An extracellular Cu/Zn superoxide dismutase from Neocaridina denticulata sinensis: cDNA cloning, mRNA expression and characterizations of recombinant protein.

Neocaridina denticulata sinensis possesses characters of rapid growth, tenacious vitality, short growth cycle, transparent, and easy feeding. Therefore, it is gradually being developed into an animal model for basic research on decapod crustaceans. Herein, a Cu/Zn superoxide dismutase (Cu/Zn-SOD), named as Nd-ecCu/Zn-SOD, was identified and characterized from N. denticulata sinensis. The full-length cDNA sequence of Nd-ecCu/Zn-SOD is 829 bp containing a 684 bp open reading frame, which encodes a protein of 227 amino acid residues with a typical Sod_Cu domain. The quantitative real-time PCR analysis showed that Nd-ecCu/Zn-SOD mRNA was expressed in all the tested tissues. Under challenge with copper, the mRNA expression of Nd-ecCu/Zn-SOD reached the maximum at 6 h, and decreased until 24 h. After 24 h of exposure, its expression was up-regulated significantly at 36 h. After then its expression sharply decreased with a comeback at 48 h. The result indicated that Nd-ecCu/Zn-SOD might play an important role in the stress response of N. denticulata sinensis. The expression of Nd-ecCu/Zn-SOD in gills challenged with Vibrio parahaemolyticus changed in a time-dependent manner. Nd-ecCu/Zn-SOD was lowly expressed in early developmental stages by RNA-Seq technology, yet it showed that a cyclical rise and fall occurred between middle stages and late stages. In addition, Nd-ecCu/Zn-SOD was recombinantly expressed using E. coli and the recombinant protein was purified as a single band on SDS-PAGE. The recombinant Nd-ecCu/Zn-SOD (rNd-ecCu/Zn-SOD) existed enzymatic activity under a wide range of temperature and pH. The exposure of metal ions was found that Zn2+, Mg2+, Ca2+, Ba2+, and Cu2+ could inhibit the enzymatic activity of rNd-ecCu/Zn-SOD, and Mn2+ increased the enzymatic activity of rNd-ecCu/Zn-SOD. These results indicate that Nd-ecCu/Zn-SOD may play a pivotal role in resistant against oxidative damage and act as a biomarker under stressful environment.

Study on Purification and Characterization of Polyphenol Oxidase from Acetes chinensis.

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant Km was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn2+ and K+ could enhance the activity of PPO; that Ba2+ and Ca2+ could inhibit the activity of PPO; and that Cu2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.

Effect of infrared radiation on activity and conformation of polyphenol oxidase from Acetes chinensis.

The aim of this paper was to study the effect of infrared radiation (IR) on the activity and conformation of polyphenol oxidase (PPO) in Acetes chinensis. In this paper, the specific activity of PPO was increased from 21.2 to 643.4 U/mg by a four-step purification. The results showed that IR treatment had greater effect on the enzyme activity and conformation of PPO than hot air (HA) treatment. After IR treatment at 70°C, the relative enzyme activity of PPO was 9.28%, the surface hydrophobicity index increased by 80.42%, and the content of sulfhydryl group decreased to 96.99% of the control group. The results of circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) showed that the α-helix of PPO treated by IR decreased and the random coil increased. The intrinsic fluorescence intensity of PPO decreased after IR treatment, indicating that the tertiary structure of PPO was destroyed. Scanning electron microscopy (SEM) results showed that the surface microstructure of PPO after IR treatment became clear and compact. In conclusion, IR treatment can completely destroy the secondary structure and tertiary structure of PPO and cause enzyme inactivation. This study provides a treatment for reducing the activity of PPO from A. chinensis during the production and processing. PRACTICAL APPLICATION: This study shows that IR treatment has a better inhibitory effect on the activity of PPO than HA treatment. It provides a better treatment method for inactivating the activity of PPO from Acetes chinensis during the production and processing.

Characterization of a novel extracellular CuZn superoxide dismutase from Rimicaris exoculata living around deep-sea hydrothermal vent.

Superoxide dismutase (SOD, EC 1.15.1.1) is a member of metalloenzyme that plays a key role in protecting organisms from oxidative damage. A novel extracellular CuZn superoxide dismutase RESOD was identified from Rimicaris exoculata, a dominant species that lives in close proximity to the deep-sea hydrothermal vents. It encoded a protein consisting of 227 amino acids with a signal peptide of 22 amino acids. Sequence analysis revealed that it had the characteristics of CuZn superoxide dismutase, and had low homology with the known SODs. Then the recombinant RESOD was expressed successfully, and high-purity RESOD was obtained. The recombinant RESOD exhibited maximal activity and stability with a temperature range of 0 °C to 10 °C. And the optimal pH for the activity and stability was about 10. However, RESOD was sensitive to some metal ions, particularly calcium. Furthermore, the biological function of RESOD was investigated in HeLa cells. It was found that RESOD could reduce the level of oxidation, and decrease the apoptosis resulted from excessive oxidant challenge. In conclusion, a novel alkali-tolerant cold-active extracellular CuZn SOD was characterized. The characteristics make RESOD a good candidate in a wide range of applications.

Dephosphorylation of DNA Fragments with Alkaline Phosphatase.

The removal of 5' phosphates from nucleic acids is used to enhance subsequent labeling with [γ-32P]-ATP, reduce the circularization of plasmid vectors in ligation reactions, and render DNA susceptible or resistant to other enzymes that act on nucleic acids (e.g., λ exonuclease). Essentially, any nucleotide phosphatase (e.g., bacterial alkaline phosphatase, calf intestinal alkaline phosphatase [CIP], placental alkaline phosphatase, shrimp alkaline phosphatase [SAP], or several acid phosphatases such as sweet potato and prostate acid phosphatase) will catalyze the removal of 5' phosphates from nucleic acid templates. In fact, these enzymes prefer small substrates such as p-nitrophenyl phosphate (PNPP) and the exposed 5' phosphates of nucleic acids to bulky globular protein substrates.

Alkaline Phosphatase.

Several types of alkaline phosphatases (or alkaline phosphomonoesterase) are commonly used in molecular cloning, including bacterial alkaline phosphatase (BAP) and calf intestinal alkaline phosphatase (CIP, CIAP, or CAP). Similar enzymes isolated from more esoteric cold-blooded organisms (e.g., SAP from shrimp) have become available in recent years and have the advantage of being easier to inactivate than BAP or CIP at the end of dephosphorylation reactions. The uses and properties of these enzymes are introduced here.

Dual Reporter Bioluminescence Imaging with NanoLuc and Firefly Luciferase.

Bioluminescence imaging is a powerful, broadly utilized method for noninvasive imaging studies in cell-based assays and small animal models of normal physiology and multiple diseases. In combination with molecular engineering of cells and entire organisms using luciferase enzymes, bioluminescence imaging has enabled novel applications including studies of protein-protein interactions, ligand-receptor interactions, cell trafficking, and drug targeting in mouse models. We describe use of a novel luciferase enzyme derived from Oplophorus gracilirostris, NanoLuc, in cell-based assays bioluminescence imaging of tumor-bearing mice. We also combine NanoLuc with another luciferase enzyme, firefly luciferase, to image multiple signal transduction events in one imaging session.

Is digestive cathepsin D the rule in decapod crustaceans?

Cathepsin D is an aspartic endopetidase with typical characteristics of lysosomal enzymes. Cathepsin D activity has been reported in the gastric fluid of clawed lobsters where it acts as an extracellular digestive enzyme. Here we investigate whether cathepsin D is unique in clawed lobsters or, instead, common in decapod crustaceans. Eleven species of decapods belonging to six infraorders were tested for cathepsin D activity in the midgut gland, the muscle tissue, the gills, and when technically possible, in the gastric fluid. Cathepsin D activity was present in the midgut gland of all 11 species and in the gastric fluid from the seven species from which samples could be taken. All sampled species showed higher activities in the midgut glands than in non-digestive organs and the activity was highest in the clawed lobster. Cathepsin D mRNA was obtained from tissue samples of midgut gland, muscle, and gills. Analyses of deduced amino acid sequence confirmed molecular features of lysosomal cathepsin D and revealed high similarity between the enzymes from Astacidea and Caridea on one side, and the enzymes from Penaeoidea, Anomura, and Brachyura on the other side. Our results support the presence of cathepsin D activity in the midgut glands and in the gastric fluids of several decapod species suggesting an extracellular function of this lysosomal enzyme. We discuss whether cathepsin D may derive from the lysosomal-like vacuoles of the midgut gland B-cells and is released into the gastric lumen upon secretion by these cells.

First record of deep-sea caridean shrimp Acanthephyra fimbriata Alcock Anderson, 1894 (Crustacea: Decapoda: Acanthephyridae) from southwest coast of India.

The present work reports the new occurrence of deep-sea shrimp Acanthephyra fimbriata Alcock Anderson, 1894 from southwestern Indian waters. The samples were caught in bottom trawls conducted between the depths of 200 and 350 m from two fish landing centers off Kerala along Arabian Sea from the southwest coast of India during 2015. Additionally, a phylogenetic analysis was used to explore the relationships of the genus Acanthephyra based on two genes: mitochondrial cytochrome c oxidase subunit 1 (COI) and 16S DNA (16S) with the present specimen and sequences retrieved from NCBI GenBank. The results revealed intraspecies (COI: 0-3 % 16S: 0-0.3 %) and interspecies divergence (COI: 17.5-20.9 % 16S: 5.2-9.5 %) among A. fimbriata.

Evaluating the non-lethal effects of organophosphorous and carbamate insecticides on the yabby (Cherax destructor) using cholinesterase (AChE, BChE), Glutathione S-Transferase and ATPase as biomarkers.

The toxicity of two organophosphorus insecticides, chlorpyrifos (CPF), malathion (MAL), and one carbamate insecticide, methomyl (METH), to the yabby (Cherax destructor) was assessed by measuring cholinesterase (AChE, BChE), Glutathione S-Transferase (GST) and Na+/K+ATPase activity after 96h of exposure. Yabbies exposed to all three insecticides at 2 and 5µgL-1 exhibited significant AChE, BChE, GST and Na+/K+ATPase inhibition. Based on these enzyme inhibition tests, the toxicity of the three insecticides to C. destructor was CPF > MAL > METH. After 14 days of recovery the yabbies enzymatic activities of AChE, BChE, GST and Na+/K+ATPase was measured. Recovery of The enzyme activity recovery was faster after the exposure to METH than for the yabbies exposed to CPF and MAL. Slow recovery of enzyme activity could affect the physical activities of organisms and produce indirect effects on populations if such crayfish are less able to elude predators or search for food.

Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways.

Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis.

Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep-sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity, and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bifunctional nature of these LumiFluor reporters enables greatly improved spatiotemporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, noninvasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes.

Phylogeny and New Classification of Hydrothermal Vent and Seep Shrimps of the Family Alvinocarididae (Decapoda).

The paper addresses the phylogeny and classification of the hydrothermal vent shrimp family Alvinocarididae. Two morphological cladistic analyses were carried out, which use all 31 recognized species of Alvinocarididae as terminal taxa. As outgroups, two species were included, both representing major caridean clades: Acanthephyra purpurea (Acanthephyridae) and Alpheus echiurophilus (Alpheidae). For additional support of the clades we utilised available data on mitochondrial Cytochrome c Oxidase I gene (CO1) and 16S ribosomal markers. Both morphological and molecular methods resulted in similar tree topologies and nearly identical clades. We consider these clades as evolutionary units and thus erect two new subfamilies: Rimicaridinae (Alvinocaridinides, Manuscaris, Opaepele, Shinkaicaris, Rimicaris), Alvinocaridinae (Alvinocaris), whilst recognising Mirocaridinae (with genera Mirocaris and Nautilocaris) at subfamily level. One genus, Keldyshicaris could not be assigned to any subfamily and is thus left as incertae sedis. The monophyly of Alvinocardinae was supported by morphological data, but not supported by molecular data (two analyses); the monophyly of all subfamilies was supported both by morphological and molecular data. Chorocaris is herein synonymized with Rimicaris, whilst Opaepele vavilovi is herein transferred to a new genus Keldyshicaris. Morphological trends within Alvinocarididae are discussed and short biogeographical remarks are given. We provide emended diagnoses for all subfamilies and genera along with keys to all recognized species.

Molecular characterization and organ-specific expression of the gene that encodes betaine aldehyde dehydrogenase from the white shrimp Litopenaeus vannamei in response to osmotic stress.

Crustaceans overcome osmotic disturbances by regulating their intracellular concentration of ions and osmolytes. Glycine betaine (GB), an osmolyte accumulated in response to hyperosmotic stress, is synthesized by betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) through the oxidation of betaine aldehyde. A partial BADH cDNA sequence from the white shrimp Litopenaeus vannamei was obtained and its organ-specific expression during osmotic stress (low and high salinity) was evaluated. The partial BADH cDNA sequence (LvBADH) is 1103bp long and encodes an open reading frame for 217 protein residues. The amino acid sequence of LvBADH is related to that of other BADHs, TMABA-DH and ALDH9 from invertebrate and vertebrate homologues, and includes the essential domains of their function and regulation. LvBADH activity and mRNA expression were detected in the gills, hepatopancreas and muscle with the highest levels in the hepatopancreas. LvBADH mRNA expression increased 2-3-fold in the hepatopancreas and gills after 7days of osmotic variation (25 and 40ppt). In contrast, LvBADH mRNA expression in muscle decreased 4-fold and 15-fold after 7days at low and high salinity, respectively. The results indicate that LvBADH is ubiquitously expressed, but its levels are organ-specific and regulated by osmotic stress, and that LvBADH is involved in the cellular response of crustaceans to variations in environmental salinity.

The Inhibitory Effects of Cu(2+) on Exopalaemon carinicauda Arginine Kinase via Inhibition Kinetics and Molecular Dynamics Simulations.

We studied the Cu(2+)-mediated inhibition and aggregation of Exopalaemon carinicauda arginine kinase (ECAK). We found that Cu(2+) significantly inactivated ECAK activity and double-reciprocal kinetics demonstrated that Cu(2+) induced noncompetitive inhibition of arginine and ATP (IC50 = 2.27 ± 0.16 µM; K i for arginine = 13.53 ± 3.76; K i for ATP = 4.02 ± 0.56). Spectrofluorometry results showed that Cu(2+) induced ECAK tertiary structural changes including the exposure of hydrophobic surfaces that directly induced ECAK aggregation. The addition of osmolytes such as glycine and proline successfully blocked ECAK aggregation induced by Cu(2+) and recovered ECAK activity. We built a 3D structure for ECAK using the ECAK ORF gene sequence. Molecular dynamics (MD) and docking simulations between ECAK and Cu(2+) were conducted to elucidate the binding mechanisms. The results showed that Cu(2+) blocked the entrance to the ATP active site; these results are consistent with the experimental result that Cu(2+) induced ECAK inactivation. Since arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrates, our study can provide new information about the effect of Cu(2+) on ECAK enzymatic function and unfolding, including aggregation, and the protective effects of osmolytes on ECAK folding to better understand the role of the invertebrate ECAK metabolic enzyme in marine environments.

A glycosyl hydrolase family 16 gene is responsible for the endogenous production of ß-1,3-glucanases within decapod crustaceans.

To identify the gene responsible for the production of a ß-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor. An open reading frame of 1098 bp for G. natalis and 1095 bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5 kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor. These putative proteins contained binding and catalytic domains that are characteristic of a ß-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8-9 amino acid residue peptides from a previously purified ß-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active ß-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious (Mictyris platycheles and Paragrapsus laevis) and terrestrial decapod species (Coenobita rugosus, Coenobita perlatus, Coenobita brevimanus and Birgus latro) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the ß-1,3-glucanase: 1) a digestive enzyme which hydrolyses ß-1,3-glucans, 2) an enzyme which cleaves ß-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms.

Inhibitory effects of pain relief drugs on neurological enzymes: implications on their potential neurotoxicity to aquatic animals.

Pain relief medications commonly occur in the aquatic environment at measurable levels. While the neurotoxicity of pain relievers to higher vertebrates is currently known, little is known about their effects on aquatic animals. This study investigated the neurotoxicity of pain relievers to aquatic animals. We used three neurological enzymes, cholinesterase (ChE), adenosine triphosphatase (ATPase), and monoamine oxidase (MAO), from a freshwater planarian (Dugesia japonica) and green neon shrimp (Neocaridina denticulata) as biomarkers to examine the effects of pain relievers on in vitro activity. The activity of MAO and ChE, but not ATPase, was significantly inhibited by acetaminophen, but not by other pain relievers examined. It was likely that the inhibitory effects of acetaminophen on shrimp neurological enzymes were more severe than on the planarian. These findings suggest that acetaminophen is potentially neurotoxic to aquatic animals, at least in terms of neurotransmission disturbance.

Taking their breath away: metabolic responses to low-oxygen levels in anchialine shrimps (Crustacea: Atyidae and Alpheidae).

Crustaceans generally act as oxy-regulators, maintaining constant oxygen uptake as oxygen partial pressures decrease, but when a critical low level is reached, ventilation and aerobic metabolism shut down. Cave-adapted animals, including crustaceans, often show a reduced metabolic rate possibly owing in part to the hypoxic nature of such environments. However, metabolic rates have not been thoroughly explored in crustaceans from anchialine habitats (coastal ponds and caves), which can experience variable oxygenic regimes. Here, an atypical oxy-conforming pattern of oxygen uptake is reported in the Hawaiian anchialine atyid Halocaridina rubra, along with other unusual metabolic characteristics. Ventilatory rates are near-maximal in normoxia and did not increase appreciably as PO2 declined, resulting in a decline in VO2 during progressive hypoxia. Halocaridina rubra maintained in anoxic waters survived for seven days (the duration of the experiment) with no measureable oxygen uptake, suggesting a reliance on anaerobic metabolism. Supporting this, lactate dehydrogenase activity was high, even in normoxia, and oxygen debts were quickly repaid by an unusually extreme increase in oxygen uptake upon exposure to normoxia. In contrast, four related anchialine shrimp species from the Ryukyu Islands, Japan, exhibited physiological properties consistent with previously studied crustaceans. The unusual respiratory patterns found in H. rubra are discussed in the context of a trade-off in gill morphology for osmoregulatory ion transport vs. diffusion of respiratory gasses. Future focus on anchialine species may offer novel insight into the diversity of metabolic responses to hypoxia and other physiological challenges experienced by crustaceans.

Unconventional secretion of the mutated 19 kDa protein of Oplophorus luciferase (nanoKAZ) in mammalian cells.

The putative amino-terminal signal peptide of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase was found to be a functional secretory peptide in mammalian cells. A 16 amino acid substituted mutant of KAZ (nanoKAZ) could be secreted from mammalian cells using the amino-terminal signal peptide of KAZ, but KAZ could not be secreted at all. Notably, nanoKAZ lacking the amino-terminal signal peptide could be secreted from mammalian cells, and the distribution of nanoKAZ on the cell membrane was confirmed by video-rate bioluminescence imaging. Thus, nanoKAZ lacking the amino-terminal signal peptide was expressed in the cytoplasm, translocated to the cell membrane, and released into the culture medium through an endoplasmic reticulum-Golgi-independent pathway.

Luminescence enhancement of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase by three amino acid substitutions.

To characterize the luminescence properties of nanoKAZ, a 16 amino acid substituted mutant of the catalytic 19kDa protein (KAZ) of Oplophorus luciferase, the effects of each mutated amino acid were investigated by site-specific mutagenesis. All 16 single substituted KAZ mutants were expressed in Escherichia coli cells and their secretory expressions in CHO-K1 cells were also examined using the signal peptide sequence of Gaussia luciferase. Luminescence activity of KAZ was significantly enhanced by single amino acid substitutions at V44I, A54I, or Y138I. Further, the triple mutant KAZ-V44I/A54I/Y138I, named eKAZ, was prepared and these substitutions synergistically enhanced luminescence activity, showing 66-fold higher activity than wild-KAZ and also 7-fold higher activity than nanoKAZ using coelenterazine as a substrate. Substrate specificity of eKAZ for C2- and/or C6-modified coelenterazine analogues was different from that of nanoKAZ, indicating that three amino acid substitutions may be responsible for the substrate recognition of coelenterazine to increase luminescence activity. In contrast, these substitutions did not stimulate protein secretion from CHO-K1 cells, suggesting that the folded-protein structure of KAZ might be different from that of nanoKAZ.