Heterologous expression of a papain-like protease inhibitor (SnuCalCpI17) in the E. coli and its mode of inhibition.

The effect of the Escherichia coli (E. coli) Rosetta (DE3) system on the expression of recombinant papain-like cysteine protease inhibitors (SnuCalCpIs) was evaluated, and the inhibition mode of the expressed inhibitor was determined. SnuCalCpI08 and SnuCalCpI17, which previously had not been expressed in the E. coli BL21 (DE3) system due to rare codons of more than 10%, were successfully expressed in E. coli Rosetta (DE3) since the strain provides tRNAs for six rare codons. Initially, both inhibitors were expressed as inclusion bodies; however, the water solubility of SnuCalCpI17 could be improved by lowering the incubation temperature, reducing the IPTG concentration, and increasing the induction time. In contrast, the other inhibitor could not be solubilized in water. To validate whether the inhibitor was expressed with correct protein folding, a papain inhibition assay was performed with SnuCalCpI17. SnuCalCpI17 showed a half-maximal inhibitory concentration (IC50) of 105.671 ± 9.857 µg/mL and a slow-binding inhibition mode against papain at pH 7.0 with a Kiapp of 75.80 µg/mL. The slow-binding inhibitor has a slow dissociation from the inhibitor-target complex, resulting in a long residence time in vivo, and thus can effectively inhibit the target at doses far below the IC50 of the inhibitor. KEY POINTS: • Propeptide inhibitor (SnuCalCpI17) containing rare codons was expressed in E. coli Rosetta (DE3). • The slow-binding inhibition was shown by plotting the apparent first-order rate constant (kobs). • Protein-protein interaction between SnuCalCpIs and papain was verified by docking simulation.

Role of Endogenous Cathepsin L in Muscle Protein Degradation in Olive Flounder (Paralichthys olivaceus) Surimi Gel.

We investigated the effect of endogenous cathepsin L on surimi gel produced from olive flounder (Paralichthys olivaceus). The amino acid sequences of six proteins predicted or identified as cathepsin L were obtained from the olive flounder genome database, and a phylogenetic analysis was conducted. Next, cathepsin L activity toward N-α-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) (Z-F-R-AMC) was detected in crude olive flounder extract and a crude enzyme preparation. A considerable decrease in the level of myosin heavy chain (MHC) in surimi occurred during autolysis at 60 °C. In contrast, the levels of actin, troponin-T, and tropomyosin decreased only slightly. To prevent protein degradation by cathepsin L, a protease inhibitor was added to surimi. In the presence of 1.0% protease inhibitor, the autolysis of olive flounder surimi at 60 °C was inhibited by 12.2%; the degree of inhibition increased to 44.2% as the inhibitor concentration increased to 3.0%. In addition, the deformation and hardness of modori gel increased as the inhibitor concentration increased to 2.0%. Therefore, cathepsin L plays an important role in protein degradation in surimi, and the quality of surimi gel could be enhanced by inhibiting its activity.

Molecular cloning and anti-invasive activity of cathepsin L propeptide-like protein from Calotropis procera R. Br. against cancer cells.

Cathepsin L of cancer cells has been shown to play an important role in degradation of extracellular matrix for metastasis. In order to reduce cell invasion, cathepsin L propeptide-like proteins which are classified as the I29 family in the MEROPS peptidase database were characterized from Calotropis procera R. Br., rich in cysteine protease. Of 19 candidates, the cloned and expressed recombinant SnuCalCp03-propeptide (rSnuCalCp03-propeptide) showed a low nanomolar Ki value of 2.3 ± 0.2 nM against cathepsin L. A significant inhibition of tumor cell invasion was observed with H1975, HT29, MDA-BM-231, PANC1, and PC3 with a 76, 67, 67, 63, and 79% reduction, respectively, in invasion observed in the presence of 400 nM of the rSnuCalCp03-propeptide. In addition, thermal and pH study showed rSnuCalCp03-propeptide consisting of secondary structures was stable at a broad range of temperatures (30-70 °C) and pH (2-10, except for 5 which is close to the isoelectric point of 5.2).

Impacts of the Dynamic High-Pressure Pre-Treatment and Post-Treatment of Whey Protein Aggregates on Their Physicochemical Properties and Emulsifying Activities.

The impacts of dynamic high-pressure (DHP) pretreatment and post-treatment (100 MPa) on the physicochemical and functional properties of whey protein isolate (WPI) aggregates formed by thermal treatment were investigated in this study. When WPI aggregates were formed by thermal treatment, the size of the aggregates formed with the DHP pretreated WPI was smaller than that of the aggregates formed with the original WPI. The size of the WPI aggregates formed by thermal treatment decreased with DHP post-treatment. The conformational parameters (ζ-potential, surface hydrophobicity, and intrinsic fluorescence intensity) of the WPI subjected to DHP pretreatment were not significantly influenced by thermal treatment. However, DHP post-treatment affected these parameters for the WPI aggregates formed during thermal treatment because of dissociation caused by intense shear and cavitation forces during DHP treatment. The emulsifying activity index (EAI) of the WPI aggregates slightly improved with DHP treatment, but its order had little effect on the magnitude of the EAI increase. DHP pretreatment or post-treatment can modulate the conformational structures and the physicochemical properties of protein aggregates.

Characterization and molecular docking study of cathepsin L inhibitory peptides (SnuCalCpIs) from Calotropis procera R. Br.

Propeptides, released from the autocatalytic activation of its zymogen, are potential inhibitors against proteases involved in cancer cell invasion and migration. Our research team previously obtained novel propeptides (SnuCalCpIs) from transcriptome analysis of the medicinal plant Calotropis procera R. Br. and reported them as promising candidates for cancer therapeutics due to their cathepsin L inhibition activity. In the present study, inhibitory activity among SnuCalCpIs was compared with inhibition efficiency and verified by in silico molecular docking analysis. Only SnuCalCpI03 and SnuCalCpI15, expressed in Escherichia coli, showed inhibitory activity against cathepsin L as competitive inhibitors, and the half-maximal inhibitory concentrations (IC50) values of 2.1 nM and 1.6 nM, respectively. They were stable below 70 °C, maintaining more than 90% inhibitory activity over a wide range of pH (2.0-10.0), except at the isoelectric point (pI). The template-based docking simulation models showed that SnuCalCpI02, SnuCalCpI12, and SnuCalCpI16 could not interact with the substrate-binding cleft of cathepsin L even though they possessed the same conserved domain. In contrast, SnuCalCpI03 and SnuCalCpI15 interacted with cathepsin L along the propeptide binding loop and substrate-binding cleft, resulting in obstruction of substrate access to the active site.

A jacalin-related lectin domain-containing lipase from chestnut (Castanea crenata): Purification, characterization, and protein identification.

A novel lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was discovered from Korean chestnut (Castanea crenata). The lipase was isolated and purified by ammonium sulfate precipitation and a fast protein liquid chromatography system equipped with HiTrap DEAE-Sepharose Fast Flow, HiTrap Q-Sepharose Fast Flow, and HiPrep Sephacryl S-100 Hi-Resolution columns. The purified C. crenata lipase showed a 15.8% yield, purification fold number of 465.8, and specific activity against triolein of 88.5 mU/mg. The enzyme exhibited hydrolytic activity toward tributyrin, trilaurin, and triolein, and was maximally active at pH 8.0 and 35 °C, with triolein used as the substrate. The activation energy (Ea) and deactivation energy (Ed) of triolein hydrolysis were 38.41 and 83.35 kJ/mol, respectively. In the enzyme kinetic study, Vmax, Km, and k cat were 110.58 mU/mg, 0.11 mM, and 0.221 min-1, respectively. The relatively low Km value indicated that the lipase has high affinity for its substrate. Moreover, Mg2+ and Ca2+ increased the lipase activity to 115.4% and 108.3%, respectively. The results of peptide fingerprinting revealed that the C. crenata lipase with a molecular weight of 33.3 kDa was structurally similar to the mannose-binding lectin of the jacalin-related lectin domain superfamily, implying that it has potential as a therapeutic agent for use in the biomedical industry.

Divergent substrate specificities and regioselectivities of three lipase isoforms from Cordyceps militaris: Combinatorial advantages for entomopathogenicity and prospects as biocatalysts.

Cordyceps militaris, an entomopathogenic Cordyceps mushroom, is a crucial ethnopharmacological agricultural product with applications in traditional oriental remedies in East Asia. Since lipases are reported to serve as key enzymatic equipment for entomopathogenic fungi during the host infection, the presence of various lipases with different biochemical features in C. militaris was elucidated. Three lipases from C. militaris (CML) of 60-70 kDa were isolated according to protein hydrophobicity; isoform relationships were identified by peptide mapping using liquid chromatography-electrospray ionization-tandem mass spectrometry. The CML isoforms exhibited distinct substrate specificities, which were related to the hydrophobicity of each isoform. Furthermore, the integral stereoselectivity of each lipase towards trioleoylglycerol diverged into two classes (sn-1,3 and sn-2 regioselectivity) that are rare in canonical fungal lipases. Overall, our results demonstrate that C. militaris secretes lipase isoforms with cocktail-like enzyme functions that may contribute to the entomopathogenic life cycle of C. militaris. Each CML isoform has distinct advantages for biocatalyst applications in the food and oleochemical industries.

Purification and characterization of a novel acid-tolerant and heterodimeric ß-glucosidase from pumpkin (Cucurbita moschata) seed.

A novel ß-glucosidase was purified from pumpkin (Cucurbita moschata) seed by anion exchange chromatography and gel permeation chromatography, and its molecular mass was determined to be 42.8 kDa by gel permeation chromatography. The heterodimeric structure consisting of two subunits, free from disulfide bonds, was determined by native-PAGE analysis followed by zymography. The enzyme was maximally active at pH 4.0 and 70°C, and Vmax, Km, and kcat values were 0.078 units mg-1 protein, 2.22 mM, and 13.29 min-1, respectively, employing p-nitrophenyl-ß-d-glucopyranoside as the substrate. The high content of glycine determined by amino acid analysis implies that the enzyme possesses flexible conformations and interacts with cell membranes and walls in nature. Circular dichroism studies revealed that the high stability of the enzyme within the pH range of 2.0-10.0 is due to its reversible pH-responsive characteristics for α-helix-antiparallel ß-sheet interconversion.

Erythorbyl fatty acid ester as a multi-functional food emulsifier: Enzymatic synthesis, chemical identification, and functional characterization of erythorbyl myristate.

Erythorbyl myristate (EM), a potential multi-functional food emulsifier, was newly synthesized by immobilized lipase-catalyzed esterification between antioxidative erythorbic acid and antibacterial myristic acid. The yield and productivity of EM were 56.13 ± 2.51 mg EM/g myristic acid and 1.76 ± 0.08 mM/h, respectively. The molecular structure of EM was identified as (R)-2-((R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-2-hydroxyethyl tetradecanoate using HPLC-ESI/MS and 2D [1H-1H] NMR COSY. The hydrophilic-lipophilic balance of EM was 11.5, suggesting that EM could be proper to stabilize oil-in-water emulsions. Moreover, isothermal titration calorimetry demonstrated the micellar thermodynamic behavior of EM and determined its critical micelle concentration (0.36 mM). In terms of antioxidative property, EM exhibited the radical scavenging activity against DPPH (EC50: 35.47 ± 0.13 µM) and ABTS (EC50: 36.45 ± 1.98 µM) radicals. Finally, EM showed bacteriostatic and bactericidal activities against Gram-positive foodborne pathogens (minimum inhibitory concentration: 0.06-0.60 mM; minimum bactericidal concentration: 0.07-0.93 mM).

Expression of barley HvCBF4 enhances tolerance to abiotic stress in transgenic rice.

C-repeat/dehydration-responsive element binding factors (CBF/DREBs) are a family of transcription factors that regulate freezing tolerance in Arabidopsis. As a step towards understanding the stress response of monocotyledonous plants, we isolated a barley gene HvCBF4 whose expression is induced by low-temperature stress. Transgenic over-expression of HvCBF4 in rice resulted in an increase in tolerance to drought, high-salinity and low-temperature stresses without stunting growth. Interestingly, under low-temperature conditions, the maximum photochemical efficiency of photosystem II in the dark-adapted state (F(v)/F(m), where F(v) is the variable fluorescence and F(m) is the maximum fluorescence) in HvCBF4 plants was higher by 20% and 10% than that in non-transgenic and CBF3/DREB1A plants, respectively. Using the 60K Rice Whole Genome microarray, 15 rice genes were identified that were activated by HvCBF4. When compared with 12 target rice genes of CBF3/DREB1A, five genes were common to both HvCBF4 and CBF3/DREB1A, and 10 and seven genes were specific to HvCBF4 and CBF3/DREB1A, respectively. Interestingly, HvCBF4 did not activate Dip1 and Lip5, two important target genes of CBF3/DREB1A, in transgenic rice under normal growth conditions, but their expression was enhanced by HvCBF4 under low-temperature conditions. Our results suggest that CBF/DREBs of barley act differently from those of Arabidopsis in transgenic rice.

Selective production of 1-monocaprin by porcine liver carboxylesterase-catalyzed esterification: Its enzyme kinetics and catalytic performance.

Porcine liver carboxylesterase (PLE) belongs to carboxylesterase family (EC 3.1.1.1) as a serine-type esterase. The PLE-catalyzed esterification of capric acid with glycerol in reverse micelles was investigated on the catalytic performance and enzyme kinetics. The most suitable structure of reverse micelles was comprised of isooctane (reaction medium) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT, anionic surfactant) with 0.1 of R-value ([water]/[surfactant]) and 3.0 of G/F-value ([glycerol]/[fatty acid]) for the PLE-catalyzed esterification. In the aspect of regio-selectivity, the PLE mainly produced 1-monocaprin without any other products (di- and/or tricaprins of subsequent reactions). Furthermore, the degree of esterification at equilibrium state (after 4 h from the initiation) was 62.7% under the optimum conditions at pH 7.0 and 60 °C. Based on Hanes-Woolf plot, the apparent Km and Vmax values were calculated to be 16.44 mM and 38.91 µM/min/mg protein, respectively.

A reliable and reproducible method for the lipase assay in an AOT/isooctane reversed micellar system: modification of the copper-soap colorimetric method.

The copper-soap method, which is based on the absorbance of a fatty acid-copper complex at 715 nm, is a widely used colorimetric assay to determine the lipase activity in reversed micellar system. However, the absorbance of the bis(2-ethylhexyl) sodium sulfosuccinate (AOT)-copper complex prevents the use of an AOT/isooctane reversed micellar system. An extraction step was added to the original procedure to remove AOT and eliminate interference from the AOT-copper complex. Among the solvents tested, acetonitrile was determined to be the most suitable because it allows for the generation of a reproducible calibration curve with oleic acid that is independent of the AOT concentrations. Based on the validation data, the modified method, which does not experience interference from the AOT-copper complex, could be a useful method with enhanced accuracy and reproducibility for the lipase assay.

Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. revealed by de novo transcriptome analysis.

Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.

Thermal deactivation kinetics of Pseudomonas fluorescens lipase entrapped in AOT/isooctane reverse micelles.

Thermostability of the lipase (EC 3.1.1.3) was found to be increased by the enzyme-entrapment in 50 mM AOT/isooctane reverse micelles. The half-life (15.75 h) of Pseudomonas fluorescens lipase entrapped in reverse micelles at 70 °C was 9.72- and 11.41-fold longer than those solubilized in a glycerol pool or in 10 mM phosphate buffer (pH 8.0), respectively. The enzyme deactivation model considering a two-step series-type was employed, and deactivation constants for the second step (k2) at all temperatures were drastically decreased after the lipase was entrapped in reverse micelles. In particular, k2 (0.0354 h⁻¹) at 70 °C in reverse micelles was 12.33- and 13.14-fold lower than in a glycerol pool or in the phosphate buffer, respectively. The deactivation energies (from k1, k2) for the lipase entrapped in the reverse micelles, solubilized in a glycerol pool, or in the aqueous buffer were 7.51, 26.35 kcal/mol, 5.93, 21.08 kcal/mol, and 5.53, 17.57 kcal/mol, respectively.

Overexpression of the transcription factor AP37 in rice improves grain yield under drought conditions.

Transcription factors with an APETELA2 (AP2) domain have been implicated in various cellular processes involved in plant development and stress responses. Of the 139 AP2 genes predicted in rice (Oryza sativa), we identified 42 genes in our current study that are induced by one or more stress conditions, including drought, high salinity, low temperature, and abscisic acid. Phylogenic analysis of these 42 stress-inducible AP2 genes revealed the presence of six subgroups (I-VI) with distinct signature motifs. Two genes, AP37 and AP59, representing subgroups I and II, respectively, were functionally characterized. Both genes were found to be induced upon 2 h of exposure to drought and high-salinity conditions but to differ in their expression profile upon exposure to low temperature and abscisic acid. The overexpression of AP37 and AP59 in rice under the control of the constitutive promoter OsCc1 increased the tolerance to drought and high salinity at the vegetative stage. Increased tolerance to low temperatures was observed only in OsCc1:AP37 plants. More importantly, the OsCc1:AP37 plants showed significantly enhanced drought tolerance in the field, which increased grain yield by 16% to 57% over controls under severe drought conditions, yet exhibited no significant difference under normal growth conditions. In contrast, grain yield in OsCc1:AP59 plants in the field was reduced by 23% to 43% compared with controls under both normal and drought stress conditions. Microarray experiments identified 10 and 38 genes that are up-regulated by AP37 and AP59, respectively, in addition to 37 genes that are commonly induced by both factors. Our results suggest that the AP37 gene has the potential to improve drought tolerance in rice without causing undesirable growth phenotypes.