提高的...     

Enhancing the stability and solubility of TEV protease using in silico design

The ability to rationally increase the stability and solubility of recombinant proteins has long been a goal of biotechnology and has significant implications for biomedical research. Poorly soluble enzymes, for example, result in the need for larger reaction volumes, longer incubation times, and more restricted reaction conditions, all of which increase the cost and have a negative impact on the feasibility of the process. Rational design is achieved here by means of the PoPMuSiC program, which performs in silico predictions of stability changes upon single-site mutations. We have used this program to increase the stability of the tobacco etch virus (TEV) protein. TEV is a 27-kDa nuclear inclusion protease with stringent specificity that is commonly used for the removal of solubility tags during protein purification protocols. However, while recombinant TEV can be produced in large quantities, a limitation is its relatively poor solubility (generally approximately 1 mg/mL), which means that large volumes and often long incubation times are required for efficient cleavage. Following PoPMuSiC analysis of TEV, five variants predicted to be more stable than the wild type were selected for experimental analysis of their stability, solubility, and activity. Of these, two were found to enhance the solubility of TEV without compromising its functional activity. In addition, a fully active double mutant was found to remain soluble at concentrations in excess of 40 mg/mL. This modified TEV appears thus as an interesting candidate to be used in recombinant protein technology.     

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.     

Structure-based rational design of self-inhibitory peptides to disrupt the intermolecular interaction between the troponin subunits C and I in neuropathic pain

The troponin (Tn) is a ternary complex consisting of three subunits TnC, TnI and TnT; molecular disruption of the Tn complex has been recognized as an attractive strategy against neuropathic pain. Here, a self-inhibitory peptide is stripped from the switch region of TnI interaction interface with TnC, which is considered as a lead molecular entity and then used to generate potential peptide disruptors of TnC–TnI interaction based on a rational molecular design protocol. The region is a helical peptide segment capped by N- and C-terminal disorders. Molecular dynamics simulation and binding free energy analysis suggests that the switch peptide can interact with TnC in a structurally and energetically independent manner. Terminal truncation of the peptide results in a number of potent TnC binders with considerably simplified structure and moderately decreased activity relative to the native switch. We also employ fluorescence polarization assays to substantiate the computational findings; it is found that the rationally designed peptides exhibit moderate or high affinity to TnC with dissociation constants K_D at micromolar level.     

Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach

Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a V_H and V_L domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in V_H and V_L domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the V_L domain to one that best pairs with the existing V_H. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the V_L domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.