Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method

α-Amino ester hydrolases (AEH, E.C. 3.1.1.43) catalyze the synthesis and hydrolysis of α-amino β-lactam antibiotics. The AEH enzymes have been shown to feature excellent synthetic capability but suffer from poor thermostability. AEH from Xanthomonas campestris exhibits an optimal activity temperature of 25 °C, an observed half-life of 5 min at 30 °C, and a "T-50" value, the temperature at which the half-life is 30 min, of 27 °C. To improve the thermostability of AEH, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures of AEH from Xanthomonas citri. A family of stabilized variants was created including a consensus-driven triple variant, A275P/N186D/V622I. Independent NNK saturation of two high B-factor sites, K34 and E143, on the triple variant resulted in our best variant, the quadruple mutant E143H/A275P/N186D/V622I, with a "T-50" value of 34 °C (7 °C improvement) and 1.3-fold activity compared to wild-type.     

Rationally selected single-site mutants of the Thermoascus aurantiacus endoglucanase increase hydrolytic activity on cellulosic substrates

Variants of the Thermoascus aurantiacus Eg1 enzyme with higher catalytic efficiency than wild-type were obtained via site-directed mutagenesis. Using a rational mutagenesis approach based on structural bioinformatics and evolutionary analysis, two positions (F16S and Y95F) were identified as priority sites for mutagenesis. The mutant and parent enzymes were expressed and secreted from Pichia pastoris and the single site mutants F16S and Y95F showed 1.7- and 4.0-fold increases in k(cat) and 1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic substrates, respectively, while maintaining thermostability. Similar to the parent enzyme, the two variants were active between pH 4.0 and 8.0 and showed optimal activity at temperature 70°C at pH 5.0. The purified enzymes were active at 50°C for over 12 h and retained at least 80% of initial activity for 2 h at 70°C. In contrast to the improved hydrolysis seen with the single mutation enzymes, no improvement was observed with a third variant carrying a combination of both mutations, which instead showed a 60% reduction in catalytic efficiency. This work further demonstrates that non-catalytic amino acid residues can be engineered to enhance catalytic efficiency in pretreatment enzymes of interest.     

Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 α-amylase

Asparagine deamidation is one of the important determinants of protein thermostability. Here, structure based mutagenesis has been done in order to probe the role of Asn residues in thermostability of a Ca independent Bacillus sp. KR-8104 α-amylase (BKA). Residues involved in potential deamidation processes have been selected and replaced using a site directed mutagenesis. Fourteen different variants were tested for thermostability by measuring residual activities after incubation at high temperature. In comparison to the wild-type enzyme, four mutated variants are able to increase the half life of the protein at high temperatures. The highest stabilization resulted from the substitution of asparatate in place of asparagine at position 112, leading to a nearly fivefold increase of the enzyme's half-life at 70°C. Also replacement of Asn129 to aspartic acid and Asn312 to serine markedly increased the half-life of the enzyme at 70°C indicating that the deamination of these residues may have a deleterious effect on BKA.     

Enhancement of thermostability of fungal deglycating enzymes by directed evolution

Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50°C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50°C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.     

Engineering a large protein by combined rational and random approaches: stabilizing the Clostridium thermocellum cellobiose phosphorylase

The Clostridium thermocellum cellobiose phosphorylase (CtCBP) is a large protein consisting of 812 amino acids and has great potential in the production of sugar phosphates, novel glycosides, and biofuels. It is relatively stable at 50 °C, but is rapidly inactivated at 70 °C. To stabilize CtCBP at elevated temperatures, two protein-engineering approaches were applied, i.e. site-directed mutagenesis based on structure-guided homology analysis and random mutagenesis at various mutation rates. The former chose substitutions by comparison of the protein sequences of CBP homologs, utilized structural information to identify key amino acid residues responsible for enhanced stability, and then created a few variants accurately. The latter constructed large libraries of random mutants at different mutagenesis frequencies. A novel combinational selection/screening strategy was employed to quickly isolate thermostability-enhanced and active variants. Several stability-enhanced mutants were obtained by both methods. Manually combining the stabilizing mutations identified from both rational and random approaches led to the best mutant (CM3) with the halftime of inactivation at 70 °C extended from 8.3 to 24.6 min. The temperature optimum of CM3 was increased from 60 to 80 °C. These results suggested that a combination of rational design and random mutagenesis could have a solid basis for engineering large proteins.     

Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants

Recombinant microbial transglutaminase (rMTG), an enzyme useful for the cross-linking or the posttranslational modification of (therapeutic) proteins, was optimized by random mutagenesis for the first time. A screening method was developed which, in addition to state-of-the-art procedures, includes a proteolytic activation step of the expressed soluble pro-enzyme. The library of 5,500 clones was screened for variants with increased thermostability and heat-sensitivity, respectively. Mutant enzymes were overproduced, isolated and characterized. After just one round of mutagenesis, nine variants with a single amino acid exchange showed a remarkably increased thermostability at 60 degrees C. The exchange of a serine residue close to the N-terminus against proline resulted in an rMTG mutant (S2P) with 270% increased half-life. Seven variants exhibited an increased heat-sensitivity at 60 degrees C of which one mutant (G25S) retained its specific activity between 10 and 40 degrees C. The mutations responsible for the increased thermostability and the heat-sensitivity were identified and assigned to the three-dimensional (3D) structure. All single point mutations related to changed thermal properties of rMTG are located in the N-terminal domain (i.e. the left side wall of the active site cleft of the front view of the MTG as defined by the literature [Kashiwagi, T., Yokoyama, K., Ishikawa, K., Ono, K., Ejima, D., Matsui, H., Suzuki, E., 2002. Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J. Biol. Chem. 277, 44252-44260] showing the importance of this part of the protein.     

一个耐热碱性磷酸酯酶突变型的研究

为了进一步揭示蛋白质的耐热机制,对含有耐热碱性磷酸酯酶（ＦＤ－ＴＡＰ）的表达质粒ｐＴＡＰ５０３Ｆ进行了随机诱变,用菌落原位显色法从约５０００个转化子中筛选到４个耐热性下降的突变型克隆,并对其中１克隆（ＴＡＰＭ３）进行了部分酶学性质、ＤＮＡ和氨基酸序列的研究。酶学性质研究表明,与野生型相比,该突变型酶的耐热性有较大幅度的下降,而热激活性无明显改变。ＤＮＡ序列分析表明在１２３９位ＴＡＰＭ３发生Ｇ→Ａ转     

Further improvement of phosphite dehydrogenase thermostability by saturation mutagenesis

Phosphite dehydrogenase represents a new enzymatic system for regenerating reduced nicotinamide cofactors for industrial biocatalysis. We previously engineered a variant of phosphite dehydrogenase with relaxed cofactor specificity and significantly increased activity and stability. Here we performed one round of random mutagenesis followed by comprehensive saturation mutagenesis to further improve the enzyme thermostability while maintaining its activity. Two new thermostabilizing mutations were identified. These, along with the 12 mutations previously identified, were subjected to saturation mutagenesis using the parent enzyme or the engineered thermostable variant 12x as a template, followed by screening of variants with increased thermostability. Of the 12 previously identified sites, 6 yielded new variants with improved stability over the parent enzyme. Several mutations were found to be context-dependent. On the basis of molecular modeling and biochemical analysis, various mechanisms of thermostabilization were identified. Combining the most thermostabilizing mutation at each site resulted in a variant that showed a 100-fold increase in half-life at 62 degrees C over the 12x mutant. The final mutant has improved the half-life of thermal inactivation at 45 degrees C by 23,000-fold over the parent enzyme. The engineered phosphite dehydrogenase will be useful in NAD(P)H regeneration.     

Enhancing RGI lyase thermostability by targeted single point mutations

Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering approach involving single amino acid substitution. Nine individual amino acids were selected as targets for site-saturated mutagenesis by the use of a predictive consensus approach in combination with prediction of protein mutant stability changes and B-factor iteration testing. After extensive experimental verification of the thermal stability of the designed mutants versus the original wild-type RGI lyase, several promising single point mutations were obtained, particularly in position Glu434 on the surface of the enzyme protein. The best mutant, Glu434Leu, produced a half-life of 31 min at 60 °C, corresponding to a 1.6-fold improvement of the thermal stability compared to the original RGI lyase. Gly55Val was the second best mutation with a thermostability half-life increase of 27 min at 60 °C, and the best mutations following were Glu434Trp, Glu434Phe, and Glu434Tyr, respectively. The data verify the applicability of a combinatorial predictive approach for designing a small site saturation library for improving enzyme thermostability. In addition, new thermostable RGI lyases suitable for enzymatic upgrading of pectinaceous plant biomass materials at elevated temperatures were produced.     

Introduction of glycine and proline residues onto protein surface increases the thermostability of endoglucanase CelA from Clostridium thermocellum

A saturation mutagenesis library was constructed at the position 329 of the endoglucanase CelA from Clostridium thermocellum based on previous results (Yi and Wu, 2010), and one mutation, S329G, was identified to contribute to the enhanced thermostability. The result inspired a rational design approach focusing on the introduction of Gly or Pro residue onto the protein surface, which led to the identification of two additional beneficial mutations, H194G and S269P. Combination of these three mutations resulted in a mutant with a 10-fold increase in half-life of inactivation (60 min) at 86°C without compromising activity compared with the wild-type. Its reaction temperature for maximum activity increased from 75 to 85°C. The results provide valuable thermostability-related structural information on this thermophilic enzyme.     

Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure

An efficient random mutagenesis procedure coupled to a replica plate screen facilitated the isolation of mutant subtilisins from Bacillus amyloliquefaciens that had altered autolytic stability under alkaline conditions. Out of about 4000 clones screened, approximately 70 produced subtilisins with reduced stability (negatives). Two clones produced a more stable subtilisin (positives) and were identified as having a single mutation, either Ile107Val or Lys213Arg (the wild-type amino acid is followed by the codon position and the mutant amino acid). One of the negative mutants, Met50Val, was at a site where other homologous subtilisins contained a Phe. When the Met50Phe mutation was introduced into the B. amyloliquefaciens gene, the mutant subtilisin was more alkaline stable. The double mutant (Ile107Val/Lys213Arg) was more stable than the isolated single mutant parents. The triple mutant (Met50Phe/Ile107Val/Lys213Arg) was even more stable than Ile107Val/Lys213Arg (up to two times the autolytic half-time of wild-type at pH 12). These studies demonstrate the feasibility for improving the alkaline stability of proteins by random mutagenesis and identifying potential sites where substitutions from homologous proteins can improve alkaline stability.     

Improving the thermostability of <Emphasis Type="Italic">N</Emphasis>-carbamyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolase by error-prone PCR

To facilitate the easier production of d-amino acids using N-carbamyl-d-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 °C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of d-amino acids.     

Thermostability improvement of maltogenic amylase MAUS149 by error prone PCR

The thermostability of maltogenic amylase from Bacillus sp. US149 (MAUS149) was improved by random mutagenesis using error prone PCR. The library constructed for the mutants obtained was subjected to screening, leading to the selection of a thermostable mutant enzyme named MA-A27. The latter was noted to contain four single mutations, namely D46V, P78L, V145A, and K548E. The half-life times recorded for MA-A27 at 50 °C and 55 °C were 70 min and 25 min, compared to 30 min and 13 min for the wild type, respectively. The results from molecular modeling attributed the increase in thermostability observed for MA-A27 to P78L and K548E substitutions that led to new hydrogen bond and salt bridge formations. Further site-directed mutagenesis studies showed that the P78L and K548E single mutations underwent an increase in thermostability, thus confirming the joint contribution of both substitutions to the increase in thermostability observed for MA-A27.     

Three amino acid changes contribute markedly to the thermostability of β-glucosidase BglC from Thermobifida fusca

Thermostability of β-glucosidase was enhanced by family shuffling, site saturation mutagenesis, and site-directed mutagenesis. Family shuffling was carried out based on β-glucosidase BglC from Thermobifida fusca and β-glucosidase BglB from Paebibacillus polymxyxa with the help of synthetic primers. High-throughput screening revealed mutants with higher thermostability than both parental enzymes. The most thermostable mutant VM2 containing three key amino acid changes in L444Y/G447S/A433V had a 144-fold increase in half-life of inactivation as compared to the parental enzyme BglC. The mutant VM2 showed 28% and 94% increase in k(cat) towards p-nitrophenyl-β-D-glucopyranoside (pNPG) and cellobiose, respectively. The mutant with enhanced stability would facilitate the recycle of β-glucosidase in the bioconversion of cellulosic biomass.     

Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase

Bacillus licheniformis alpha-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism. Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood. Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability. Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression. A total of 175 amylase variants were created and analysed in vitro. Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature. Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes. By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme. The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme's half-life at 80 degrees C (pH 5.6, 0.1 mM CaCl(2)).These results, combined with those of previous mutational analyses, show that the structural determinants contributing to the overall thermostability of BLA concentrate in domain B and at its interface with the central A domain. This region contains a triadic Ca-Na-Ca metal-binding site that appears extremely sensitive to any modification that may alter or reinforce the network of electrostatic interactions entrapping the metal ions. In particular, a loop spanning from residue 178 to 199, which undergoes pronounced conformational changes upon removal of calcium, appears to be the key feature for maintaining the enzyme structural integrity. Outside this region, most salt-bridges that were destroyed by mutations were found to be dispensable, except for an Asp121-Arg127 salt-bridge that contributes to the enhanced thermostability of BLA compared to other homologous bacterial alpha-amylases. Finally, our studies demonstrate that the natural resistance of BLA against high temperature is not optimized and can be enhanced further through various means, including the removal of possibly deamidating residues.     

Exploring the thermostable properties of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by a combinatorial directed evolution strategy

As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained. Those mutation sites were subsequently combined by adopting several rounds of iterative saturation mutagenesis (ISM) approach. After four rounds of saturation mutagenesis, one best mutant ISM-4 with a 3400-fold improvement in half-life (t _1/2) inactivation at 65 °C, 18 °C increase in apparent T _m value, and 20 °C increase in optimum temperature was obtained, compared to wild-type HheC. To the best of our knowledge, the mutant represents the most thermostable HheC variant reported up to now. Moreover, the mutant was as active as wild-type enzyme for the substrate 1,3-dichloro-2-propanol, and they remained most enantioselectivity of wild-type enzyme in the kinetic resolution of rac-2-chloro-1-phenolethanol, exhibiting a great potential for industrial applications. Our structural investigation highlights that surface loop regions are hot spots for modulating the thermostability of HheC, the residues located at these regions contribute to the thermostability of HheC in a cooperative way, and protein rigidity and oligomeric interface connections contribute to the thermostability of HheC. All of these essential factors could be used for further design of an even more thermostable HheC, which, in turn, could greatly facilitate the application of the enzyme as a biocatalyst.

     

Directed evolution of the thermostable xylanase from Thermomyces lanuginosus

The thermostability of the endo-beta-1,4-xylanase from Thermomyces lanuginosus (xynA) was improved by directed evolution using error-prone PCR. Transformants expressing the variant xylanases were first selected on 0.4% Remazol Brilliant Blue-xylan and then exposed to 80 degrees C. Whereas the wild type XynA lost 90% activity after 10 min at 80 degrees C, five mutants displayed both higher stabilities and activities than XynA. Four mutants were subjected to further mutagenesis to improve the stability and activity of the xylanase. Subsequent screening revealed three mutants with enhanced thermostability. Mutant 2B7-10 retained 71% of its activity after treatment at 80 degrees C for 60 min and had a half-life of 215 min at 70 degrees C, which is higher than that attained by XynA. Sequence analysis of second generation mutants revealed that mutations were not concentrated in any particular region of the protein and exhibited much variation. The best mutant obtained from this study was variant 2B7-10, which had a single substitution (Y58F) in beta-sheet A of the protein, which is the hydrophilic, solvent-accessible outer surface of the enzyme. Most of the mutants obtained in this study displayed a compromise between stability and activity, the only exception being mutant 2B7-10. This variant showed increased activity and thermostability.     

Enhancement of proteinase inhibitory activity of recombinant human cystatin C using random-centroid optimization

We had previously written a random-centroid optimization computer program for genetics (RCG) to optimize protein engineering, which was successfully applied to modify single site of the 16 amino acid residues at the active site of B. stearothermophilys neutral protease for improving thermostability [J. Agric. Food Chem., 46 (1998) 1655]. The same program was applied in this study to double-site mutation of the entire sequence of human cystatin C (HCC) with 120 residues for improving its protease inhibitory activity. The RCG program selected two sites simultaneously and amino acid residues to replace the sites selected in the sequence in order to find the best papain-inhibitory activity and stability of the protease inhibitor. Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris. Of the total 45 mutants, G12W/H86V mutant showed a 5-fold increase in the bioactivity over the recombinant wild-type (WT) cystatin. Also, P13F mutant exhibited a half-life temperature (T1/2) 5.2 degrees C higher than 68.2 degrees C of WT in addition to a 56% greater papain inhibitory activity. Mutation for diminishing beta-sheet content reduced polymerization of cystatin C, thus improving papain-inhibitory activity. The approach using RCG was able to improve the functional properties of cystatin by least relying on the prior knowledge of its molecular structure.     

Structural perturbation and compensation by directed evolution at physiological temperature leads to thermostabilization of beta-lactamase

The choice of protein for use in technical and medical applications is limited by stability issues, making understanding and engineering of stability key. Here, enzyme destabilization by truncation was combined with directed evolution to create stable variants of TEM-1 beta-lactamase. This enzyme was chosen because of its implication in prodrug activation therapy, pathogen resistance to lactam antibiotics, and reporter enzyme bioassays. Removal of five N-terminal residues generated a mutant which did not confer antibiotic resistance at 37 degrees C. Accordingly, the half-life time in vitro was only 7 s at 40 degrees C. However, three cycles comprising random mutagenesis, DNA shuffling, and metabolic selection at 37 degrees C yielded mutants providing resistance levels significantly higher than that of the wild type. These mutants demonstrated increased thermoactivity and thermostability in time-resolved kinetics at various temperatures. Chemical denaturation revealed improved thermodynamic stabilities of a three-state unfolding pathway exceeding wild-type construct stability. Elongation of one optimized deletion mutant to full length increased its stability even further. Compared to that of the wild type, the temperature optimum was shifted from 35 to 50 degrees C, and the beginning of heat inactivation increased by 20 degrees C while full activity at low temperatures was maintained. We attribute these effects mainly to two independently acting boundary interface residue exchanges (M182T and A224V). Structural perturbation by terminal truncation, evolutionary compensation at physiological temperatures, and elongation is an efficient way to analyze and improve thermostability without the need for high-temperature selection, structural information, or homologous proteins.     

Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution

To expand the functionality of lipase from Rhizopus arrhizus (RAL) we have used error-prone PCR and DNA shuffling methods to create RAL mutants with improved thermostability and the optimum temperature. One desirable mutant with three amino acids substitution was obtained. The mutated lipase was purified and characterized. The optimum temperature of the mutant lipase was higher by 10 °C than that of the wild-type RAL (WT-RAL). In addition, the thermostability characteristic of the mutant was also improved as the result of directed evolution. The half-life (T_1/2) at 50 °C of the mutant exceeded those of WT-RAL by 12-fold. To confirm which substitution contributed to enhance thermostability and the optimum temperature for lipase activity, three chimeric lipases: chimeric lipase 1(CL-1; A9T), chimeric lipase 2 (CL-2; E190V) and chimeric lipase 3 (CL-3; M225I) from the WT-RAL gene were constructed. Each of the chimeric enzymes was purified and characterized. Amino acid substitution at position 190 was determined to be critical for lipase thermostability and the optimum temperature, while the residue at position 9 and 225 had only marginal effect. The mutational effect is interpreted according to a simulated three-dimensional structure for the mutant lipase.