Synthesis of covalent organic frameworks via Kabachnik-Fields reaction for water treatment.

Providing safe and clean domestic water for people is currently one of the greatest worldwide issues. In this context, heavy metal ions and pathogenic microbes are the two major factors in water pollution. The conventional water treatment methods, however, are generally high-energy and high-resource consumptive. Herein, we report, the first of its kind, the room-temperature synthesis of α-aminophosphonate-linked COFs via three-component one-pot in situ Kabachnik-Fields reaction (KF-3CR). Due to the coexistent bioactive α-aminophosphonate and photosensitive porphyrin, the obtained APCOF-1 exhibits highly efficient solar-powered bactericidal and heavy metal ion removal abilities, which allows it to be a promising COF-based multifunctional material for water treatment in an energy- and resource-saving way. Specifically, by incorporating APCOF-1 (up to 50 wt%) with eco-friendly and low-cost chitosan, an APCOF-1 @chitosan aerogel-based helical setup is fabricated via a facile templated freeze-drying approach and it can be a continuous flow-through water purifier model to achieve scaled-up water treatment through adsorptive removal of heavy metal ions and sunlight-driven sterilization. We believe that this research not only can significantly enrich the synthetic methodology of COFs, but also will hopefully bring COFs one step closer to the practical application.

Synergistic Antibacterial and Anti-Inflammatory Effects of a Drug-Loaded Self-Standing Porphyrin-COF Membrane for Efficient Skin Wound Healing.

Chronic wound infections resulting from severe bacterial invasion have become a major medical threat worldwide. Herein, we report a large-area, homogeneous, and self-standing porphyrin-covalent organic framework (COF)-based membrane with encapsulated ibuprofen (IBU) via an in situ interfacial polymerization and impregnation approach. The obtained IBU@DhaTph-membrane exhibits highly effective antibacterial and anti-inflammatory effects via synergistic light-induced singlet oxygen (1 O2 ) generation and controllable IBU release, which is well supported by in vitro experiments. In addition, the IBU@DhaTph-membrane-based biocompatible "band-aid" type dressing is fabricated, and its excellent anti-infection and tissue remodeling activities are fully evidenced by in vivo chronic wound-healing experiments. This study may inspire and promote the fabrication of many more new types of COF-based multifunctional biomaterials for various skin injuries in clinical medicine.

Nucleic Acid Vaccine Targeting Nogo-66 Receptor and Paired Immunoglobulin-Like Receptor B as an Immunotherapy Strategy for Spinal Cord Injury in Rats.

Nogo-66 receptor (NgR) and paired immunoglobulin-like receptor B (PirB) are two common receptors of various myelin-associated inhibitors (MAIs) and, thus, play an important role in MAIs-induced inhibitory signalling of regeneration following spinal cord injury (SCI). Based on the concept of protective autoimmunity, vaccine approaches could induce the production of antibodies against inhibitors in myelin, such as using purified myelin, spinal cord homogenates, or MAIs receptor NgR, in order to block the inhibitory effects and promote functional recovery in SCI models. However, due to the complication of the molecules and the mechanisms involved in MAIs-mediated inhibitory signalling, these immunotherapy strategies have yielded inconsistent outcomes. Therefore, we hypothesized that the choice and modification of self-antigens, and co-regulating multiple targets, may be more effective in repairing the injured spinal cord and improving functional recovery. In this study, NgR and PirB were selected to construct a double-targeted granulocyte-macrophage colony stimulating factor-NgR-PirB (GMCSF-NgR-PirB) nucleic acid vaccine, and investigate the efficacy of this immunotherapy in a spinal cord injury model in rats. The results showed that this vaccination could stimulate the production of antibodies against NgR and PirB, block the inhibitory effects mediated by various MAIs, and promote nerve regeneration and functional recovery after spinal cord injury. These findings suggest that nucleic acid vaccination against NgR and PirB can be a promising therapeutic strategy for SCI and other central nervous system diseases and injuries.

Synaptic Plasticity in PTSD and associated Comorbidities: The Function and Mechanism for Diagnostics and Therapy.

The studying of synaptic plasticity, the ability of synaptic connections between neurons to be weakened or strengthened and specifically long-term potentiation (LTP) and long-term depression (LTD), is one of the most active areas of research in neuroscience. The process of synaptic connections playing a crucial role in improving cognitive processes is important to the processing of information in brain. In general, the dysfunction of synaptic plasticity was involved in a wide spectrum of central nervous system (CNS) disorders, including some neurodegenerative disorders. Thus, synaptic plasticity which is a dysfunction reported in neurodegenerative disorders may also be involved in posttraumatic stress disorder (PTSD), an anxiety and/or memory disorder developed after experiencing natural disasters, domestic violence or combat-related trauma. In this review, we mainly focus on discussing the biological function and mechanism for diagnostics and therapy of synaptic plasticity in PTSD and associated comorbidities, such as schizophrenia, depression, sleep disturbances and alcohol dependence, and further studying the molecular mechanisms of PTSD with a particular focus on the LTP/LTD, glutamatergic ligand-receptor systems, voltage-gated calcium channels (VGCCs) and brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (TrkB). The summarized function and mechanism of synaptic plasticity in PTSD and its comorbidities may help us further understand PTSD and provide insight into novel neuroplasticity modifying for diagnostics and treatment for PTSD.

Luteimonas rhizosphaerae sp. nov., isolated from the rhizosphere of Triticum aestivum L.

A Gram-stain-negative, rod-shaped bacterium, strain 4-12T, was isolated from the rhizosphere of Triticum aestivum L. from the Xiaokai River irrigation area, China. The isolate grew optimally at 30 °C, pH 7.5-8.0 and with 1.0 % (w/v) NaCl. Based on the 16S rRNA gene sequence and phylogenetic analysis, strain 4-12T belonged to the genus Luteimonas with the highest degree of 16S rRNA gene sequence similarity to Luteimonas tolerans UM1T (97.68 %), followed by Luteimonas terrae THG-MD21T (97.67 %), Lysobacter panaciterrae Gsoil 068T (97.21 %) and Luteimonas aestuarii B9T (97.16 %). However, the DNA-DNA relatedness values between strain 4-12T and closely related Luteimonas strains were well below 40 %. The average nucleotide identity and the Genome-to-Genome Distance Calculator also showed low relatedness (below 95 and 70 %, respectively) between strain 4-12T and the type strains in genus Luteimonas. Ubiquinone-8 was the predominant quinone. The major fatty acids were iso-C15 : 0, iso-C11 : 0, iso-C17 : 0 and iso-C17 : 1ω9c. Polar lipids were dominated by diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unidentified phospholipids. The DNA G+C content was 69.5 %. According to the phenotypic, genetic and chemotaxonomic data, strain 4-12T is considered to represent a novel species in the genus Luteimonas, for which the name >Luteimonas rhizosphaerae sp. nov. is proposed, with strain 4-12T (=CCTCC AB 2016261T=KCTC 52585T) as the type strain.

Dissecting the Native Architecture and Dynamics of Cyanobacterial Photosynthetic Machinery.

The structural dynamics and flexibility of cell membranes play fundamental roles in the functions of the cells, i.e., signaling, energy transduction, and physiological adaptation. The cyanobacterial thylakoid membrane represents a model membrane that can conduct both oxygenic photosynthesis and respiration simultaneously. In this study, we conducted direct visualization of the global organization and mobility of photosynthetic complexes in thylakoid membranes from a model cyanobacterium, Synechococcus elongatus PCC 7942, using high-resolution atomic force, confocal, and total internal reflection fluorescence microscopy. We visualized the native arrangement and dense packing of photosystem I (PSI), photosystem II (PSII), and cytochrome (Cyt) b6f within thylakoid membranes at the molecular level. Furthermore, we functionally tagged PSI, PSII, Cyt b6f, and ATP synthase individually with fluorescent proteins, and revealed the heterogeneous distribution of these four photosynthetic complexes and determined their dynamic features within the crowding membrane environment using live-cell fluorescence imaging. We characterized red light-induced clustering localization and adjustable diffusion of photosynthetic complexes in thylakoid membranes, representative of the reorganization of photosynthetic apparatus in response to environmental changes. Understanding the organization and dynamics of photosynthetic membranes is essential for rational design and construction of artificial photosynthetic systems to underpin bioenergy development. Knowledge of cyanobacterial thylakoid membranes could also be extended to other cell membranes, such as chloroplast and mitochondrial membranes.

Salinicola tamaricis sp. nov., a heavy-metal-tolerant, endophytic bacterium isolated from the halophyte Tamarix chinensis Lour.

A Gram-stain-negative, rod-shaped bacterium, strain F01T, was isolated from leaves of Tamarix chinensis Lour. The isolate grew optimally at 30 °C, at pH 7.0 and with 5.0 % (w/v) NaCl, and showed a high tolerance to manganese, lead, nickel, ferrous ions and copper ions. The major fatty acids were C18 : 1ω7c and C16 : 0, and the predominant respiratory quinone was Q-9. Polar lipids were dominated by diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, unidentified aminoglycolipids and phospholipids. The DNA G+C content was 65.8 %. Based on multilocus phylogenetic analysis, strain F01T belonged to the genus Salinicola, with highest 16S rRNA gene sequence similarity to Salinicola peritrichatus CGMCC 1.12381T (97.7 %). The level of DNA-DNA hybridization between strain F01T and closely related Salinicola strains was well below 70 %. According to the phenotypic, genetic and chemotaxonomic data, strain F01T is considered to represent a novel species in the genus Salinicola, for which the name Salinicola tamaricis sp. nov. is proposed. The type strain is F01T (=CCTCC AB 2015304T=KCTC 42855T).

Nicotine enhances GABAergic inhibition of oxytocin mRNA-expressing neuron in the hypothalamic paraventricular nucleus in vitro in rats.

We recently found that extracellular administration of nicotine indirectly excited hypothalamic paraventricular nucleus (PVN) corticotropin-releasing hormone (CRH) mRNA-expressing neurons. In this study, we studied the effect of nicotine on PVN oxytocin (OT) mRNA-expressing neuron in vitro in rats, by whole-cell patch-clamp recording technique, immunohistochemistry methods and single-cell reverse-transcription multiplex polymerase chain reaction (SC-RT-mPCR) methods Our results showed that 79.3% (73/92) of the 92 PVN putative magnocellular neurons co-expressed GAPDH mRNA and OT mRNA. Under current-clamp recording conditions, local micro application of nicotine (1-300µM) induced a decrease in spontaneous firing rate accompanied with a hyperpolarization of membrane potential in 76.7% (56/73) of PVN OT mRNA-expressing magnocellular neurons. The nicotine induced inhibition in spontaneous activity of PVN OT mRNA-expressing magnocellular neurons was dose-dependent. The half-inhibitory concentration (IC50) is 2.9µM. The nicotine induced hyperpolarization of PVN OT mRNA-expressing magnocellular neurons was sensitive to GABAA receptor antagonist, SR95531 (10µM) and tetrodotoxin (TTX, 1µM). In addition, local micro application of nicotine induced a significant increase in frequency of spontaneous inhibitory postsynaptic potentials (sIPSPs), but without changes in the sEPSPs amplitude of the OT-mRNA expressing neurons. Biocytin staining confirmed that the nicotine-sensitive OT-mRNA expressing neurons were the PVN magnocellular neurons. These results demonstrated that nicotine enhances the GABAergic inhibition, resulting in a decrease in spontaneous firing rate of the PVN OT-mRNA expressing neurons. These findings suggested that nicotine modulated PVN OT secretion via enhancement of both presynaptic action potential drive and quantal GABA release.

Larsenimonas suaedae sp. nov., a moderately halophilic, endophytic bacterium isolated from the halophyte Suaeda salsa.

A moderately halophilic, Gram-stain-negative, non-endospore-forming endophytic bacterium designated strain ST307T was isolated from the euhalophyte Suaeda salsa in Dongying, China. Strain ST307T was aerobic, rod-shaped, motile and orange-yellow-pigmented. The organism grew at NaCl concentrations of 0.6-20 % (w/v) (optimum 5-6 %, w/v), at temperatures of 5-45 °C (optimum 35 °C) and at pH 5-9 (optimum pH 7-8). It accumulated poly-ß-hydroxybutyric acid and produced exopolysaccharides. The major fatty acids were C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and C16 : 1ω7c/C16 : 1ω6c. The predominant lipoquinone was ubiquinone Q-9. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, a glycoaminolipid and a phosphoglycoaminolipid. The DNA G+C content was 60.5 mol%. Phylogenetic analyses of 16S rRNA gene sequences and concatenated atpA, rpoD and secA gene sequences revealed that the strain represents a member of the genus Larsenimonas. The closest related type strain was Larsenimonas salina M1-18T. Mean DNA-DNA relatedness values between strain ST307T and the related species L. salina M1-18T, Chromohalobacter beijerinckii DSM 7218T, C. canadensis DSM 6769T, C. israelensis DSM 6768T, C. marismortui CGMCC 1.2321T, C. nigrandesensis DSM 14323T, C. salexigens DSM 3043T and C. sarecensis DSM 15547T were 15±2-45±1 %. On the basis of phenotypic, chemotaxonomic and molecular features, strain ST307T clearly represents a novel species of the genus Larsenimonas. The name Larsenimonassuaedae sp. nov. is proposed, with ST307T (=CGMCC 1.8902T=DSM 22428T) as the type strain.

New ansamycin derivatives generated by simultaneous mutasynthesis.

The conversion from triene- to diene-typed ansamycins is clarified step by step in Streptomyces seoulensis IFB-A01. Such an intertype convertibility is adopted to establish for the first time the simultaneous mutasynthesis of both types of C17-benzene ansamycins (C17BAs). Three of the newly generated unnatural compounds showed potent cytotoxicity.

Properties of 4 Hz stimulation-induced parallel fiber-Purkinje cell presynaptic long-term plasticity in mouse cerebellar cortex in vivo.

Cerebellar parallel fiber-Purkinje cell (PF-PC) long-term synaptic plasticity is important for the formation and stability of cerebellar neuronal circuits, and provides substrates for motor learning and memory. We previously reported both presynaptic long-term potentiation (LTP) and long-term depression (LTD) in cerebellar PF-PC synapses in vitro. However, the expression and mechanisms of cerebellar PF-PC synaptic plasticity in the cerebellar cortex in vivo are poorly understood. In the present study, we studied the properties of 4 Hz stimulation-induced PF-PC presynaptic long-term plasticity using in vivo the whole-cell patch-clamp recording technique and pharmacological methods in urethane-anesthetised mice. Our results demonstrated that 4 Hz PF stimulation induced presynaptic LTD of PF-PC synaptic transmission in the intact cerebellar cortex in living mice. The PF-PC presynaptic LTD was attenuated by either the N-methyl-D-aspartate receptor antagonist, D-aminophosphonovaleric acid, or the group 1 metabotropic glutamate receptor antagonist, JNJ16259685, and was abolished by combined D-aminophosphonovaleric acid and JNJ16259685, but enhanced by inhibition of nitric oxide synthase. Blockade of cannabinoid type 1 receptor activity abolished the PF-PC LTD and revealed a presynaptic PF-PC LTP. These data indicate that both endocannabinoids and nitric oxide synthase are involved in the 4 Hz stimulation-induced PF-PC presynaptic plasticity, but the endocannabinoid-dependent PF-PC presynaptic LTD masked the nitric oxide-mediated PF-PC presynaptic LTP in the cerebellar cortex in urethane-anesthetised mice.

N-methyl-d-aspartate inhibits cerebellar Purkinje cell activity via the excitation of molecular layer interneurons under in vivo conditions in mice.

N-methyl-d-aspartate (NMDA) receptors play a key role in synaptic transmission, and are widely expressed on the membrane of granule cells, parallel fibers, and molecular layer interneurons (MLIs) in the cerebellar cortex of mammals. In cerebellar slices, activation of NMDA receptors increases inhibitory postsynaptic currents (IPSCs) of Purkinje cells (PCs). However, the effects of NMDA on the cerebellar network under in vivo conditions are currently unclear. In the present study, we examined the effects of NMDA on the spontaneous activity of PCs and MLIs in urethane-anesthetized mice by electrophysiological, pharmacological, and juxtacellular labeling methods. Our results revealed that cerebellar surface application of NMDA (5-200µM) reduced the PC simple spike (SS) firing rate in a dose-dependent manner. Application of GABAA receptor antagonist, SR95531 (20µM) abolished NMDA-induced inhibition of PCs spontaneous activity, and revealed NMDA-induced excitation of cerebellar PCs. NMDA receptor antagonist, DAP-V (250µM) did not affect the mean frequency of SS firing, but the SS firing rate of PCs became more regular than the control. In addition, NMDA increased the spike firing of both basket-type and stellate-type MLIs. Overall, these results indicated that NMDA-induced excitation of MLIs at the cerebellar surface may inhibit PC activity. Thus, NMDA receptors of MLIs may play a key role in regulating the spontaneous activity of PCs, and in information transmission and integration in cerebellar cortex.

Structural and mechanistic insights into collagen degradation by a bacterial collagenolytic serine protease in the subtilisin family.

A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP-01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP-01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate-binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP-01 displayed a non-strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1' site in collagen. His211 is a key residue for the P1-basic-residue preference of MCP-01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms.

Naphthol radical couplings determine structural features and enantiomeric excess of dalesconols in Daldinia eschscholzii.

Understanding how simple molecules are pieced together in organisms may aid biotechnological manipulation and synthetic approaches to complex natural products. The mantis-associated fungus Daldinia eschscholzii IFB-TL01 produces the unusually structured immunosuppressants (±)-dalesconols A and B, along with their congener (±)-dalesconol C, with the (-)-enantiomers in excess. Here we report that these structural and stereochemical peculiarities of dalesconols A-C are a result of promiscuous and atropselective couplings of radicals derived from 1,3,6,8-tetrahydroxynaphthalene, 1,3,8-trihydroxynaphthalene and 1,8-dihydroxynaphthalene. The observed (-)-enantiomeric excess is found to depend on the dominance of particular conformers of naphthol dimer intermediates, which are ligands of laccase.

Tenderization effect of cold-adapted collagenolytic protease MCP-01 on beef meat at low temperature and its mechanism.

The enzymes currently used to increase meat tenderness are all mesophilic or thermophilic proteases. This study provides insight into the tenderization effect and the mechanism of a cold-adapted collagenolytic enzyme MCP-01 on beef meat at low temperatures. MCP-01 (10 U of caseinolytic activity) reduced the meat shear force by 23% and increased the relative myofibrillar fragmentation index of the meat by 91.7% at 4 °C, and it also kept the fresh colour and moisture of the meat. Compared to the commercially used tenderizers papain and bromelain, MCP-01 showed a unique tenderization mechanism. MCP-01 had a strong selectivity for degrading collagen at 4 °C, showed a distinct digestion pattern on the myofibrillar proteins, and had a different disruption pattern on the muscle fibres under scanning electron micrograph. These results suggest that the cold-adapted collagenolytic protease MCP-01 may be promising for use as a meat tenderizer at low and moderate temperatures.

Mechanistic insight into the function of the C-terminal PKD domain of the collagenolytic serine protease deseasin MCP-01 from deep sea Pseudoalteromonas sp. SM9913: binding of the PKD domain to collagen results in collagen swelling but does not unwind the collagen triple helix.

Deseasin MCP-01 is a bacterial collagenolytic serine protease. Its catalytic domain alone can degrade collagen, and its C-terminal PKD domain is a collagen-binding domain (CBD) that can improve the collagenolytic efficiency of the catalytic domain by an unknown mechanism. Here, scanning electron microscopy (SEM), atomic force microscopy (AFM), zeta potential, and circular dichroism spectroscopy were used to clarify the functional mechanism of the PKD domain in MCP-01 collagenolysis. The PKD domain observably swelled insoluble collagen. Its collagen-swelling ability and its improvement to the collagenolysis of the catalytic domain are both temperature-dependent. SEM observation showed the PKD domain swelled collagen fascicles with an increase of their diameter from 5.3 mum to 8.8 mum after 1 h of treatment, and the fibrils forming the fascicles were dispersed. AFM observation directly showed that the PKD domain bound collagen, swelled the microfibrils, and exposed the monomers. The PKD mutant W36A neither bound collagen nor disturbed its structure. Zeta potential results demonstrated that PKD treatment increased the net positive charges of the collagen surface. PKD treatment caused no change in the content or the thermostability of the collagen triple helix. Furthermore, the PKD-treated collagen could not be degraded by gelatinase. Therefore, though the triple helix monomers were exposed, the PKD domain could not unwind the collagen triple helix. Our study reveals the functional mechanism of the PKD domain of the collagenolytic serine protease MCP-01 in collagen degradation, which is distinct from that of the CBDs of mammalian matrix metalloproteases.

Determination of ofloxacin and gatifloxacin by mixed micelle-mediated cloud point extraction-fluorimetry combined methodology.

A cloud point extraction process using mixed micelle of the anionic surfactant sodium dodecyl sulfate (SDS) and the non-ionic surfactant polyoxyethylene(7.5)nonylphenylether (PONPE 7.5) to extract two fluoroquinolone antimicrobial agents, ofloxacin and gatifloxacin, from aqueous media was investigated. The method is based on the mixed micelle-mediated extraction of fluoroquinolones in the presence of NaCl as an inducing agent in phase separation, followed by spectrofluorimetric determination. The effect of different variables such as pH, PONPE7.5 concentration, SDS concentration, NaCl concentration, cloud point temperature, and time was investigated, and optimum conditions were established. At optimum conditions, the rectilinear calibration graphs were obtained in the concentration range of 0.1-150 and 0.1-250ngmL(-1) for ofloxacin and gatifloxacin, and the limits of detection were 0.04 and 0.06ngmL(-1), respectively. The proposed procedure was applied successfully for the detection of the investigated drugs in their pharmaceutical dosage forms, in spiked plasma, spiked urine, and urine samples, with good precision and accuracy.

Ecological function of myroilysin, a novel bacterial M12 metalloprotease with elastinolytic activity and a synergistic role in collagen hydrolysis, in biodegradation of deep-sea high-molecular-weight organic nitrogen.

Nearly all high-molecular-weight (HMW) dissolved organic nitrogen and part of the particulate organic nitrogen in the deep sea are present in hydrolysis-resistant amides, and so far the mechanisms of biodegradation of these types of nitrogen have not been resolved. The M12 family is the second largest family in subclan MA(M) of Zn-containing metalloproteases and includes most enzymes from animals and only one enzyme (flavastacin) from a human-pathogenic bacterium (Flavobacterium meningosepticum). Here, we characterized the novel M12 protease myroilysin with elastinolytic activity and collagen-swelling ability from the newly described deep-sea bacterium Myroides profundi D25. Myroilysin is a monomer enzyme with 205 amino acid residues and a molecular mass of 22,936 Da. It has the same conserved residues at the four zinc ligands as astacin and very low levels of identity (

Hydrolysis of insoluble collagen by deseasin MCP-01 from deep-sea Pseudoalteromonas sp. SM9913: collagenolytic characters, collagen-binding ability of C-terminal polycystic kidney disease domain, and implication for its novel role in deep-sea sedimentary particulate organic nitrogen degradation.

Collagens are the most abundant proteins in marine animals and their degradation is important for the recycling of marine nitrogen. However, it is rather unclear how marine collagens are degraded because few marine collagenolytic proteases are studied in detail. Deseasins are a new type of multidomain subtilases. Here, the collagenolytic activity of deseasin MCP-01, the type example of deseasins, was studied. MCP-01 had broad substrate specificity to various type collagens from terrestrial and marine animals. It completely decomposed insoluble collagen into soluble peptides and amino acids, and was more prone to degrade marine collagen than terrestrial collagen. Thirty-seven cleavage sites of MCP-01 on bovine collagen chains were elucidated, showing the cleavage is various but specific. As the main extracellular cold-adapted protease from deep-sea bacterium Pseudoalteromonas sp. SM9913, MCP-01 displayed high activity at low temperature and alkaline range. Our data also showed that the C-terminal polycystic kidney disease (PKD) domain of MCP-01 was able to bind insoluble collagen and facilitate the insoluble collagen digestion by MCP-01. Site-directed mutagenesis demonstrated that Trp-36 of the PKD domain played a key role in its binding to insoluble collagen. It is the first time that the structure and function of a marine collagenolytic protease, deseasin MCP-01, has been studied in detail. Moreover, the PKD domain was experimentally proven to bind to insoluble protein for the first time. These results imply that MCP-01 would play an important role in the degradation of deep-sea sedimentary particulate organic nitrogen.