Improvement Of Thermostability Research Articles

Improved thermostability of creatinase from Alcaligenes Faecalis through non-biased phylogenetic consensus-guided mutagenesis

BackgroundEnzymatic quantification of creatinine has become an essential method for clinical evaluation of renal function. Although creatinase (CR) is frequently used for this purpose, its poor thermostability severely limits industrial applications. Herein, we report a novel creatinase from Alcaligenes faecalis (afCR) with higher catalytic activity and lower KM value, than currently used creatinases. Furthermore, we developed a non-biased phylogenetic consensus method to improve the thermostability of afCR.ResultsWe applied a non-biased phylogenetic consensus method to identify 59 candidate consensus residues from 24 creatinase family homologs for screening afCR mutants with improved thermostability. Twenty-one amino acids of afCR were selected to mutagenesis and 11 of them exhibited improved thermostability compared to the parent enzyme (afCR-M0). Combination of single-site mutations in sequential screens resulted in a quadruple mutant D17V/T199S/L6P/T251C (M4-2) which showed ~ 1700-fold enhanced half-life at 57 °C and a 4.2 °C higher T5015 than that of afCR-M0. The mutant retained catalytic activity equivalent to afCR-M0, and thus showed strong promise for application in creatinine detection. Structural homology modeling revealed a wide range of potential molecular interactions associated with individual mutations that contributed to improving afCR thermostability.ConclusionsResults of this study clearly demonstrated that the non-biased-phylogenetic consensus design for improvement of thermostability in afCR is effective and promising in improving the thermostability of more enzymes.

Immobilization studies of a pectinase produced by Aspergillus terreus.

Aspergillus terreus can produce different holocellulose-degrading enzymes when grown in sugarcane bagasse, with predominant pectinase activity. Thus, pectinase was selected for purification and immobilization studies. Ion exchange and molecular exclusion chromatography studies were performed, after which it was possible to semipurify the enzyme with a yield of 80%. The crude extract pectinase (PECEB) and the partially purified enzyme (PEC2) were immobilized on monoamino-N-aminoethyl (MANAE)-agarose with pectinase activity yields of 66% and 98%, respectively. After immobilization in MANAE-agarose, the pectinase showed higher activity at acidic pH (pH 4.0) when compared to the nonimmobilized enzyme. It was also found that after the immobilization process, there was a threefold improvement in the enzyme's thermostability. Also, it was possible to reuse the immobilized enzyme for up to five cycles of hydrolysis with effective production of reducing sugars (0.196 mg/g of substrate). The industrial application test revealed a significant decrease in the viscosity of guava juice when the immobilized enzyme was used. PECEB, immobilized on MANAE-agarose, was the enzyme sample that generated the highest pulp viscosity reduction (approximately 47%). Although additional studies are needed for practical industrial application, the results obtained herein reveal the potential of application of immobilized pectinase in the industry.

Computation-aided engineering of starch-debranching pullulanase from Bacillus thermoleovorans for enhanced thermostability.

Pullulanases are widely used in food, medicine, and other industries because they specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. In addition, high-temperature thermostable pullulanase has multiple advantages, including decreasing saccharification solution viscosity accompanied with enhanced mass transfer and reducing microbial contamination in starch hydrolysis. However, thermophilic pullulanase availability remains limited. Additionally, most do not meet starch-manufacturing requirements due to weak thermostability. Here, we developed a computation-aided strategy to engineer the thermophilic pullulanase from Bacillus thermoleovorans. First, three computational design predictors (FoldX, I-Mutant 3.0, and dDFIRE) were combined to predict stability changes introduced by mutations. After excluding conserved and catalytic sites, 17 mutants were identified. After further experimental verification, we confirmed six positive mutants. Among them, the G692M mutant had the highest thermostability improvement, with 3.8°C increased Tm and 2.1-fold longer half-life than the wild type at 70°C. We then characterized the mechanism underlying increased thermostability, such as rigidity enhancement, closer conformation, and strengthened motion correlation using root mean square fluctuation (RMSF), principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. KEY POINTS: • A computation-aided strategy was developed to engineer pullulanase thermostability. • Seventeen mutants were identified by combining three computational design predictors. • The G692M mutant was obtained with increased Tmand half-life at 70°C.

Rational design and structure insights for thermostability improvement of Penicillium verruculosum Cel7A cellobiohydrolase

Thermostability is a fundamental characteristic of enzymes that is of high importance for industrial implementation of enzymatic catalysis. Cellobiohydrolases are enzymes capable to hydrolyze the most abundant natural polysaccharide – cellulose. These enzymes are widely applied in industry for processing of cellulose containing materials. However, structural and functional engineering of cellobiohydrolases for improving their properties is a challenging task. In this study, the thermostability of Penicillium verruculosum Cel7A cellobiohydrolase was increased through rational design of substitutions with proline. The stabilizing substitution G415P resulted in 3.4-fold increase in half-life time at 60 °C compared to wild-type enzyme. Molecular dynamics simulations indicated a clear effect of the stabilizing substitution G415P and the destabilizing substitutions D62P, S191P, and S273P on the stability of the enzyme tertiary structure. The stabilizing substitution G415P decreased flexibility of the lateral sides of the enzyme active site tunnel, while the considered destabilizing substitutions increased their flexibility.

Enhancing the thermostability of Rhizopus chinensis lipase by rational design and MD simulations

To improve the thermostability of r27RCL from Rhizopus chinensis and broaden its industrial applications, we used rational design (FoldX) according to ΔΔG calculation to predict mutations. Four thermostable variants S142A, D217V, Q239F, and S250Y were screened out and then combined together to generate a quadruple-mutation (S142A/D217V/Q239F/S250Y) variant, called m31. m31 exhibited enhanced thermostability with a 41.7-fold longer half-life at 60 °C, a 5 °C higher of topt, and 15.8 °C higher of T5030 compared to that of r27RCL expressed in Pichiapastoris. Molecular dynamics simulations were conducted to analyze the mechanism of the thermostable mutant. The results indicated that the rigidity of m31 was improved due to the decreased solvent accessible surface area, a newly formed salt bridge of Glu292:His171, and the increased ΔΔG of m31. According to the root-mean-square-fluctuation analysis, three positive mutations S142A, D217V, and Q239F located in the thermal weak regions and greatly decreased the distribution of thermal-fluctuated regions of m31, compared to that of r27RCL. These results suggested that to simultaneously implement MD simulations and ΔΔG-based rational approaches will be more accurate and efficient for the improvement of enzyme thermostability.

Computational design of oligosaccharide producing levansucrase from Bacillus licheniformis RN-01 to improve its thermostability for production of levan-type fructooligosaccharides from sucrose

Levansucrase catalyzes production of levan and levan-type fructooligosaccharides (LFOs) with potential applications in food and pharmaceutical industries such as prebiotics and anti-tumor agents. Previous study found that Y246S mutant of Bacillus licheniformis RN-01 levansucrase (oligosaccharide producing levansucrase, OPL) could effectively produce LFOs but its thermostability is limited at high temperature. In this study, molecular dynamics (MD) and computational protein design were used to create mutants with higher thermostability than OPL by rigidifying highly flexible residues on enzyme surface. MD results show that highly flexible residues suitable for design are K82, N83, D179, and Q308. Two approaches were employed to improve their interactions by allowing them to be amino acids that could potentially form favorable interactions with their neighboring residues or natural amino acids except G, P and C. Flexibilities of designed residues of K82H, N83R, Q308S and K82H/N83R mutants are lower than those of OPL. Experimental results show that characteristics and product patterns of designed mutants are relatively similar to those of OPL. K82H/N83R mutant has higher thermostability than OPL with 1.7-fold increase in t1/2. Circular dichroism result suggests that designed mutations do not drastically affect secondary structures. This study shows how computational technique can engineer enzyme for thermostability improvement.

Improving the thermostability and acid resistance of Rhizopus oryzae α-amylase by using multiple sequence alignment based site-directed mutagenesis.

Higher thermostability or acid resistance for fungal α-amylase will help to improve the sugar-making process and cut down the production costs. Here, the thermostability or acid resistance of Rhizopus oryzae α-amylase (ROAmy) was significantly enhanced by site-directed evolution based on multiple sequence alignment (MSA) method. For instance, compared with the wild-type ROAmy, the optimum temperature of mutants G136D and A144Y was increased from 50 to 55°C, whereas for mutants V174R and I276P, the optimum temperature was increased from 50 to 60°C. The optimum pH of mutants G136D and A144Y shifted from 5.5 to 5.0, whereas for mutants V174R and T253E, the optimum pH changed from 5.5 to 4.5. The results showed that mutant V174R had a 2.52-fold increase in half-life at 55°C, a 2.55-fold increase in half-life at pH 4.5, and a 1.61-fold increase in catalytic efficiency (kcat /Km ) on soluble starch. The three-dimensional model simulation revealed that changes of hydrophilicity, hydrogen bond, salt bridge, or rigidity observed in mutants might mainly account for the improvement of thermostability and acid resistance. The mutants with improved catalytic properties attained in this work may render an accessible and operable approach for directed evolution of fungal α-amylase aimed at interesting functions.

Catalytic and structural effects of flexible loop deletion in organophosphorus hydrolase enzyme: A thermostability improvement mechanism

Thermostability improvement of enzymes used industrially or commercially would develop their capacity and commercial potential due to increased enzymatic competence and cost-effectiveness. Several stabilizing factors have been suggested to be the base of thermal stability, like proline replacements, disulfide bonds, surface loop truncation and ionic pair networks creation. This research evaluated the mechanism of increasing the rigidity of organophosphorus hydrolase enzyme by flexible loop truncation. Bioinformatics analysis revealed that the mutated protein retains its stability after loop truncation (five amino acids deleted). The thermostability of the wild-type (OPH-wt) and mutated (OPH-D5) enzymes were investigated by half-life, Delta Gi, and fluorescence and far-UV CD analysis. Results demonstrated an increase half-life and Delta Gi in OPH-D5 compared to OPH-wt. These results were confirmed by extrinsic fluorescence and circular dichroism (CD) spectrometry experiments, therefore, as rigidity increased in OPHD5 after loop truncation, half-life and Delta Gi also increased. Based on these findings, a strong case is presented for thermostability improvement of OPH enzyme by flexible loop truncation after bioinformatics analysis.

Simultaneously Improved Thermostability and Hydrolytic Pattern of Alpha-Amylase by Engineering Central Beta Strands of TIM Barrel.

This study reported simultaneously improved thermostability and hydrolytic pattern of α-amylase from Bacillus subtilis CN7 by rationally engineering the mostly conserved central beta strands in TIM barrel fold. Nine single point mutations and a double mutation were introduced at the 2nd site of the β7 strand and 3rd site of the β5 strand to rationalize the weak interactions in the beta strands of the TIM barrel of α-amylase. All the five active mutants changed the compositions and percentages of maltooligosaccharides in final hydrolytic products compared to the product spectrum of the wild-type. A mutant Y204V produced only maltose, maltotriose, and maltopentaose without any glucose and maltotetraose, indicating a conversion from typical endo-amylase to novel maltooligosaccharide-producing amylase. A mutant V260I enhanced the thermal stability by 7.1°C. To our best knowledge, this is the first report on the simultaneous improvement of thermostability and hydrolytic pattern of α-amylase by engineering central beta strands of TIM barrel and the novel "beta strands" strategy proposed here may be useful for the protein engineering of other TIM barrel proteins.

Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis.

γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). In this study, stabilized variants of the GAD from Lactobacillus brevis were constructed by consensus mutagenesis. Using Consensus Finder ( http://cbs-kazlab.oit.umn.edu/ ), eight positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these eight residues were individually mutated to match the consensus sequence using site-directed mutagenesis. Compared to the wild-type, T383K variant displayed the largest shift in thermostability among the single variants, with a 3.0°C increase in semi-inactivation temperature (T5015), a 1.7-fold improvement of half-life (t1/2) at 55°C, and a 1.2-fold improvement of t1/2 at 37°C, respectively, while its catalytic efficiency (kcat/Km) was reduced. To obtain the mutant with improvement in both thermostability and catalytic activity, we performed a site-saturation mutation at T383. Notably, mutants T383V and T383G exhibited an increasement in thermostability and kcat/Km than that of wild-type. This study not only emphasizes the value of consensus mutagenesis for improving the thermostability of GAD but also sheds a powerful guidance to study the thermal stability of other enzymes.

Improving low-temperature activity and thermostability of exo-inulinase InuAGN25 on the basis of increasing rigidity of the terminus and flexibility of the catalytic domain

ABSTRACT Enzymes displaying high activity at low temperatures and good thermostability are attracting attention in many studies. However, improving low-temperature activity along with the thermostability of enzymes remains challenging. In this study, the mutant Mut8S, including eight sites (N61E, K156R, P236E, T243K, D268E, T277D, Q390K, and R409D) mutated from the exo-inulinase InuAGN25, was designed on the basis of increasing the number of salt bridges through comparison between the low-temperature-active InuAGN25 and thermophilic exo-inulinases. The recombinant Mut8S, which was expressed in Escherichia coli, was digested by human rhinovirus 3 C protease to remove the amino acid fusion sequence at N-terminus, producing RfsMut8S. Compared with wild-type RfsMInuAGN25, the mutant RfsMut8S showed (1) lower root mean square deviation values, (2) lower root mean square fluctuation (RMSF) values of residues in six regions of the N and C termini but higher RMSF values in five regions of the catalytic pocket, (3) higher activity at 0–40°C, and (4) better thermostability at 50°C. This study proposes a way to increase low-temperature activity along with a thermostability improvement of exo-inulinase on the basis of increasing the rigidity of the terminus and the flexibility of the catalytic domain. These findings may prove useful in formulating rational designs for increasing the thermal performance of enzymes.

Thermostability improvement of Aspergillus awamori glucoamylase via directed evolution of its gene located on episomal expression vector in Pichia pastoris cells.

Novel thermostable variants of glucoamylase (GA) from filamentous fungus Aspergillus awamori X100 were constructed using the directed evolution approach based on random mutagenesis by error-prone PCR of the catalytic domain region of glucoamylase gene located on a new episomal expression vector pPEHα in Pichia pastoris cells. Out of 3000 yeast transformants screened, six new thermostable GA variants with amino acid substitutions Val301Asp, Thr390Ala, Thr390Ala/Ser436Pro, Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu were identified and studied. To estimate the effect of each substitution in the double mutants, we have constructed the relevant single mutants of GA by site-directed mutagenesis and analyzed their thermal properties. Results of the analysis showed that only Ile82Phe and Ser8Arg substitutions by themselves increased enzyme thermostability. While the substitutions Leu7Met, Asn9His and Gln338Leu decreased the thermal stability of GA, the synergistic effect of double mutant variants Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu resulted in significant thermostability improvement as compared to the wild type GA. Thr390Ala and Thr390Ala/Ser436Pro mutant variants revealed the highest thermostability with free activation energy changes ΔΔG of 2.99 and 3.1kJ/mol at 80°C, respectively.

Nano co-immobilization of α-amylase and maltogenic amylase by nanomagnetic combi-cross-linked enzyme aggregates method for maltose production from corn starch

Starch hydrolysis to maltose by nano-magnetic combined cross-linked enzyme aggregates of α-amylase and maltogenic amylase (NM-Combi-CLEAs) is an important step to open new perspectives for special food and pharmaceutic production. Improvement of mass transfer, thermostability, functional specificity, and reusability of combined enzymes was performed. The obtained results exhibited that, 1:9 ratio of α-amylase/maltogenic amylase, use of tert-butanol as precipitant, 2 mM glutardialdehyde, 1:0.75 ratios of combined enzymes to lysine, 20 h crosslinking at 3–4 °C are well-suited conditions. The dynamic light scattering (DLS) results implied that the nanomagnetites diameter was about 81.9–88.9 nm, with polydispersity index (PDI) of 0.242 and a Ȥ-potential of −21 mV. Moreover, the particle size, PDI, and Ȥ-potential of NM-Combi-CLEAs were around 99.6 nm, 0.088, and −32 mV respectively. The NM-Combi-CLEAs kept 80.4% of its original activity after 10 cycles, its Km value exhibited about 1.5 folds reduction with about 1.5 times enhance in thermostability at 95 °C than free one. Immobilization activity yield revealed about 84% of activity retaining by NM-Combi-CLEAs strategy. Accordingly, this efficacious nanobiocatalyst with high thermostability and reusability recommended for starch conversion to maltose.

Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

AbstractHalogenases are valuable biocatalysts for selective C−H activation, but despite recent efforts to broaden their application scope by means of protein engineering, improvement of thermostability and catalytic efficiency is still desired. A directed evolution campaign aimed at generating a thermostable flavin‐dependent tryptophan 6‐halogenase with reasonable activity suitable for chemoenzymatic purposes. These characteristics were tackled by combining successive rounds of epPCR along with semi‐rational mutagenesis leading to a triple mutant (Thal‐GLV) with substantially increased thermostability (▵TM=23.5 K) and higher activity at 25 °C than the wild type enzyme. Moreover, an active‐site mutation has a striking impact on thermostability but also on enantioselectivity. Our data contribute to a detailed understanding of biohalogenation and provide a profound basis for future engineering strategies to facilitate chemoenzymatic application of these attractive biocatalysts.

Improvement in catalytic activity and thermostability of a GH10 xylanase and its synergistic degradation of biomass with cellulase

BackgroundXylanase is one of the most extensively used biocatalysts for biomass degradation. However, its low catalytic efficiency and poor thermostability limit its applications. Therefore, improving the properties of xylanases to enable synergistic degradation of lignocellulosic biomass with cellulase is of considerable significance in the field of bioenergy.ResultsUsing fragment replacement, we improved the catalytic performance and thermostability of a GH10 xylanase, XylE. Of the ten hybrid enzymes obtained, seven showed xylanase activity. Substitution of fragments, M3, M6, M9, and their combinations enhanced the catalytic efficiency (by 2.4- to fourfold) as well as the specific activity (by 1.2- to 3.3-fold) of XylE. The hybrids, XylE-M3, XylE-M3/M6, XylE-M3/M9, and XylE-M3/M6/M9, showed enhanced thermostability, as observed by the increase in the T50 (3–4.7 °C) and Tm (1.1–4.7 °C), and extended t1/2 (by 1.8–2.3 h). In addition, the synergistic effect of the mutant xylanase and cellulase on the degradation of mulberry bark showed that treatment with both XylE-M3/M6 and cellulase exhibited the highest synergistic effect. In this case, the degree of synergy reached 1.3, and the reducing sugar production and dry matter reduction increased by 148% and 185%, respectively, compared to treatment with only cellulase.ConclusionsThis study provides a successful strategy to improve the catalytic properties and thermostability of enzymes. We identified several xylanase candidates for applications in bioenergy and biorefinery. Synergistic degradation experiments elucidated a possible mechanism of cellulase inhibition by xylan and xylo-oligomers.

Natural rubber–based composites filled with bioglasses from a CaO‐SiO2‐P2O5‐Ag2O system. Effect of Ag2O concentration in the filler on composite properties

Bioactive glass was first synthesized by L. Hench in 1971. There are many studies on the properties of several metals and metal ions dopants used in the SiO2‐CaO‐P2O5 system of bioglasses, such as Ag, Cu, Zn, and Fe. A number of authors have carried out research related to the influence of silver oxide on the properties of bioglasses. However, publications on the properties of elastomer‐based composites containing bioactive glasses are relatively scarce. We have not found in the literature studies discussing how silver oxide concentration in bioglasses of the CaO‐SiO2‐P2O5‐Ag2O system affects the significant properties of a natural rubber biocomposite. In this regard, the purpose of the present work is to investigate the aforementioned influence on the properties of this type of composites, namely, vulcanization, physicomechanical, thermal, dynamic, dielectric, electric, and thermoconductive characteristics. We have established those parameters of the composites to be impacted considerably by both degree of filling with bioglass and the silver oxide content in the latter. The improvement in the composites thermostability and some of their physicomechanical performance is the most significant. The volume resistance decreases, and the thermal conductivity coefficients increase. Results from scanning electron microscopy and energy‐dispersive X‐ray (EDX) analyses have confirmed the influence of silver oxide initially on the phase composition of the bioglass, hence on the properties of the biocomposites through changes in the bioglass used as filler. The dielectric characteristics of some of the biocomposites suggest that they can be used as substrates and insulating layers in flexible antennas for short‐range wireless communications.

Improving the catalytic performance of Proteinase K from Parengyodontium album for use in feather degradation

Proteinase K (PROK) from Parengyodontium album hydrolyzes keratin, a major protein component of poultry feathers, which are an inexpensive and renewable protein resource. Based on structural studies for analysis of amino acid flexibility near the catalytic center, identification of highly conserved residues, and experimental screening, we obtained a mutant R218S with residual activity 1.6-fold higher than that of PROK after incubation at 60 °C for 1 h. Molecular dynamics simulation indicated that substitution of Arg218 with Ser leads to three hydrogen bonds being introduced into the structure, stabilizing the β-sheet in which Ser218 is located, and thus improvement of thermostability. Additionally, the mutant R218S had a 15% increase in specific activity compared to PROK and improvement in the rate and thoroughness of feather degradation compared with PROK. We confirmed the positive effects of enhancing catalytic center rigidity on enzyme thermostability, a finding which may have broad applications.

Protein surface engineering of endoglucanase Penicillium verruculosum for improvement in thermostability and stability in the presence of 1-butyl-3-methylimidazolium chloride ionic liquid

Thermostability and stability in ionic liquids are essential properties of cellulases that are applied in industrial processes of bioconversion. Engineering of protein surface of endoglucanase II from Penicillium verruculosum was used to improve the enzyme thermostability and stability in 1-butyl-3-methylimidazolium chloride ([Bmim]Cl). The engineering was based on analysis of the protein surface topography and enhanced by multiple sequence alignment and ΔΔG calculations. In the case of the thermostability, half-life time was improved in 1.3–1.6 times at 70 °C and 1.2–1.4 times at 80 °C. In the case of the stability in [Bmim]Cl, the residual activity after 72 h of incubation in the presence of [Bmim]Cl (50 g/L, 50 °C, pH 4.5) was 1.7–1.9 times greater for the tailored enzyme. The yield of reducing sugars after enzymatic hydrolysis of aspen wood pretreated with [Bmim]Cl was 10–20% higher with the tailored endoglucanase.

Engineered disulfide bonds improve thermostability and activity of L-isoleucine hydroxylase for efficient 4-HIL production in Bacillus subtilis 168.

4‐Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l‐Isoleucine hydroxylase specifically converts l‐Ile to 4‐hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4‐hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l‐isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half‐life of variant T181C was 4.03 h at 50°C, 10.27‐fold the half‐life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56‐fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole‐cell biotransformation, Bacillus subtilis 168/pMA5‐ido T181C recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4‐hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4‐hydroxyisoleucine. Rational design of disulfide bond improved l‐isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases.

Critical effect of proline on thermostability of endoglucanase II from Penicillium verruculosum

Thermostability is an important protein property essential for the protein industrial application. Different approaches of rational design are used for protein thermostability improvement. In this research, the effect of proline substitutions on the protein thermostability was tested. Several types of available data on protein characteristics were used for substitution position choosing in order to find those that are critical for thermostability improvement while maintaining the specific activity. The approach used was based on combined analysis of protein structure, structure-function relationship, multiple sequence alignment, and folding free energy. Proline substitutions that were carried out had a critical effect on thermostability of endoglucanase II from Penicillium verruculosum. The most stabilizing substitution S308P provided a 4- and 2.4-fold increase in time of half-life at 70 °C and 80 °C with maintaining the specific activity. Molecular dynamics simulations revealed the observed effects on the protein structure and indicated principal changes in flexibility and hydrogen bonds networks.