Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents

ABSTRACT Cellulases from glycoside hydrolase family 5 (GH5) are key endoglucanase enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα)8-barrel structure, where each (βα) module is essential for the enzyme’s stability and activity. Despite their shared topology, the thermostability of GH5 endoglucanase enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on the previously characterized thermophilic GH5 endoglucanase Egl5A from Talaromyces emersonii (TeEgl5A), which has an optimal temperature of 90°C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 from Stegonsporium opalus (SoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optimum (10 and 20°C), the temperature at which the protein lost 50% of its activity (T50) (15 and 19°C), and the melting temperature (Tm) (16.5 and 22.9°C) and extended half-lives (t1/2) (∼240- and 650-fold at 55°C) relative to the values for the mesophilic parent enzyme and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase, Cel5 from Aspergillus niger (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both the SoCel5 and TeEgl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between α2 and α3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parent SoCel5. IMPORTANCE Thermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM barrel fragments, and even fragments from unrelated folds, to generate new structures. However, little research has been done on GH5 endoglucanases. In this study, two GH5 endoglucanases exhibiting TIM barrel structure, SoCel5 and TeEgl5A, with different thermal properties, were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure-guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

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Characterizing Activity and Thermostability of GH5 Cellulase Chimeras from Mesophilic and Thermophilic Parents

10.1101/382069 ◽

2018 ◽

Author(s):

Fei Zheng ◽

Josh V. Vermaas ◽

Jie Zheng ◽

Yuan Wang ◽

Tao Tu ◽

...

Keyword(s):

Thermal Properties ◽

Rational Design ◽

Enzyme Stability ◽

Catalytic Efficiency ◽

Industrial Applications ◽

Essential Property ◽

Tim Barrel ◽

Dynamics Simulations ◽

Temperature Optima ◽

Hybrid Enzymes

ABSTRACTCellulases from glycoside hydrolase (GH) family 5 are key enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα)8-barrel structure, where each (βα) module is essential for the enzyme stability and activity. Despite their shared topology, the thermostability of GH5 enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on a previously characterized thermophilic GH5 cellulase from Talaromyces emersonii (TeEgl5A, with an optimal temperature of 90°C), we created ten hybrid enzymes with the mesophilic cellulase from Prosthecium opalus (PoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optima (10 and 20°C), T50 (15 and 19°C), Tm (16.5 and 22.9°C), and extended half life, t1/2 (~240- and 650-fold at 55°C) relative to the mesophilic parent enzyme, and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 cellulase from Aspergillus nidulans (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics simulations (MD) of both PoCel5 and TeEgl5A parent enzymes as well as their hybrids, we hypothesize that improved hydrophobic packing of the interface between α2 and α3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to the mesophilic parent PoCel5.IMPORTANCEThermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM-barrel fragments and even fragments from unrelated folds, to generate new structures. However, there are not many research on GH5 cellulases. In this study, two GH5 cellulases, which showed TIM-barrel structure, PoCel5 and TeEgl5A with different thermal properties were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

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The N-Terminal β-Sheet of the Hyperthermophilic Endoglucanase from Pyrococcus horikoshii Is Critical for Thermostability

Applied and Environmental Microbiology ◽

10.1128/aem.07576-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3059-3067 ◽

Cited By ~ 10

Author(s):

Trent C. Yang ◽

Steve Legault ◽

Emery A. Kayiranga ◽

Jyothi Kumaran ◽

Kazuhiko Ishikawa ◽

...

Keyword(s):

Triosephosphate Isomerase ◽

Industrial Applications ◽

Crystalline Cellulose ◽

Bioinformatics Analyses ◽

Pyrococcus Horikoshii ◽

Content Type ◽

Enzyme Thermostability ◽

Tim Barrel ◽

Key Residues ◽

Β Sheet

ABSTRACTThe β-1,4-endoglucanase (EC 3.2.1.4) from the hyperthermophilic archaeonPyrococcus horikoshii(EGPh) has strong hydrolyzing activity toward crystalline cellulose. When EGPh is used in combination with β-glucosidase (EC 3.2.1.21), cellulose is completely hydrolyzed to glucose at high temperature, suggesting great potential for EGPh in bioethanol industrial applications. The crystal structure of EGPh shows a triosephosphate isomerase (TIM) (β/α)8-barrel fold with an N-terminal antiparallel β-sheet at the opposite side of the active site and a very short C-terminal sequence outside of the barrel structure. We describe here the function of the peripheral sequences outside of the TIM barrel core structure. Sequential deletions were performed from both N and C termini. The activity, thermostability, and pH stability of the expressed mutants were assessed and compared to the wild-type EGPh enzyme. Our results demonstrate that the TIM barrel core is essential for enzyme activity and that the N-terminal β-sheet is critical for enzyme thermostability. Bioinformatics analyses identified potential key residues which may contribute to enzyme hyperthermostability.

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Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Applied and Environmental Microbiology ◽

10.1128/aem.03123-16 ◽

2016 ◽

Vol 83 (6) ◽

Cited By ~ 5

Author(s):

Hong Yang ◽

Pengjun Shi ◽

Yun Liu ◽

Wei Xia ◽

Xiaoyu Wang ◽

...

Keyword(s):

Substrate Specificity ◽

Glycoside Hydrolase ◽

Turnover Rate ◽

Catalytic Efficiency ◽

Glycoside Hydrolase Family ◽

Multiple Sequence ◽

Content Type ◽

Hydrogen Bond Interactions ◽

Wide Range ◽

Hydrogen Network

ABSTRACT Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4 ) from Aspergillus niger. Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (k cat/Km , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (k cat/Km ) of these enzymes (in descending order, NfEG12A [100%], NfEG12A-FN [140%], and NfEG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (k cat). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis. IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the −1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (k cat). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.

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Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

Applied and Environmental Microbiology ◽

10.1128/aem.06814-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2230-2240 ◽

Cited By ~ 40

Author(s):

Xiaoyun Su ◽

Roderick I. Mackie ◽

Isaac K. O. Cann

Keyword(s):

Catalytic Efficiency ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Wild Type ◽

Ph Optimum ◽

Content Type ◽

Caldicellulosiruptor Bescii ◽

Type Protein ◽

Wild Type Protein ◽

Carbohydrate Binding Modules

ABSTRACTThermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses ofCaldicellulosiruptor besciiCel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins ofC. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s−1ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparentkcatof the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in theKmled to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

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Improved Transferase/Hydrolase Ratio through Rational Design of a Family 1 β-Glucosidase from Thermotoga neapolitana

Applied and Environmental Microbiology ◽

10.1128/aem.00359-13 ◽

2013 ◽

Vol 79 (11) ◽

pp. 3400-3405 ◽

Cited By ~ 29

Author(s):

Pontus Lundemo ◽

Patrick Adlercreutz ◽

Eva Nordberg Karlsson

Keyword(s):

Rational Design ◽

High Surface ◽

Great Influence ◽

Single Amino Acid ◽

Glycoside Hydrolase Family ◽

Aqueous Environment ◽

Thermotoga Neapolitana ◽

Potential Yield ◽

Content Type ◽

Alkyl Glycosides

ABSTRACTAlkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. Through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very difficult to do using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process. A thermostable β-glucosidase from the well-studied glycoside hydrolase family 1 fromThermotoga neapolitana,TnBgl1A, was mutated in an attempt to improve its value for synthesis of alkyl glycosides. This was done by rational design using prior knowledge from structural homologues together with a recently generated model of the enzyme in question. Three out of four studied mutations increased the hydrolytic reaction rate in an aqueous environment, while none displayed this property in the presence of an alcohol acceptor. This shows that even if the enzyme resides in a separate aqueous phase, the presence of an organic solvent has a great influence. We could also show that a single amino acid replacement in a less studied part of the aglycone subsite, N220F, improves the specificity for transglycosylation 7-fold and thereby increases the potential yield of alkyl glycoside from 17% to 58%.

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A C-Terminal Proline-Rich Sequence Simultaneously Broadens the Optimal Temperature and pH Ranges and Improves the Catalytic Efficiency of Glycosyl Hydrolase Family 10 Ruminal Xylanases

Applied and Environmental Microbiology ◽

10.1128/aem.00016-14 ◽

2014 ◽

Vol 80 (11) ◽

pp. 3426-3432 ◽

Cited By ~ 9

Author(s):

Zhongyuan Li ◽

Xianli Xue ◽

Heng Zhao ◽

Peilong Yang ◽

Huiying Luo ◽

...

Keyword(s):

Specific Activity ◽

Glycosyl Hydrolase ◽

Beech Wood ◽

Catalytic Efficiency ◽

Cd Spectroscopy ◽

Titration Calorimetry ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Sheep Rumen ◽

Hydrolase Family

ABSTRACTEfficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed inEscherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.

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Improvement of the Catalytic Ability of a Thermostable and Acidophilic β-Mannanase Using a Consensus Sequence Design Strategy

Frontiers in Microbiology ◽

10.3389/fmicb.2021.722347 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qingping Liang ◽

Yuming Zhan ◽

Mingxue Yuan ◽

Linyuan Cao ◽

Changliang Zhu ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Rational Design ◽

Consensus Sequence ◽

Catalytic Efficiency ◽

Design Strategy ◽

Glycoside Hydrolase Family ◽

Biochemical Characteristics ◽

Consensus Analysis ◽

Aspergillus Kawachii ◽

Hydrolase Family

In order to improve the catalytic efficiency of a thermostable and acidophilic β-mannanase (ManAK; derived from marine Aspergillus kawachii IFO 4308), three mutants were designed by amino acid sequence consensus analysis with a second β-mannanase (ManCbs), which also belongs to the glycoside hydrolase family 5 (GH5) and has excellent catalytic efficiency. Three mutants were constructed and their biochemical characteristics were measured after heterologous expression in Pichia pastoris. The results revealed that the kcat/Km values of the three recombinant mannanases ManAKC292V, ManAKL293V, and ManAKL294H were enhanced by 303.0, 280.4, and 210.1%, respectively. Furthermore, ManAKL293V showed greater thermostability than ManAK, retaining 36.5% of the initial enzyme activity after incubation at 80°C for 5min. This study therefore provides a rational design strategy based on consensus sequence analysis to develop industrially valuable β-mannanase for future applications in marine aquafeed.

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A Novel Glycoside Hydrolase Family 113 Endo-β-1,4-Mannanase from Alicyclobacillus sp. Strain A4 and Insight into the Substrate Recognition and Catalytic Mechanism of This Family

Applied and Environmental Microbiology ◽

10.1128/aem.04071-15 ◽

2016 ◽

Vol 82 (9) ◽

pp. 2718-2727 ◽

Cited By ~ 14

Author(s):

Wei Xia ◽

Haiqiang Lu ◽

Mengjuan Xia ◽

Ying Cui ◽

Yingguo Bai ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Catalytic Mechanism ◽

Substrate Recognition ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Binding Pockets ◽

Alicyclobacillus Acidocaldarius ◽

Key Residues

ABSTRACTFew members of glycoside hydrolase (GH) family 113 have been characterized, and information on substrate recognition by and the catalytic mechanism of this family is extremely limited. In the present study, a novel endo-β-1,4-mannanase of GH 113, Man113A, was identified in thermoacidophilicAlicyclobacillussp. strain A4 and found to exhibit both hydrolytic and transglycosylation activities. The enzyme had a broad substrate spectrum, showed higher activities on glucomannan than on galactomannan, and released mannobiose and mannotriose as the main hydrolysis products after an extended incubation. Compared to the only functionally characterized and structure-resolved counterpartAlicyclobacillus acidocaldariusManA (AaManA) of GH 113, Man113A showed much higher catalytic efficiency on mannooligosaccharides, in the order mannohexaose ≈ mannopentaose > mannotetraose > mannotriose, and required at least four sugar units for efficient catalysis. Homology modeling, molecular docking analysis, and site-directed mutagenesis revealed the vital roles of eight residues (Trp13, Asn90, Trp96, Arg97, Tyr196, Trp274, Tyr292, and Cys143) related to substrate recognition by and catalytic mechanism of GH 113. Comparison of the binding pockets and key residues of β-mannanases of different families indicated that members of GH 113 and GH 5 have more residues serving as stacking platforms to support −4 to −1 subsites than those of GH 26 and that the residues preceding the acid/base catalyst are quite different. Taken as a whole, this study elucidates substrate recognition by and the catalytic mechanism of GH 113 β-mannanases and distinguishes them from counterparts of other families.

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Quantifying Methane and Methanol Metabolism of “Methylotuvimicrobium buryatense” 5GB1C under Substrate Limitation

mSystems ◽

10.1128/msystems.00748-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 3

Author(s):

Lian He ◽

Yanfen Fu ◽

Mary E. Lidstrom

Keyword(s):

Growth Rates ◽

Rational Design ◽

Metabolic Flux ◽

Tricarboxylic Acid Cycle ◽

Industrial Applications ◽

Flux Analysis ◽

Type I ◽

Methanol Metabolism ◽

Content Type ◽

Substrate Limitation

ABSTRACT Methanotrophic bacteria are a group of prokaryotes capable of using methane as their sole carbon and energy source. Although efforts have been made to simulate and elucidate their metabolism via computational approaches or 13C tracer analysis, major gaps still exist in our understanding of methanotrophic metabolism at the systems level. Particularly, direct measurements of system-wide fluxes are required to understand metabolic network function. Here, we quantified the central metabolic fluxes of a type I methanotroph, “Methylotuvimicrobium buryatense” 5GB1C, formerly Methylomicrobium buryatense 5GB1C, via 13C isotopically nonstationary metabolic flux analysis (INST-MFA). We performed labeling experiments on chemostat cultures by switching substrates from 12C to 13C input. Following the switch, we measured dynamic changes of labeling patterns and intracellular pool sizes of several intermediates, which were later used for data fitting and flux calculations. Through computational optimizations, we quantified methane and methanol metabolism at two growth rates (0.1 h−1 and 0.05 h−1). The resulting flux maps reveal a core consensus central metabolic flux phenotype across different growth conditions: a strong ribulose monophosphate cycle, a preference for the Embden-Meyerhof-Parnas pathway as the primary glycolytic pathway, and a tricarboxylic acid cycle showing small yet significant fluxes. This central metabolic consistency is further supported by a good linear correlation between fluxes at the two growth rates. Specific differences between methane and methanol growth observed previously are maintained under substrate limitation, albeit with smaller changes. The substrate oxidation and glycolysis pathways together contribute over 80% of total energy production, while other pathways play less important roles. IMPORTANCE Methanotrophic metabolism has been under investigation for decades using biochemical and genetic approaches. Recently, a further step has been taken toward understanding methanotrophic metabolism in a quantitative manner by means of flux balance analysis (FBA), a mathematical approach that predicts fluxes constrained by mass balance and a few experimental measurements. However, no study has previously been undertaken to experimentally quantitate the complete methanotrophic central metabolism. The significance of this study is to fill such a gap by performing 13C INST-MFA on a fast-growing methanotroph. Our quantitative insights into the methanotrophic carbon and energy metabolism will pave the way for future FBA studies and set the stage for rational design of methanotrophic strains for industrial applications. Further, the experimental strategies can be applied to other methane or methanol utilizers, and the results will offer a unique and quantitative perspective of diverse methylotrophic metabolism.

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Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

Applied and Environmental Microbiology ◽

10.1128/aem.00449-17 ◽

2017 ◽

Vol 83 (13) ◽

Cited By ~ 13

Author(s):

Guang Yang ◽

Hua Yao ◽

Matteo Mozzicafreddo ◽

Patrizia Ballarini ◽

Sandra Pucciarelli ◽

...

Keyword(s):

Catalytic Activity ◽

Catalytic Efficiency ◽

Industrial Applications ◽

Enzyme Engineering ◽

Content Type ◽

Cold Adapted ◽

Wide Range ◽

Cold Active ◽

Surface Loops ◽

The Antarctic

ABSTRACT The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.

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