Retained Enzyme Activity Research Articles

Site-directed mutagenesis of nattokinase: Unveiling structure-function relationship for enhanced functionality

Site-directed mutagenesis was employed to investigate the structure-function relationship of nattokinase (NK) and its effect on the enzymatic activity, thermostability, pH tolerance, and fibrinolytic properties of NK. Specific mutations (T270S, V271I, E262D, and A259T) were introduced within the nk gene, targeting regions predicted to be involved in substrate binding. The NK(E262D) mutant exhibited a significant increase in enzymatic activity (2-fold) and catalytic efficiency (2.2-fold) as assessed by N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) hydrolysis, compared to the wild type. In silico analysis supported these findings, demonstrating lower binding energy for the NK(E262D) mutant, suggesting stronger fibrin affinity. Thermostability assays revealed that NK(E262D) and NK(A259T) displayed exceptional stability, retaining enzyme activity at 60 °C. All mutants exhibited a broader pH tolerance range (pH 5.0–10.0) compared to the wild-type NK. The fibrinolytic activity assay revealed that the E262D mutant possessed the highest fibrinolytic activity (2414 U/mg), surpassing the wild-type. This study reported an NK variant with improved enzymatic activity, thermostability, and fibrinolytic properties.

Immobilized glucoamylase based on ZIF-8: Preparation, response surface optimization, characterization.

The glucoamylase@ZIF-8 was prepared using ZIF-8 material as the carrier in this study. The preparation process was optimized by response surface methodology, and the stability of glucoamylase@ZIF-8 was determined. The material was characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The results showed that the optimum preparation process of glucoamylase@ZIF-8 was 1.65mol 2-methylimidazole, 5.85mL glucoamylase, 33°C stirring temperature, 90min stirring time, and 84.0230%±0.6006% embedding rate. At 100°C, the free glucoamylase completely lost its activity, whereas the glucoamylase@ZIF-8 still had a retained enzyme activity of 12.0123%±0.86158%; at pH 3-6, the highest activity of glucoamylase@ZIF-8 was 95.9531%±0.96181%, and about 80% of glucoamylase activity could be retained under alkaline conditions. When the ethanol concentration was 13%, the retained enzyme activity was 7.9316%±0.19805%, significantly higher than free enzymes. The Km of glucoamylase@ZIF-8 and free enzyme were 1235.6825 and 80.317mg/mL, respectively. Vmax was 0.2453 and 0.149mg/(mLmin), respectively. The appearance, crystal strength, and thermal stability of glucoamylase@ZIF-8 were improved after optimization, and they had high reusability.

Trypsin/Zn3(PO4)2 Hybrid Nanoflowers: Controlled Synthesis and Excellent Performance as an Immobilized Enzyme.

Immobilized enzymes are a significant technological approach to retain enzyme activity and reduce enzyme catalytic cost. In this work, trypsin-incorporated Zn3(PO4)2 hybrid nanoflowers were prepared via mild precipitation and coordination reactions. The controllable preparation of hybrid nanoflowers was achieved by systematically investigating the effects of the raw-material ratio, material concentration and reaction temperature on product morphology and physicochemical properties. The enzyme content of hybrid nanoflowers was about 6.5%, and the maximum specific surface area reached 68.35 m2/g. The hybrid nanoflowers exhibit excellent catalytic activity and environmental tolerance compared to free trypsin, which was attributed to the orderly accumulation of nanosheets and proper anchoring formation. Further, the enzyme activity retention rate was still higher than 80% after 12 repeated uses. Therefore, trypsin/Zn3(PO4)2 hybrid nanoflowers—which combine functionalities of excellent heat resistance, storage stability and reusability—exhibit potential industrial application prospects.

Immobilization of 2‐Deoxy‐d–ribose‐5‐phosphate Aldolase on Membrane Supports by Spray‐Coating: Optimization by Design‐of‐Experiment

AbstractA strategy for the immobilization of the enzyme 2‐deoxy‐d‐ribose‐5‐phosphate aldolase (DERA) is presented via spray‐coating on membrane support material to generate an enzymatically active membrane which can be used in continuously run synthesis modules. A functional, water‐soluble copolymer containing addressable units to covalently bind DERA is mixed with the enzyme, followed by spray‐coating of the mixture onto polyacrylonitrile/polyethylene imine‐ (PAN/PEI) membranes and a subsequent post‐processing to stabilize the coating. Confirmation of successful immobilization was achieved by atomic force microscopy (AFM) imaging and assessment of retained enzyme activity. Through variation of relevant parameters, improvements in stability and activity were seen, which formed the basis for the following optimization by Design‐of‐Experiment (DoE). A first fractional factorial design yielded additional performance improvements and important insights into the impacts of experimental parameters and their interaction. A second full factorial design did not result in further improvements, but validated the results of the first design.

A Multicomponent Butyrylcholinesterase Preparation for Enzyme Inhibition-Based Assay of Organophosphorus Pesticides

A new method of producing butyrylcholinesterase (BChE) preparations, stable in storage and use, has been proposed. The BChE preparation is the enzyme co-immobilized with 0.2 M 5-5′-dithiobis (2-nitrobenzoic acid) in starch or gelatin gel. All experimental preparations retain enzyme activity for at least 300 d. The preparations based on gelatin gel show higher activity but lower sensitivity to the toxicants tested in this study compared to the starch gel-based preparations. A method has been proposed for integrated detection of anti-cholinesterase substances in aqueous solutions using the experimental preparation with immobilized BChE. After the additional incubation of the preparation with the immobilized enzyme in the solution of the analyte, the detection limits of malathion and pirimiphos-methyl determined using the IC20 values were below their maximum allowable concentrations—0.005 µM and 0.03 µM, respectively.

Piezoelectric inkjet printing of tyrosinase (polyphenol oxidase) enzyme on atmospheric plasma treated polyamide fabric

Tyrosinase enzyme was digitally printed on plasma pretreated polyamide-6,6 fabric using several sustainable technologies. Ink containing carboxymethyl cellulose was found to be the most suitable viscosity modifier for this enzyme. Before and after being deposited on the fabric surface, the printed inks retained enzyme activity of 69% and 60%, respectively, compared to activity prior printing process. A good number of the printed enzyme was found to be strongly adsorbed on the fabric surface even after several rinsing cycles due to surface activation by plasma treatment. Rinsed out fabrics retained a maximum activity of 34% resulting from the well-adsorbed enzymes. The activity of tyrosinase on printed fabrics was more stable than ink solution for at least 60 days. Effects of pH, temperature and enzyme kinetics on ink solution and printed fabrics were assessed. Tyrosinase printed synthetic fabrics can be utilized for a range of applications from biosensing and wastewater treatment to cultural heritage works.

Bioengineering of Halobacterium sp. NRC-1 gas vesicle nanoparticles with GvpC fusion protein produced in E. coli

Gas vesicle nanoparticles (GVNPs) are hollow, buoyant prokaryotic organelles used for cell flotation. GVNPs are encoded by a large gas vesicle protein (gvp) gene cluster in the haloarchaeon, Halobacterium sp. NRC-1, including one gene, gvpC, specifying a protein bound to the surface of the nanoparticles. Genetically engineered GVNPs in the Halobacterium sp. have been produced by fusion of foreign sequences to gvpC. To improve the versatility of the GVNP platform, we developed a method for displaying exogenously produced GvpC fusion proteins on the haloarchaeal nanoparticles. The streptococcal IgG-binding protein domain was fused at or near the C-terminus of GvpC, expressed and purified from E. coli, and shown to bind to wild-type GVNPs. The two fusion proteins, GvpC3GB and GvpC4GB, without or with a highly acidic GvpC C-terminal region, were found to be able to bind nanoparticles equally well. The GVNP-bound GvpC-IgG-binding fusion protein was also capable of binding to an enzyme-linked IgG-HRP complex which retained enzyme activity, demonstrating the hybrid system capability for display and delivery of protein complexes. This is the first report demonstrating functional binding of exogenously produced GvpC fusion proteins to wild-type haloarchaeal GVNPs which significantly expands the capability of the platform to produce bioengineered nanoparticles for biomedical applications.Key points • Haloarchaeal gas vesicle nanoparticles (GVNPs) constitute a versatile display system. • GvpC-streptococcal IgG-binding fusion proteins expressed in E. coli bind to GVNPs. • IgG-binding proteins displayed on floating GVNPs bind and display IgG-HRP complex. Graphical abstract

Machine-Assisted Discovery of Chondroitinase ABC Complexes toward Sustained Neural Regeneration.

Among the many molecules that contribute to glial scarring, chondroitin sulfate proteoglycans (CSPGs) are known to be potent inhibitors of neuronal regeneration. Chondroitinase ABC (ChABC), a bacterial lyase, degrades the glycosaminoglycan (GAG) side chains of CSPGs and promotes tissue regeneration. However, ChABC is thermally unstable and loses all activity within a few hours at 37°C under dilute conditions. To overcome this limitation, the discovery of a diverse set of tailor-made random copolymers that complex and stabilize ChABC at physiological temperature is reported. The copolymer designs, which are based on chain length and composition of the copolymers, are identified using an active machine learning paradigm, which involves iterative copolymer synthesis, testing for ChABC thermostability upon copolymer complexation, Gaussian process regression modeling, and Bayesian optimization. Copolymers are synthesized by automated PET-RAFT and thermostability of ChABC is assessed by retained enzyme activity (REA) after 24h at 37°C. Significant improvements in REA in three iterations of active learning are demonstrated while identifying exceptionally high-performing copolymers. Most remarkably, one designed copolymer promotes residual ChABC activity near 30%, even after one week and notably outperforms other common stabilization methods for ChABC. Together, these results highlight a promising pathway toward sustained tissue regeneration.

In‐Situ Encapsulation of Protein into Nanoscale Hydrogen‐Bonded Organic Frameworks for Intracellular Biocatalysis

AbstractHydrogen‐bonded organic frameworks (HOFs) are porous materials with great potential for biological applications. The self‐assembly of HOFs and biomacromolecules, however, is challenging. We report herein the self‐assembly of nanoscale HOFs (nHOFs) to encapsulate protein for intracellular biocatalysis. The self‐assembly of tetrakis(4‐amidiniumphenyl)methane and azobenzenedicarboxylate can encapsulate protein in situ to form protein@nHOFs under mild conditions. This strategy is applicable to proteins with different surface charge and molecular weight, showing a high protein encapsulation efficiency and minimal effect on protein activity. A cellular delivery study shows that the protein@TA‐HOFs can efficiently enter cells and retain enzyme activity for biochemical catalysis in living cells for neuroprotection. Our strategy paves new avenues for interfacing nHOFs with biological settings and sheds light on expanding nHOFs as a platform for biomacromolecule delivery and disease treatment.

A Modified Brewing Procedure Informed by the Enzymatic Profiles of Gluten-Free Malts Significantly Improves Fermentable Sugar Generation in Gluten-Free Brewing

The mashing step underpins the brewing process, during which the endogenous amylolytic enzymes in the malt, chiefly β-amylase, α-amylase, and limit dextrinase, act concurrently to rapidly hydrolyze malt starch to fermentable sugars. With barley malts, the mashing step is relatively straightforward, due in part to malted barley’s high enzyme activity, enzyme thermostabilities, and gelatinization properties. However, barley beers also contain gluten and individuals with celiac disease or other gluten intolerances should avoid consuming these beers. Producing gluten-free beer from gluten-free malts is difficult, generally because gluten-free malts have lower enzyme activities. Strategies to produce gluten-free beers commonly rely on exogenous enzymes to perform the hydrolysis. In this study, it was determined that the pH optima of the enzymes from gluten-free malts correspond to regions already typically targeted for barley mashes, but that a lower mashing temperature was required as the enzymes exhibited low thermostability at common mashing temperatures. The ExGM decoction mashing procedure was developed to retain enzyme activity, but ensure starch gelatinization, and demonstrates a modified brewing procedure using gluten-free malts, or a combination of malts with sub-optimal enzyme profiles, that produces high fermentable sugar concentrations. This study demonstrates that gluten-free malts can produce high fermentable sugar concentrations without requiring enzyme supplementation.

Superoxide Dismutase 1 Nanoparticles (Nano-SOD1) as a Potential Drug for the Treatment of Inflammatory Eye Diseases.

Inflammatory eye diseases remain the most common clinical problem in ophthalmology. The secondary processes associated with inflammation, such as overproduction of reactive oxygen species (ROS) and exhaustion of the endogenous antioxidant system, frequently lead to tissue degeneration, vision blurring, and even blindness. Antioxidant enzymes, such as copper–zinc superoxide dismutase (SOD1), could serve as potent scavengers of ROS. However, their delivery into the eye compartments represents a major challenge due to the limited ocular penetration. This work presents a new therapeutic modality specifically formulated for the eye on the basis of multilayer polyion complex nanoparticles of SOD1 (Nano-SOD1), which is characterized by appropriate storage stability and pronounced therapeutic effect without side reactions such as eye irritation; acute, chronic, and reproductive toxicity; allergenicity; immunogenicity; mutagenicity even at high doses. The ability of Nano-SOD1 to reduce inflammatory processes in the eye was examined in vivo in rabbits with a model immunogenic uveitis—the inflammation of the inner vascular tract of the eye. It was shown during preclinical studies that topical instillations of Nano-SOD1 were much more effective compared to the free enzyme in decreasing uveitis manifestations. In particular, we noted statistically significant differences in such inflammatory signs in the eye as corneal and conjunctival edema, iris hyperemia, and fibrin clots. Moreover, Nano-SOD1 penetrates into interior eye structures more effectively than SOD itself and retains enzyme activity in the eye for a much longer period of time, decreasing inflammation and restoring antioxidant activity in the eye. Thus, the presented Nano-SOD1 can be considered as a potentially useful therapeutic agent for the treatment of ocular inflammatory disorders.

Cell-autonomous expression of the acid hydrolase galactocerebrosidase

Lysosomal storage diseases (LSDs) are typically caused by a deficiency in a soluble acid hydrolase and are characterized by the accumulation of undegraded substrates in the lysosome. Determining the role of specific cell types in the pathogenesis of LSDs is a major challenge due to the secretion and subsequent uptake of lysosomal hydrolases by adjacent cells, often referred to as "cross-correction." Here we create and validate a conditional mouse model for cell-autonomous expression of galactocerebrosidase (GALC), the lysosomal enzyme deficient in Krabbe disease. We show that lysosomal membrane-tethered GALC (GALCLAMP1) retains enzyme activity, is able to cleave galactosylsphingosine, and is unable to cross-correct. Ubiquitous expression of GALCLAMP1 fully rescues the phenotype of the GALC-deficient mouse (Twitcher), and widespread deletion of GALCLAMP1 recapitulates the Twitcher phenotype. We demonstrate the utility of this model by deleting GALCLAMP1 specifically in myelinating Schwann cells in order to characterize the peripheral neuropathy seen in Krabbe disease.

Revealing the important role of allosteric property in sucrose phosphate synthase from sugarcane with N-terminal domain deletion

Sucrose occupies many essential roles to control regulation of carbon partitioning in plants, including prokaryotic cells. Sucrose phosphate synthase (SPS; EC 2.4.1.14) is a key enzyme to catalyze the form of sucrose in primary sucrose synthesis pathway. Plants SPS has a molecular size around 120 kDa, which consists of N-terminal domain, C-terminal domain, and central domain. We produced the recombinant sugarcane SPS (SoSPS1) in Escherichia coli, however, the expression often appears to be a shorter form with retained enzyme activity. In our result, we reported that the shorter form is suggested to have a truncated N-terminal 20-kDa region. The truncated form of So SPS1 (ΔN-SPS) tends to enhance the specific activity 10-fold compared to full-length SoSPS1. The full-lenght SoSPS1 showed a remarkable allosteric activation by glucose-6-phosphate (G6P), while none of the N-terminal truncated form had such a characteristic. By kinetic analysis of full-length SoSPS1, a higher substrate affinity was shown in the presence of G6P. Conversely, the ΔN-SPS showed a similar substrate affinity whether G6P was added or not. Based on these results, we revealed that N-terminal region of SoSPS1 has essential role for allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity.

Congenic mapping and candidate gene analysis for streptozotocin-induced diabetes susceptibility locus on mouse chromosome 11.

Streptozotocin (STZ) has been widely used to induce diabetes in rodents. Strain-dependent variation in susceptibility to STZ has been reported; however, the gene(s) responsible for STZ susceptibility has not been identified. Here, we utilized the A/J-11SM consomic strain and a set of chromosome 11 (Chr. 11) congenic strains developed from A/J-11SM to identify a candidate STZ-induced diabetes susceptibility gene. The A/J strain exhibited significantly higher susceptibility to STZ-induced diabetes than the A/J-11SM strain, confirming the existence of a susceptibility locus on Chr. 11. We named this locus Stzds1 (STZ-induced diabetes susceptibility 1). Congenic mapping using the Chr. 11 congenic strains indicated that the Stzds1 locus was located between D11Mit163 (27.72Mb) and D11Mit51 (36.39Mb). The Mpg gene, which encodes N-methylpurine DNA glycosylase (MPG), a ubiquitous DNA repair enzyme responsible for the removal of alkylated base lesions in DNA, is located within the Stzds1 region. There is a close relationship between DNA alkylation at an early stage of STZ action and the function of MPG. A Sanger sequence analysis of the Mpg gene revealed five polymorphic sites in the A/J genome. One variant, p.Ala132Ser, was located in a highly conserved region among rodent species and in the minimal region for retained enzyme activity of MPG. It is likely that structural alteration of MPG caused by the p.Ala132Ser mutation elicits increased recognition and excision of alkylated base lesions in DNA by STZ.

Improvement of enzyme stability for alkyl esters synthesis in miniemulsion systems by using media engineering

AbstractBACKGROUNDOil‐in‐water miniemulsions have been suggested to display potential for application as a ‘green’ system for enzymatic alkyl ester synthesis. Still, a realistic assessment of the feasibility of this approach for practical applications requires further insight, namely the relation between enzyme stability and reaction operational conditions. The objective of this work is within such scope. Accordingly, it extends previous research on esterification catalyzed by Fusarium solani pisi cutinase, by addressing medium‐chain length substrates, and aims to contribute to the optimization of reaction conditions and towards the design of an efficient set‐up.RESULTSThe esterification yield was significantly enhanced (e.g. 50% increase for octyl octanoate system) through the correction of the initial pH of the reaction medium to pH 6. Under such environment, conformational changes of the enzyme were negligible after 24 h, resulting in improved enzyme stability for all systems under study. Maximum esterification rate (3.59 mmol min‐1) and product yield (91%) were achieved for octyl decanoate synthesis. Fed‐batch mode of operation allowed for the production of 1 mol L‐1 hexyl octanoate, while retaining enzyme activity over 5 days. Consecutive batch runs allowed for the cumulative production of 1.1 mol L‐1 hexyl octanoate after four cycles.CONCLUSIONSMedium engineering enabled the use of miniemulsion systems for the synthesis of medium‐chain alkyl esters and increase in the final concentration of the intended product. Through a fed‐batch mode the enzyme stability and reutilization were tuned allowing long‐term utilization of the free enzyme which could not be achieved through a repeated batch system. © 2017 Society of Chemical Industry

Generation of therapeutic protein variants with the human serum albumin binding capacity via site-specific fatty acid conjugation

Extension of the serum half-life is an important issue in developing new therapeutic proteins and expanding applications of existing therapeutic proteins. Conjugation of fatty acid, a natural human serum albumin ligand, to a therapeutic protein/peptide was developed as a technique to extend the serum half-life in vivo by taking advantages of unusually long serum half-life of human serum albumin (HSA). However, for broad applications of fatty acid-conjugation, several issues should be addressed, including a poor solubility of fatty acid and a substantial loss in the therapeutic activity. Therefore, herein we systematically investigate the conditions and components in conjugation of fatty acid to a therapeutic protein resulting in the HSA binding capacity without compromising therapeutic activities. By examining the crystal structure and performing dye conjugation assay, two sites (W160 and D112) of urate oxidase (Uox), a model therapeutic protein, were selected as sites for fatty acid-conjugation. Combination of site-specific incorporation of a clickable p-azido-L-phenylalanine to Uox and strain-promoted azide-alkyne cycloaddition allowed the conjugation of fatty acid (palmitic acid analog) to Uox with the HSA binding capacity and retained enzyme activity. Deoxycholic acid, a strong detergent, greatly enhanced the conjugation yield likely due to the enhanced solubility of palmitic acid analog.

Variant Amino Acid Residues Alter the Enzyme Activity of Peanut Type 2 Diacylglycerol Acyltransferases.

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triacylglycerol (TAG) biosynthesis via the acyl-CoA-dependent acylation of diacylglycerol. This reaction is a major control point in the Kennedy pathway for biosynthesis of TAG, which is the most important form of stored metabolic energy in most oil-producing plants. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar ‘Luhua 14.’ Sequence analysis of 11 different peanut cultivars revealed a gene family of 8 peanut DGAT2 genes (designated AhDGAT2a-h). Sequence alignments revealed 21 nucleotide differences between the eight ORFs, but only six differences result in changes to the predicted amino acid (AA) sequences. A representative full-length cDNA clone (AhDGAT2a) was characterized in detail. The biochemical effects of altering the AhDGAT2a sequence to include single variable AA residues were tested by mutagenesis and functional complementation assays in transgenic yeast systems. All six mutant variants retained enzyme activity and produced lipid droplets in vivo. The N6D and A26P mutants also displayed increased enzyme activity and/or total cellular fatty acid (FA) content. N6D mutant mainly increased the content of palmitoleic acid, and A26P mutant mainly increased the content of palmitic acid. The A26P mutant grew well both in the presence of oleic and C18:2, but the other mutants grew better in the presence of C18:2. AhDGAT2 is expressed in all peanut organs analyzed, with high transcript levels in leaves and flowers. These levels are comparable to that found in immature seeds, where DGAT2 expression is most abundant in other plants. Over-expression of AhDGAT2a in tobacco substantially increased the FA content of transformed tobacco seeds. Expression of AhDGAT2a also altered transcription levels of endogenous tobacco lipid metabolic genes in transgenic tobacco, apparently creating a larger carbon ‘sink’ that supports increased FA levels.

Serial deletion reveals structural basis and stability for the core enzyme activity of human glutaminase 1 isoforms: relevance to excitotoxic neurodegeneration

BackgroundGlutaminase 1 is a phosphate-activated metabolic enzyme that catalyzes the first step of glutaminolysis, which converts glutamine into glutamate. Glutamate is the major neurotransmitter of excitatory synapses, executing important physiological functions in the central nervous system. There are two isoforms of glutaminase 1, KGA and GAC, both of which are generated through alternative splicing from the same gene. KGA and GAC both transcribe 1–14 exons in the N-terminal, but each has its unique C-terminal in the coding sequence. We have previously identified that KGA and GAC are differentially regulated during inflammatory stimulation and HIV infection. Furthermore, glutaminase 1 has been linked to brain diseases such as amyotrophic lateral sclerosis, Alzheimer’s disease, and hepatic encephalopathy. Core enzyme structure of KGA and GAC has been published recently. However, how other coding sequences affect their functional enzyme activity remains unclear.MethodsWe cloned and performed serial deletions of human full-length KGA and GAC from the N-terminal and the C-terminal at an interval of approximately 100 amino acids (AAs). Prokaryotic expressions of the mutant glutaminase 1 protein and a glutaminase enzyme activity assay were used to determine if KGA and GAC have similar efficiency and efficacy to convert glutamine into glutamate.ResultsWhen 110 AAs or 218 AAs were deleted from the N-terminal or when the unique portions of KGA and GAC that are beyond the 550 AA were deleted from the C-terminal, KGA and GAC retained enzyme activity comparable to the full length proteins. In contrast, deletion of 310 AAs or more from N-terminal or deletion of 450 AAs or more from C-terminal resulted in complete loss of enzyme activity for KGA/GAC. Consistently, when both N- and C-terminal of the KGA and GAC were removed, creating a truncated protein that expressed the central 219 AA - 550 AA, the protein retained enzyme activity. Furthermore, expression of the core 219 AA - 550 AA coding sequence in cells increased extracellular glutamate concentrations to levels comparable to those of full-length KGA and GAC expressions, suggesting that the core enzyme activity of the protein lies within the central 219 AA - 550 AA. Full-length KGA and GAC retained enzyme activities when kept at 4 °C. In contrast, 219 AA - 550 AA truncated protein lost glutaminase activities more readily compared with full-length KGA and GAC, suggesting that the N-terminal and C-terminal coding regions are required for the stability KGA and GAC.ConclusionsGlutaminase isoforms KGA and GAC have similar efficacy to catalyze the conversion of glutamine to glutamate. The core enzyme activity of glutaminase 1 protein is within the central 219 AA - 550 AA. The N-terminal and C-terminal coding regions of KGA and GAC help maintain the long-term activities of the enzymes.

Expression, purification, and characterization of an intrinsically disordered Late Embryogenesis Abundant (LEA) protein from Artemia franciscana utilizing Escherichia coli and Nicotiana tabacum

Anhydrobiosis is an astounding strategy that allows certain species (both in animals and plants) to survive severe environmental conditions such as desiccation, extreme cold, or heat in the habitat. Among different molecular strategies, expression of highly hydrophilic polypeptides termed LEA proteins has been linked to the survival of plants and animals during periods of water stress such as freezing and drying. Several classification schemes for LEA proteins have been proposed and the brine shrimp, Artemia franciscana, is the only known animal that naturally expresses LEA proteins from three different classification groups (groups 1, 3, and 6). LEA proteins occur in several subcellular compartments including the cytosol and mitochondria. To investigate the biochemical properties of LEA proteins, it is important to characterize their structure. LEA proteins are intrinsically disordered in aqueous solution and the exact structure and function of these proteins is still poorly defined and understood. We hypothesized that LEA proteins will show different protective properties depending on the target enzyme (e.g. lactate dehydrogenase vs. succinate dehydrogenase). We found, that a purified group 1 LEA protein from A. franciscana (AfrLEA 1.1) helped to retain enzyme activity after desiccation of lactate dehydrogenase (LDH) and dry storage of the enzyme for 1, 3 and 7 days in the presence or absence of bovine serum albumin or trehalose. Increased concentration of purified AfrLEA 1.1, increased the percentage of LDH activity retained after desiccation. To further characterize AfrLEA 1.1, we cloned, expressed, and purified the protein in E. coli. We purified untagged AfrLEA 1.1 protein by affinity chromatography via intein mediated purification with an affinity based chitin‐binding system; a novel protein purification system which utilizes the inducible self‐cleavage activity of protein splicing elements to separate the target protein from the affinity tag. Furthermore, AfrLEA1.1 was expressed in Nicotiana tabacum to investigate if the protein increases drought tolerance of this model plant.Support or Funding InformationSupported by NSF IOS‐1456809

Expression, purification and enzymatic characterization of undecaprenyl pyrophosphate phosphatase from Vibrio vulnificus

Undecaprenyl pyrophosphate phosphatase (UppP), a cell membrane integral enzyme, catalyzes the dephosphorylation of undecaprenyl pyrophosphate to undecaprenyl phosphate, which is an essential carrier lipid in bacterial cell wall synthesis. We previously purified E. coli UppP and concluded that its catalytic site is likely located in the periplasm. To search for additional natural UppP homologs to elucidate what constitutes a common catalytic mechanism and to gain a better chance of obtaining high-resolution crystal structural information, we expressed and purified recombinant Vibrio vulnificus UppP using E. coli as a host. Mutagenesis analysis demonstrates that the proposed catalytic residues Gln-13, Glu-17, His-26 and Arg-166 are directly involved in enzyme catalysis. Additionally, mutations of most of the conserved serine and glycine residues within the proposed catalytic site (S22A, G163A and S165A) lead to complete inactivity, very low activity (<1.3% of the wild type) or no protein expression at all (G163R and G168A), whereas S23A and S167A retain enzyme activity (65% and 34%). Kinetic analysis indicates that S23A and S167A result in 1.4- and 5-fold decreases in kcat, whereas the substrate Km value exhibits only minor changes compared with wild-type UppP, implying that they are involved in enzyme catalysis. The structural modeling and molecular dynamics simulation analyses also provide a plausible structure of the catalytic core, centered on a conserved histidine (His-26) that initiates the hydrolysis of phosphate esters, rationalizing the mutagenesis data. This conclusion can be applied generally to all bacterial UppP enzymes.