| The ocean, covering more than71%of the earth’s surface, is one of the most important ecosystems on the earth. There are various and abundant microorganisms in the ocean, which offer a great potential for the discovery of new resources. Since deep sea sediments contain at least25%of the global ocean nitrogen burial, degradation and transformation of organic nitrogen mediated by extracellular enzymes from deep sea sediment bacteria may have significant effect on the global biogeochemical cycles. High molecular weight organic nitrogen is mostly present as biological and chemical hydrolysis-resistant amides on the deep sea floor. Collagen is the most abundant fibrous protein in all higher organisms, including marine animals. Due to its tight and complicated structure, collagen is resistant to common proteases and can only be degraded by a limited number of collagenolytic proteases. Collagen is therefore an important component of deep sea sedimentary particulate organic nitrogen (PON). Of all the collagenolytic proteases, mammalian matrix metalloproteinases (MMPs) have been well investigated and reports on collagenolytic proteases secreted by microorganisms are relatively fewer. Most of bacterial collagenases so far reported are pathogenic and terrestrial, and only several ones are oceanic. Thus, the degradation mechanisms of the ocean collagen are still rather unclear. Research into the function and mechanism of marine collagenases will provide important implications for global deep sea nitrogen cycling.The protease deseasin MCP-01belonging to the S8family of serine proteases is secreted by the deep sea cold-adapted bacterium Pseudoalteromonas sp. SM9913. Mature MCP-01is a multidomain protein composed of a catalytic domain (CATD), a linker, a P-proprotein domain and a ploycystic kidney disease (PKD) domain. The CATD alone can degrade type Ⅰ collagen with relatively lower efficiency than intact MCP-01. The PKD domain at its C-terminus is responsible for binding and swelling of collagen, but it cannot unwind the collagen triple helix. In this dissertation, structural analysis and biochemical experiments were performed to study the recognition and degradation mechanism of MCP-01on collagen.Myroides profundi D25is a protease-secreting bacterium isolated from the deep sea sediment of the southern Okinawa Trough at a water depth of1245m. The D25strain produces a novel elastinolytic protease of M12family, myroilysin, which cannot hydrolysis collagen but has strong collagen swelling ability and cooperates with collagenase in collagen hydrolysis. Here, a novel collagenolytic protease secreted by strain D25, designated myroicolsin, was purified from fermentation medium of strain D25and its enzymatic characteristics, gene sequence, function of domains and degradation mechanisms to collagen were studied. Moreover, the synergistic action between elastase myroilysin and collagenase myroicolsin and the pilot-scaled fermentation teechnology of myroilysin were further studied. The results are as following:(1) Structural and mechanistic insights into collagen recognition and degradation by deseasin MCP-01.In order to study the collagen recognition and degradation mechanism of MCP-01, the time-dependent progressive disintegration of collagen fiber by MCP-01, the CATD and PKD domain was observed by atomic force microscopy. Biochemical analysis was confirmed that MCP-01and the CATD, in the collagen fibers degradation process, progressively release single fibrils from collagen fibers and collagen monomers or monomer fragments from fibrils mainly by hydrolyzing the proteoglycans that interdigitate with fibrils and the telopeptides within or between fibrils. Collagen monomers are subsequently released and further degraded. To further study how the CATD binds and degrades collagen molecules, the crystal structure of the CATD (Serl-Phe333) was determined. Subtilisin Carlsberg is the prototype of the S8family, which shows a similar topology to CATD. There have been no reports that subtilisin Carlsberg is capable of degrading collagen. Structural analysis and mutational assays indicated that an enlarged substrate-binding pocket, mainly composed of loops7,9and11, was necessary for the CATD in collagen recognition. The acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for the binding of collagen which has a positively charged, hydrophobic surface.Analysis of the cleavage pattern of MCP-01on triple-helical type I collagen based on the cleavage sites was determined in our previous and present studies indicated that it displayed a non-strict preference for peptide bonds with Pro at the P1site and/or Gly at the PI’site in collagen. Moreover, MCP-01showed a different specificity from other S8proteases, tending to cleave the peptide bonds with basic residues (Lys and Arg) at the P1site in collagen. Structural analysis and mutation assays indicated that His211in the S1pocket is a key residue for the preference of MCP-01for basic residues at the P1site. In summary, our study gives structural and mechanistic insights into collagen degradation of an S8collagenolytic protease MCP-01. Because the S8collagenolytic proteases are secreted by both environmental and pathogenic microorganisms, our results are helpful in studying the environmental organic nitrogen degradation mechanism and in developing therapeutics for diseases with S8collagenolytic proteases as pathogenic factors. In addition, because deseasins (MCP-01-like proteases) are most likely widespread in marine sediment, this study sheds light on the degradation mechanism of deseasins for marine sedimentary PON.(2) Purification, gene cloning, characteristics and domain functions of collagenolytic protease myroicolsin.A protein was purified from the fermentation broth of strain D25through ammonium sulfate precipitation, DEAE-Sepharose Fast Flow chromatography and gel filtration chromatography, which was designated as myroicolsin. Substrate specificity analysis showed that myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. With insoluble bovine type I collagen fiber as the substrate, the optimal temperature of myroicolsin was60℃, and10%of the highest activity was retained at0℃. Myroicolsin displayed the greatest collagenolytic activity at pH8.5, more than60%activity was retained between pH7.0and9.5. Myroicolsin showed the highest activity in0.5M NaCl, and more than50%of the highest activity was retained in4M NaCl, showing its high salt tolerance. These results indicated that myroicolsin has evolved over time to accommodate itself to low temperature, slightly alkaline and high salt concentration of marine environment, fully participating in PON hydrolysis process in the deep-sea ecosystem. In addition,4mM Ca2+markedly increased the activity of myroicolsin. The activity of myroicolsin was almost completely abolished by the serine protease inhibitor PMSF, suggesting that myroicolsin is probably a serine proteases family.Based on the N-terminal amino acid sequence of myroicolsin and the conserved sequence in the catalytic domains of serine proteases, the complete gene of myroicolsin was cloned by a combination of PCR and thermal asymmetric interlaced PCR. The open reading frame of the whole gene is2040bp and was deduced to encode a protease precursor of679amino acid residues. The nucleotide sequence was submitted to the GenBankTM with accession number JF514144. Sequence analysis showed that myroicolsin is a novel protease of the S8family with low identity (<30%) to characterized peptidases. The precursor of myroicolsin contains a signal sequence, an N-propeptide, a catalytic domain, a linker, a β-jelly roll domain, and a C-pro-secre-tail. Based on the molecular mass of the purified myroicolsin and its N-terminal sequence, it could be deduced that the signal sequence, the N-propeptide, and the C-pro-secre-tail were cleaved spontaneously during enzyme maturation. The mature form of myroicolsin consists of507residues, including the catalytic domain, the linker, and the β-jelly roll domain. Truncated mutation assay showed that the C-pro-secre-tail is essential for the cleavage of the N-propeptide but not for the secretion and folding of the protein, the linker may be required for protein folding and the β-jelly roll domain is not required for folding and maturation. Our collagen binding assay showed that the β-jelly roll domain showed little binding ability to insoluble collagen fiber at0℃or25℃. These results suggested that myroicolsin has no C-terminal collagen-binding domain, unlike other characterized collagenolytic proteases. Therefore, it seems that myroicolsin may have a special collagen degradation mechanism.(3) Study of collagenolytic mechanism of myroicolsin.Our scanning electron microscopy and atomic force microscopy observations showed that myroicolsin released fibrils from collagen fiber and that the fibrils were further degraded into collagen monomers. Biochemical assays indicated that this stepwise degradation of collagen fiber was achieved through the hydrolysis of the proteoglycans and telopeptides in collagen fibers and fibrils. Circular dichroism spectra revealed that myroicolsin could destroy the triple helix structure of the collagen monomer, suggesting that the polypeptide chains in monomers could be released and further hydrolyzed into peptides and amino acids. The cleavage pattern of myroicolsin on collagen polypeptide chains were analyzed by high performance liquid chromatography and mass spectrometry. Myroicolsin showed different cleavage patterns against native collagen and denatured collagen. In native collagen, the P1position is often occupied by Gly, Arg, Pro, or Phe, and the P1’ position is almost always occupied by Gly. In denatured collagen, the P1position is always a basic residue (Lys or Arg), and the P1’ position is still Gly. Among serine proteases, subtilisin-like proteases are usually nonspecific peptidases with a preference to cleave after hydrophobic residues, while trypsin-like proteases always cleave the peptide bonds with a basic residue at the P1site. Therefore, it seems that myroicolsin exhibits a mixed type of PI specificity, both trypsin-like and subtilisin-like. The bacterial collagenase MCP-01of S8family showed the same P1specificity as myroicolsin, which may be the common characteristic of the S8family collagenases.(4) A model for collagen degradation by S8bacterial collagenases.Collagenolytic proteases, MCP-01and myroicolsin, from deep sea sediment bacteria are both the members of the S8family. Our results showed that they have some similarities in collagen degradation. They both first break the interfibrillar proteoglycan bridges, leading to the disassembly of the tight structure of collagen fibers and the exposure of collagen fibrils. Then telopeptides within collagen fibrils and microfibrils are hydrolyzed by them, which accelerates the unfolding of the collagen structure. Thus, these collagenases gain access to collagen monomers. Finally, the fibrillar structure of collagen is completely destroyed, and collagen monomers are degraded into peptides and free amino acids. Based on the collagenolytic mechanism of MCP-01and myroicolsin, a mechanism model for collagen fiber degradation by the S8collagenases is proposed. The results provide important evidences for the substrate degradation mechanism of the S8collagenases, and give insight into nitrogen cycling in deep-sea sediment and also provide theoretical basis for the discovery and development of novel proteases.(4) Synergistic action of myroilysin and myroicolsin in collagenolytic process and the fermentation technology of myroilysin at pilot scale.Our previous research revealed that myroilysin, secreted by strain D25, displayed little collagenolytic activity but strong collagen swelling ability. Myroilysin could play a synergistic role with other terrigenous collagenase in collagen hydrolysis through swelling collagen. In this study, we found that strain D25could secrete a collagenolytic protease myroicolsin, and myroilysin also played a synergistic role with myroicolsin in collagen hydrolysis, which could increase the collagenolytic activity of myroicolsin. The results indicated that different proteases secreted by deep sea bacteria may have ecological effect to synergistically hydrolyed the PON in the deep sea sediment. In recent years, collagen has been considered as one of the most useful biomaterials in the biomedicine field. The strong collagen swelling ability of myroilysin showed that it could be an excellent collagen modifier, which has great application potential in the production of collagen medicine materials. In order to find out the key factors significantly affecting myroilysin production of strain D25, the relative significance of variables were investigated using single factor experiments. Finally, we determined the optimal conditions for myroilysin production in shake flask culture:4%bran,2%bean pulp,1%corn powder,0.4%Na2HPO4,0.03%KH2PO4,0.1%CaCl2, pH8.0; loading volume100mL/500mL;1%inoculum density,15℃,200rpm for72-84h. We further optimized the culture condition of myroilysin in fermenters of5L and200L, and the fermentation technology of myroilysin at polit scale was developed. Under the optimal conditions, the maximum myroilysin activity in fermentation broth reached1100U/mL, which provides a foundation for developing the application potential of myroilysin in the biomedicine field. |
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