Mutant Enzymes Research Articles

Metabolic Engineering of Yarrowia lipolytica for Zeaxanthin Production.

Zeaxanthin is a carotenoid, a dihydroxy derivative of β-carotene. Zeaxanthin has antioxidant, anti-inflammatory, anticancer, and neuroprotective properties. In this study, Yarrowia lipolytica was used as a host for the efficient production of zeaxanthin. The strain Y. lipolytica PO1h was used to construct the following engineered strains for carotenoid production since it produced the highest β-carotene among the Y. lipolytica PO1h- and Y. lipolytica PEX17-HA-derived strains. By regulating the key nodes on the carotenoid pathway through wild and mutant enzyme comparison and successive modular assembly, the β-carotene concentration was improved from 19.9 to 422.0 mg/L. To provide more precursor mevalonate, heterologous genes mvaE and mvaSMT were introduced to increase the production of β-carotene by 27.2% to the yield of 536.8 mg/L. The β-carotene hydroxylase gene crtZ was then transferred, resulting in a yield of zeaxanthin of 326.5 mg/L. The oxidoreductase RFNR1 and CrtZ were then used to further enhance zeaxanthin production, and the yield of zeaxanthin was up to 775.3 mg/L in YPD shake flask.

Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q ) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.

Dynamic Behavior and Ligand Binding Properties of the Wild Type Deoxycytidine Kinase and its Characterized Gemcitabine-Resistant Variant: A Bioinformatics and Computational Study

The main challenge in using gemcitabine, a nucleoside analogue anti-cancer, includes resistance of some patients to this compound due to the genetic mutations on deoxycytidine kinase (dCK) that are found in a fraction of the human population. Here, we investigated the dynamics behavior and ligand binding properties of the wild type (WT) dCK and its characterized double mutant using a combination of bioinformatics tools and molecular dynamics (MD) simulation studies. Root-mean-square deviation (RMSD) values of the WT and mutant enzyme in complex with both ligands (deoxycytidine/gemcitabine) demonstrated that the WT enzyme forms a more stable complex with gemcitabine, as compared with its natural ligand (deoxycytidine). However, the stability of the double mutant-deoxycytidine complex is greater, when compared with gemcitabine. It was also found that Arg131 as a critical residue can affect the binding pattern of the enzyme to ligands in a manner that the WT enzyme can interact with both ligands, while, the mutant enzyme cannot establish efficient interaction with gemcitabine, and shows high affinity to deoxycytidine. These data together indicate that gemcitabine can interact with the WT enzyme in a competitive manner, while double mutant can be considered as a resistant variant against gemcitabine.

Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha.

Gibberellins (GAs) are key phytohormones that regulate growth, development, and environmental responses in angiosperms. From an evolutionary perspective, all major steps of GA biosynthesis are conserved among vascular plants, while GA biosynthesis intermediates such as ent-kaurenoic acid (KA) are also produced by bryophytes. Here, we show that in the liverwort Marchantia polymorpha, KA and GA12 are synthesized by evolutionarily conserved enzymes, which are required for developmental responses to far-red light (FR). Under FR-enriched conditions, mutants of various biosynthesis enzymes consistently exhibited altered thallus growth allometry, delayed initiation of gametogenesis, and abnormal morphology of gamete-bearing structures (gametangiophores). By chemical treatments and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, we confirmed that these phenotypes were caused by the deficiency of some GA-related compounds derived from KA, but not bioactive GAs from vascular plants. Transcriptome analysis showed that FR enrichment induced the up-regulation of genes related to stress responses and secondary metabolism in M. polymorpha, which was largely dependent on the biosynthesis of GA-related compounds. Due to the lack of canonical GA receptors in bryophytes, we hypothesize that GA-related compounds are commonly synthesized in land plants but were co-opted independently to regulate responses to light quality change in different plant lineages during the past 450 million years of evolution.

An integrative computational approach for the identification of dual inhibitors of isocitrate dehydrogenase 1 and 2 from phytocompounds of Phyllantus amarus

Genetic alterations of the genes encoding the isocitrate dehydrogenase (IDH) enzymes have been identified in about 20% of acute myeloid leukemia (AML) cases as well as many other forms of cancers. Notable among these alterations are the neomorphic IDH1_R132H and IDH2_R140Q mutations which lead to the production of an oncometabolite. Hence, their inhibition is widely considered a therapeutic strategy in the treatment of many cancers. While many inhibitors of the mutant enzymes have been developed, an inhibitor that is capable of co-inhibiting both enzymes are currently lacking while drug resistance has also limited the clinical usage of previously identified mono inhibitors. Consequently, this study employed molecular modeling approaches, such as molecular docking, molecular mechanics generalized Born Surface area (MM/GBSA), molecular dynamics (MD) simulation, and density functional theory (DFT) analysis to identify potential dual inhibitors of the previously mentioned mutant IDH1/2 from the phytocompounds of Phyllantus amarus. Of the 31 phytocompounds identified, 20 showed good binding affinities for both IDH1 _R132H and IDH2 _R140Q (ranging from −5.2 Kca/mol to −9.6 Kcal/mol) and had desirable pharmacokinetic properties. However, ellagic acid and pinoresinol possessed better pharmacokinetic properties, rendering suitable hits. Investigation of the behavior of the IDH1_R132H and IDH2_R140Q complexes with ellagic acid and pinoresinol via the RMSD, RMSF, and contact map analyses showed that all the complexes-maintained stability throughout the simulation time. Ultimately, ellagic acid and pinoresinol were identified as promising hits for the development of IDH1_R132H and IDH2_R140Q dual inhibitors. However, further experimental studies are needed to confirm their potential as therapies. Communicated by Ramaswamy H. Sarma

Binding profile of quinonoid-dihydrobiopterin to quinonoid-dihydropteridine reductase examined by in silico and in vitro analyses.

Quinonoid dihydropteridine reductase (QDPR) catalyzes the reduction of quinonoid-form dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4). BH4 metabolism is a drug target for neglected tropical disorders, as trypanosomatid protozoans, including Leishmania and Trypanosoma, require exogenous sources of biopterin for growth. Although QDPR is a key enzyme for maintaining intracellular BH4 levels, the precise catalytic properties and reaction mechanisms of QDPR are poorly understood due to the instability of quinonoid-form substrates. In this study, we analyzed the binding profile of qBH2 to human QDPR in combination with in silico and in vitro methods. First, we performed docking simulation of qBH2 to QDPR to obtain possible binding modes of qBH2 at the active site of QDPR. Then, among them, we determined the most plausible binding mode using molecular dynamics simulations revealing its atomic-level interactions and confirmed it with the in vitro assay of mutant enzymes. Moreover, it was found that not only qBH2 but also quinonoid-form dihydrofolate (qDHF) could be potential physiological substrates for QDPR, suggesting that QDPR may be a bifunctional enzyme. These findings in this study provide important insights into biopterin and folate metabolism and would be useful for developing drugs for neglected tropical diseases.

A high-throughput dual system to screen polyphosphate kinase mutants for efficient ATP regeneration in L-theanine biocatalysis

ATP, an important cofactor, is involved in many biocatalytic reactions that require energy. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We determined the catalytic properties of PPK from different sources and found that PPK from Cytophaga hutchinsonii (ChPPK) had the best catalytic activity for the substrates ADP and polyP6. An extracellular–intracellular dual system was constructed to high-throughput screen for better catalytic activity of ChPPK mutants. Finally, the specific activity of ChPPKD82N-K103E mutant was increased by 4.3 times. Therefore, we focused on the production of L-theanine catalyzed by GMAS as a model of ATP regeneration. Supplying 150 mM ATP, GMAS enzyme could produce 16.8 ± 1.3 g/L L-theanine from 100 mM glutamate. When 5 mM ATP and 5 U/mL ChPPKD82N-K103E were added, the yield of L-theanine was 16.6 ± 0.79 g/L with the conversion rate of 95.6 ± 4.5% at 4 h. Subsequently, this system was scaled up to 200 mM and 400 mM glutamate, resulting in the yields of L-theanine for 32.3 ± 1.6 g/L and 62.7 ± 1.1 g/L, with the conversion rate of 92.8 ± 4.6% and 90.1 ± 1.6%, respectively. In addition, we also constructed an efficient ATP regeneration system from glutamate to glutamine, and 13.8 ± 0.2 g/L glutamine was obtained with the conversion rate of 94.4 ± 1.4% in 4 h after adding 6 U/ mL GS enzyme and 5 U/ mL ChPPKD82N-K103E, which further laid the foundation from glutamine to L-theanine catalyzed by GGT enzyme. This proved that giving the reaction an efficient ATP supply driven by the mutant enzyme enhanced the conversion rate of substrate to product and maximized the substrate value. This is a positively combination of high yield, high conversion rate and high economic value of enzyme catalysis. The mutant enzyme will further power the ATP-consuming biocatalytic reaction platform sustainably.

Using Data Science for Mechanistic Insights and Selectivity Predictions in a Non-Natural Biocatalytic Reaction.

The study of non-natural biocatalytic transformations relies heavily on empirical methods, such as directed evolution, for identifying improved variants. Although exceptionally effective, this approach provides limited insight into the molecular mechanisms behind the transformations and necessitates multiple protein engineering campaigns for new reactants. To address this limitation, we disclose a strategy to explore the biocatalytic reaction space and garner insight into the molecular mechanisms driving enzymatic transformations. Specifically, we explored the selectivity of an "ene"-reductase, GluER-T36A, to create a data-driven toolset that explores reaction space and rationalizes the observed and predicted selectivities of substrate/mutant combinations. The resultant statistical models related structural features of the enzyme and substrate to selectivity and were used to effectively predict selectivity in reactions with out-of-sample substrates and mutants. Our approach provided a deeper understanding of enantioinduction by GluER-T36A and holds the potential to enhance the virtual screening of enzyme mutants.

Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells.

The base excision repair (BER) pathway is a frontline defender of genomic integrity and plays a central role in epigenetic regulation through its involvement in the erasure of 5-methylcytosine. This biological and clinical significance has led to a demand for analytical methods capable of monitoring BER activities, especially in living cells. Unfortunately, prevailing methods, which are primarily derived from nucleic acids, are mostly incompatible with intracellular use due to their susceptibility to nuclease degradation and other off-target interactions. These limitations preclude important biological studies of BER enzymes and many clinical applications. Herein, we report a straightforward approach for constructing biostable BER probes using a unique chimeric d/l-DNA architecture that exploits the bioorthogonal properties of mirror-image l-DNA. We show that chimeric BER probes have excellent stability within living cells, where they were successfully employed to monitor relative BER activity, evaluate the efficiency of small molecule BER inhibitors, and study enzyme mutants. Notably, we report the first example of a fluorescent probe for real-time monitoring of thymine DNA glycosylase (TDG)-mediated BER of 5-formylcytosine and 5-carboxylcytosine in living cells, providing a much-needed tool for studying DNA (de)methylation biology. Chimeric probes offer a robust and highly generalizable approach for real-time monitoring of BER activity in living cells, which should enable a broad spectrum of basic research and clinical applications.

Improved thermostability of type I pullulanase from Bacillus thermoliquefaciens by error-prone PCR

Pullulanase (PulB) is a starch-debranching enzyme. In order to improve its catalytic performance, random mutagenesis was performed on the pullulanase gene derived from Bacillus thermoliquefaciens. Two rounds of error-prone PCR were carried out. Mutant T252S was screened in the first round of error-prone library, which had the highest catalytic activity. During the second round of mutations, mutant enzyme G250P/T252S/G253T/N255K was screened, which had further improved catalytic activity and the best thermostability. Compared with the parent enzyme, the specific activity of mutant enzyme G250P/T252S/G253T/N255K increased by 1.9 times, Km decreased by 22.7 %, kcat increased by 28.7 %, and kcat/Km increased by 68.4 %. The thermostability of the mutant enzyme improved significantly, showing that the half-life at 60 °C was extended to 7.5 h, which was 87.5 % higher than that of the parent enzyme. The mutation sites in these two rounds were concentrated in the 250–255 regions, indicating that this region was an important region affecting the catalytic activity and Thermostability. The reasons for the change of enzymtic properties was also preliminarily analyzed through three-dimensional simulation.

Disease-Associated Mutation A554V Disrupts Normal Autoinhibition of DNMT1.

DNA methyltransferase 1 (DNMT1) is the enzyme primarily responsible for propagation of the methylation pattern in cells. Mutations in DNMT1 have been linked to the development of adult-onset neurodegenerative disorders; these disease-associated mutations occur in the regulatory replication foci-targeting sequence (RFTS) domain of the protein. The RFTS domain is an endogenous inhibitor of DNMT1 activity that binds to the active site and prevents DNA binding. Here, we examine the impact of the disease-associated mutation A554V on normal RFTS-mediated inhibition of DNMT1. Wild-type and mutant proteins were expressed and purified to homogeneity for biochemical characterization. The mutation increased DNA binding affinity ~8-fold. In addition, the mutant enzyme exhibited increased DNA methylation activity. Circular dichroism (CD) spectroscopy revealed that the mutation does not significantly impact the secondary structure or relative thermal stability of the isolated RFTS domain. However, the mutation resulted in changes in the CD spectrum in the context of the larger protein; a decrease in relative thermal stability was also observed. Collectively, this evidence suggests that A554V disrupts normal RFTS-mediated autoinhibition of DNMT1, resulting in a hyperactive mutant enzyme. While the disease-associated mutation does not significantly impact the isolated RFTS domain, the mutation results in a weakening of the interdomain stabilizing interactions generating a more open, active conformation of DNMT1. Hyperactive mutant DNMT1 could be responsible for the increased DNA methylation observed in affected individuals.

Highly efficient synthesis of chiral lactams by using a ω-transaminase from Bacillus megaterium and its mutant enzymes

In this study, a series of chiral lactams were successfully prepared by one-pot two-step reactions. It included a transamination reaction catalyzed by a ω-transaminase from Bacillus megaterium (ω-TABm), and subsequent spontaneous cyclisation. The different amine donors were firstly screened with ethyl levulinate as an amine acceptor, and both the excellent conversion and enantiomeric excess (e.e.p) of >99% could be achieved with 4-bromophenylethylamine as an amine donor, however, the long reaction time of 24 h was unfavorable for its application. After the amino acid residue Thr120 situated in the small pocket was mutated to Ser, the T120S variant could exhibit 10.6-fold higher activity comparing to that of wild type, and achieve the reaction with both conversion and e.e.p of >99% within 3 h. The T120S variant could also be applied to prepare the other chiral γ- and δ-lactams with excellent conversion and e.e.p. Furthermore, the inclusion bodies of ω-TABm and its variants could be aggregated as “native-like structures” to continuously prepare the chiral lactam for 18 h. The aim of the study is to provide a simply and efficiently biocatalytic route for preparing chiral lactams.

Gefitinib derivatives and drug-resistance: A perspective from molecular dynamics simulations

Epidermal-growth factor receptor (EGFR) is a transmembrane tyrosine kinase (TK) with a significant role in cell survival. EGFR is upregulated in various cancer cells and known as a druggable target. Gefitinib is a first-line TK inhibitor used against metastatic non-small cell lung cancer (NSCLC). Despite initial clinical response, a conserved therapeutic effect could not be achieved due to the occurrence of resistance mechanisms. Point mutations in EGFR genes are one of the major causes of rendered tumor sensitivity. To aid in the development of more efficient TKIs, chemical structures of prevailing drugs and their target binding patterns are very important. The aim of the present study was to propose synthetically-accessible gefitinib congeners with enhanced binding fitness to clinically frequent EGFR mutants. Docking simulations of intended molecules identified 1-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yl)-3-(oxazolidin-2-ylmethyl) thiourea (23) as a top-binder structure inside G719S, T790 M, L858R and T790 M/L858R-EGFR active sites. Superior docked complexes were subjected to the entire 400 ns molecular dynamics (MD) simulations. Analysis of data revealed the stability of mutant enzymes upon binding to molecule 23. All mutant complexes with the exception of a T790 M/L858R-EGFR, were majorly stabilized through cooperative hydrophobic contacts. Pairwise analysis of H-bonds proved Met793 as the conserved residue with stable H-bond participations as hydrogen bond donor (Frequency 63–96%). Amino acid decomposition analysis confirmed the probable role of Met793 in complex stabilization. Estimated binding free energies indicated the proper accommodation of molecule 23 inside target active sites. Pairwise energy decompositions of stable binding modes revealed the energetic contribution of key residues. Although wet lab experiments are required to unravel the mechanistic details of mEGFR inhibition, MD results provide structural basis for those events that are difficult to address experimentally. The outputs of the current study may assist to design small molecules with high potency to mEGFRs.

Reactive oxygen species linked diabetes mellitus management by natural products: A Pre-clinical study in rat

The environment has a great impact on health maintenance. Environmental stress factors generate reactive oxygen species (ROS) inour bodies. As a result, pro-oxidant status of our body is dominated over the antioxidant status that induces health disorders. Diabetesmellitus (DM), a common metabolic cum lifestyle disease, is now considered as X syndrome. Environmental stressors through ROSgeneration can impose point mutation for the production of mutant enzymes, leading to cellular metabolic disorders, especiallycarbohydrate metabolism, an important cause of diabetes. Moreover, free radicals through cellular signaling system down-regulategene expression of antioxidant enzymes that produce high levels of cellular free radicals in metabolic organs, which may lead to DM.Natural products with strong antioxidant activities have a major role in managing diabetes. Nutraceuticals in E. jumbulana, Camelliasinensis, H. antidysenterica majorly contribute to such management by increasing gene expression of antioxidant enzymes andcarbohydrate metabolic enzymes that favor glucose utilization in cell. It has also been established that such nutraceuticals can generate β cells from hepatic stem cells, so plasma insulin and c-peptide levels are elevated. Insulin receptor gene expression is also corrected by such nutraceuticals in diabetic model animals. The efficacy of such nutraceuticals is comparable with antidiabetic gold standard drugs. Such studies have been confirmed by genomics and proteomics studies using real-time PCR followed by western blotting studies. This field of research unfolds a new domain known as ‘Neutrogenomics’ in health science.It is concluded that natural products may be the parallel management process of modern therapy as per the guideline of WHO.

Development of biosensors highly responsive to N-acetylneuraminic acid in Bacillus subtilis

Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.

A very long chain fatty acid responsive transcription factor, MYB93, regulates lateral root development in Arabidopsis.

Lateral roots (LRs) are critical to root system architecture development in plants. Although the molecular mechanisms by which auxin regulates LR development have been extensively studied, several additional regulatory systems are hypothesized to be involved. Recently, the regulatory role of very long chain fatty acids (VLCFAs) has been shown in LR development. Our analysis showed that LTPG1 and LTPG2, transporters of VLCFAs, are specifically expressed in the developing LR primordium (LRP), while the number of LRs is reduced in the ltpg1/ltpg2 double mutant. Moreover, late LRP development was hindered when the VLCFA levels were reduced by the VLCFA synthesis enzyme mutant, kcs1-5. However, the details of the regulatory mechanisms of LR development controlled by VLCFAs remain unknown. In this study, we propose a novel method to analyze the LRP development stages with high temporal resolution using a deep neural network and identify a VLCFA-responsive transcription factor, MYB93, via transcriptome analysis of kcs1-5. MYB93 showed a carbon chain length-specific expression response following treatment of VLCFAs. Furthermore, myb93 transcriptome analysis suggested that MYB93 regulated the expression of cell wall organization genes. In addition, we also found that LTPG1 and LTPG2 are involved in LR development through the formation of root cap cuticle, which is different from transcriptional regulation by VLCFAs. Our results suggest that VLCFA is a regulator of LRP development through transcription factor-mediated regulation of gene expression and the transportation of VLCFAs is also involved in LR development through root cap cuticle formation.

Expression and characterization of C-terminal truncated mutants of γ-glutamyltranspeptidase II (PaGGTII) from Pseudomonas aeruginosa PAO1

The gene encoding γ-glutamyltranspeptidase II (PaGGTII) from Pseudomonas aeruginosa PAO1 was cloned in Escherichia coli. Recombinant PaGGTII showed a weak activity (0.0332 U/mg), and it can be easily inactivated. Multiple alignment of microbial GGTs showed the redundancy of the C-terminal of the small subunit of PaGGTII in length. The truncation of eight amino acid residues at the C-terminal of PaGGTII remarkably improved the activity and stability of the enzyme (PaGGTIIΔ8; 0.388 U/mg). Further truncation at the C-terminal also provided the enzyme relatively higher activity (PaGGTIIΔ9, −Δ10, −Δ11, and −Δ12). Among C-terminal truncated mutants, we focused on PaGGTIIΔ8 and examined the effect of C-terminal amino acid residues on the properties of PaGGTIIΔ8 because the activity of PaGGTII was found to be greatly improved when 8 amino acid residues were truncated. Various mutant enzymes with different C-terminal amino acid residues were constructed. They were expressed in E. coli and purified to homogeneity by ion-exchange chromatography. The properties of PaGGTIIΔ8 and the mutants obtained from mutation at E569 were characterized. Km and kcat of PaGGTIIΔ8 for γ-glutamyl-p-nitroanilide (γ-GpNA) were 8.05 mM and 15.49 s−1, respectively. PaGGTIIΔ8E569Y showed the highest catalytic efficiency for γ-GpNA with a kcat/Km of 12.55 mM−1 s−1. Mg2+, Ca2+, and Mn2+ exhibited positive effects on the catalytic activity for PaGGTIIΔ8 and its ten E569 mutants.

Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli.

Medical usage of L-asparaginase (ASNase), the first-line of acute lymphoblastic leukemia treatment, is linked to allergic responses and toxicities, which necessitates the development of new bio-better ASNases. The aim of the current study was in silico design of a novel ASNase with predicted improved enzymatic properties using strategies encompassing sequence-function analysis of known ASNase mutants. Additionally, current study aimed to show that the new enzyme is active. Based on 21 experimentally reported mutations for ASNase, a virtual library of mutated enzymes with all 7546 possible combinations of up to 4 mutations was generated. Three-dimensional models of proposed mutant enzymes were built and their in silico stabilities were calculated. The most promising mutant was selected for preparing a genetic construct suitable for expression of the designed ASNase in bacterial cells. Computational study predicted that Y176F/S241C double mutation of Escherichia coli ASNase may increase its folding stability. The designed ASNase was expressed in two different E. coli strains (Origami B(DE3) and BL21(DE3)pLysS) and then the soluble fractions prepared from the cell lysates of the host cells were used in enzyme activity assay. Results showed that enzyme activity of soluble fraction from Origami (95.4 ± 7.5 IU/0.1 mL) was four times higher than that of soluble fraction from pLysS (25.8 ± 2.5 IU/0.1 mL). A novel functional double mutant ASNase with predicted improved enzymatic properties was designed and produced in E. coli. The results of the current study suggest a great commercial potential for the identified enzyme in pharmaceutical and industrial applications.

Characterization and alteration of product specificity of Beijerinckia indica subsp. indica β-fructosyltransferase.

The trisaccharide 1-kestose, a major constituent of fructooligosaccharide, has strong prebiotic effects. We used high-performance liquid chromatography and 1H nuclear magnetic resonance spectroscopy to show that BiBftA, a β-fructosyltransferase belonging to glycoside hydrolase family 68, from Beijerinckia indica subsp. indica catalyzes transfructosylation of sucrose to produce mostly 1-kestose and levan polysaccharides. We substituted His395 and Phe473 in BiBftA with Arg and Tyr, respectively, and analyzed the reactions of the mutant enzymes with 180g/L sucrose. The ratio of the molar concentrations of glucose and 1-kestose in the reaction mixture with wild-type BiBftA was 100:8.1, whereas that in the reaction mixture with the variant H395R/F473Y was 100:45.5, indicating that H395R/F473Y predominantly accumulated 1-kestose from sucrose. The X-ray crystal structure of H395R/F473Y suggests that its catalytic pocket is unfavorable for binding of sucrose while favorable for transfructosylation.

Modification of a Thermostable β-mannanase and Its High-Efficiency Expression

A β-mannanase from Aspergillus usamii (A. usamii) belonging to glycoside hydrolase (GH) family 5 (abbreviated as AuMan5A) was selected as the research subject to enhance the enzyme protein’s thermotolerance and catalytic efficiency by replacing a random coil (loop) structure on the enzyme molecule. Furthermore, the correlation between the structure and function of β-mannanase was clarified. Homology modeling was used to simulate the three-dimensional structure of AuMan5A, and mannopentaose was docked in the AuMan5A substrate binding groove. Based on the spatial characteristics and phylogenetic analyses of the complex structure, a non-conserved loop structure (Loop FG) was speculated to be involved in enzyme-substrate interactions. Therefore, Loop FG was selected as the modified region. Additionally, the corresponding fragments of Aspergillus nidulans, Aspergillus fumigatus, and Trichoderma harzianum β-mannanases were selected to replace the Loop FG sequence (316KSPDGGN322) of AuMan5A, respectively, based on the enzymological properties and structural characteristics of other fungal GH 5 family β-mannanases. Mutant enzyme bases were constructed by PCR, and using plasmid pPICZαA, the original and mutant enzymes were expressed in Pichia pastoris GS115 to analyze the enzymological properties of the expressed products. Recombinant enzymes re-AuMan5A, re-AuMan5A-Af, re-AuMan5A-An, and re-AuMan5A-Th had an optimum temperature of 65 °C, 75 °C, 65 °C, and 70 °C, respectively. Their activity half-lives at 70 °C were 10 min, 480 min, 5 min, and 25 min, respectively, and their melting temperatures were 64.5 °C, 76.6 °C, 63.2 °C, and 69.1 °C, respectively. Re-AuMan5A-Af, re-AuMan5A-An, and re-AuMan5A-Th had a kcat/km (catalytic efficiency) value that was 12.7, 6.0, and 11.0 times higher than re-AuMan5A, respectively, with re-AuMan5A-Af exhibiting the best temperature characteristics and catalytic efficiency. The G320-to-D320 mutation of AuMan5A during loop structure replacement significantly affected AuMan5A/Af’s enzymological properties, suggesting the vital role of G320 in improving AuMan5A/Af’s thermostability, specific activity, and catalytic efficiency.