Collagenolysis Mechanism Of Deep-sea Cold-adapted Protease MCP-01 And Its Potential In Meat Tenderization

Degradation of organic nitrogen is an important part of global nitrogen cycling. In the ocean, High-Molecular-Weight Organic Nitrogen (HMWON) produced by organisms in the seawater settles and accumulates in the sediment in the form of particulate organic nitrogen (PON). Most of PON will be degraded into dissolved organic nitrogen (DON) by microorganisms, and then participates into the nitrogen cycling through ammonium regeneration, nitrification and denitrification in the sediment. Thus, the recycling of PON in deep-sea sediment would be a non-negligible part of ocean nitrogen cycling. Recent studies indicate that a significant fraction of PON is composed of amide N, including proteins and peptides. Collagens and elastins are insoluble proteins, which may be important components of sedimental PON. Collagens are polymers made up of three helically polypeptide fibrils. Because of the rigid structure, collagens are resistant to most proteases except for a limited number of collagenolytic proteases. Of all the collagenolytic proteases, mammalian matrix metalloproteinases (MMP) have been well-investigated. The reports of collagenolytic proteases from microorganisms are relatively fewer. Moreover, most of bacterial collagenolytic proteases are pathogenic and terrestrial. So far, the collagenolysis mechanisms of the ocean collagenolytic proteases are unclear. Thus, research into the function and mechanism of marine collagenases will provide important implications for deep-sea nitrogen cycling.The cold-adapted protease MCP-01 is the main protease secreted by the deep-sea cold-adapted bacterium Pseudoalteromonas sp. SM9913. The precursor of MCP-01 contains 835 amino acid residues and belongs to the S8A subfamily of serine proteases. Most mature subtilases of S8 are monodomain proteins. However, mature MCP-01 is a multidomain protein composed of a catalytic domain (CD), a linker, a P-proprotein domain and a polycystic kidney disease (PKD) domain. MCP-01 and the other 12 MCP-01-like subtilases delivered in databases are termed as deseasins. Here, with collagen as the substrate, the biochemical and enzymatic characteristics, the catalytic mechanism with a structural basis and the application of MCP-01 were studied. The results are as following:1. Expression and purification of recombinant MCP-01 and its domains in Escherichia coliMCP-01 is a typical cold-adapted protease. Due to its flexible structure, MCP-01 is very unstable at moderate or high temperatures. So it is difficult for MCP-01 to be exogenously expressed by common methods. The full-length mcp-01 gene was cloned into the expression vector pET-22b, and then was transformed into E. coli BL21 (DE3). The expression level of the full-length mcp-01 gene is low in E. coli, probably due to its multidomain structure and rare codons. Recombinant MCP-01 exists as a precursor without activity in the cytoplasm of E. coli. When the host was induced in 50% LB medium at 15?for 8 days, the active form of MCP-01 was detected in the extra cellular medium, with a production of 19.6 U/ml.Efficient methods for the expression and purification of CD and PKD domain were established. The active recombinant CD was secreted into the extra cellular medium when E. coli was induced with 0.05 mM IPTG in 50% LB medium at 15?for 7 days. The active recombinant CD in the culture was purified CD by using ion exchange chromatography and gel filtration chromatography. Recombinant PKD was expressed in the cytoplasm of E. coli by induce of 0.5 mM IPTG at 15?for 48 h. The recombinant PKD was purified by GST affinity chromatography. The molecular masses of the recombinant CD and PKD were determined using mass spectroscopy, which were consistent with those predicted from the sequences. The above results laid a foundation for further research of the catalytic properties and mechanism of MCP-01.2. Collagenolytic characteristics of MCP-01To determine the substrate specificity, the activities of MCP-01 toward some proteins and synthetic peptides were analyzed, and were compared with those of the collagenase from Clostridium histolyiicum. The result showed that deseasin MCP-01 is a collagenolytic serine protease, capable of digesting various collagens from both terrestrial and marine animals. For the insoluble collagens from different sources, MCP-01 had higher activity to fish collagen and lower activity to bovine collagen than the collagenase from C. histolyticum. For soluble collagens, the order of preferred substrate for MCP-01 was gelatin>acid-dissolved type I collagen>type IV>collagen>type II collagen. To explain the collagenolytic mechanism of MCP-01, the digestion pattern of bovine-insoluble type I collagen fiber by MCP-01 was analyzed by SDS-PAGE and N-terminal sequencing. It was shown that the insoluble collagen fiber was digested into dissolved peptides smaller than 100 kDa. According to the N-terminal sequences of some released peptides,37 possible cleavage sites of MCP-01 on bovine collagen chains were deduced, suggesting that MCP-01 had various but specific cleavage sites on type I collagen fiber, which is quite deferent from that of MMP. It is for the first time that the cleavage sites of a serine collagenolytic protease on insoluble collagens were elucidated, to our knowledge.With bovine-insoluble type I collagen fiber as substrate, MCP-01 was most active at 60?, and kept 12.4% of the highest activity at 0?. To our knowledge, MCP-01 is the first reported cold-adapted collagenolytic serine protease. MCP-01 had an optimum at pH 9.0, and over 60% activity remained between pH 6.0 to pH 10.0. Ca2+ markedly increased the enzyme activity of MCP-01, while Zn2+, Ni2+, Cu2+, and Fe2+ severely inhibited the enzyme activity.3. Collagen-binding ability of the C-terminal PKD domain of MCP-01 and its improvement for collagen degradation by the CDPKD domain is an 80-90 amino-acid module originally found in human PKD1 gene encoding a cell-surface glycoprotein polycystin-1, and later is also found in many surface layer proteins of archaebacteria. PKD domain widely lies in chitinases, cellulases, collagenases, and proteases, suggesting that it may play an important role in biopolymer degradation. The structures of three PKD domains have been solved, which show that, though their sequences are different, they all adopt a?helix fold and a conserved sequence area with two Trp packing in the hydrophobic core. However, the function of the PKD domain in proteases is still unknown.Our result showed that the recombinant CD could degrade insoluble collagen fiber with a lower efficiency (about 60%) than MCP-01. Moreover, the collagen-degraded efficiency of the recombinant CD was increased when the insoluble collagen fiber was pretreated by the recombinant PKD domain. These results suggest that the PKD domain plays an important role in the collagen degradation of MCP-01. To evaluate the function of the PKD domain in the insoluble collagen fiber digestion of MCP-01, the PKD domain were expressed in E. coli as an EGFP fusion protein (PKD-EGFP) and purified with His affinity chromatography. Then its binding ability to the insoluble collagen fiber was assayed by a spectrofluorometer with EGFP as a control. It was shown that PKD-EGFP displayed a significant binding ability to the insoluble collagen fiber, whereas EGFP had no binding ability, suggesting that the PKD domain of MCP-01 has binding ability to the insoluble collagen fiber. To clarify the important amino acid residues in PKD domain for collagen binding, the PKD domain of MCP-01 were aligned with the other reported collagen binding domains, and the alignment revealed 7 completely conserved amino acids. Then site-directed mutagenesis of the amino acids was introduced. Results showed that the mutation of Trp-36 to Ala-36 caused a significant reduction of the collagen binding ability of the PKD domain. A comparison of the CD spectra of the PKD domain and W36A indicated that change of trp36 caused little structural changes in the PKD domain. Thus, Trp-36 plays a key role in the binding of the PKD domain of MCP-01 to collagen fiber.Further results showed that PKD domain observably swelled insoluble collagen. Its collagen swelling ability is temperature-dependent. SEM observation showed the PKD domain swelled collagen fascicles with an increase of their diameter from5.3?m to 8.8?m, afterlh 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. Zeta potential results demonstrated that PKD treatment increased the net positive charges of the collagen surface, indicating that, after PKD treatment, more aggregate structures, such as fibrils, microfibrils, and monomers, are exposed. Circular dichroism spectroscopy showed that PKD treatment caused no change in the content or the thermostability of the collagen triple helix. Thus, PKD domain of MCP-01 can cause collagen swelling to expose more cleavage sites for the catalytic domain, but it cannot unwind the collagen triple helix.Based on our previous and present results, a collagenolysis model of serine protease MCP-01 is proposed, which is quite different from that of MMP.4. The crystal structure of the CD of MCP-01 and its collagenolytic mechanismOf all the collagenolytic proteases, mammalian MMP have been well-investigated. Structural studies of MMP1 indicate that the substrate-binding groove of its active site is only 5 A wide, which therefore, unable to accommodate the intact collagen triple helix (3,000 A in length and 15 A in diameter). The Hxp domain of MMP1 binds to and locally unwinds the triple helix of collagen so that each peptide of collagen may fit into the active site binding groove for the hydrolyzation of the peptide bonds. However, the catalytic mechanism of bacteria collagenases is rarely known.Different from the MMP catalytic domain, the CD of MCP-01 can independently degrade insoluble collagen. But many other subtilisins, such as Carlsberg, can not hydrolyze collagen. To gain insights into the structural basis of its collgaenolytic mechanisms, the recombinant CD was crystallized by X-ray at a resolution of 1.76 A, which represents the first reported serine collagenolytic protease structure, to our knowledge. It was shown that the CD had the same three-dimensional framework with Carlsberg. But the substrate-binding groove of the CD was 17 A, much larger than that of Carlsberg (13 A). Thus, CD can accommodate the triple-helical molecular of collagen with a diameter of 15 A. Triple-helical peptide (THP) has the same structure with native collagens. A proposed model of the CD-THP complex was obtained using docking algorithm. The mode indicates that the triple helix can be easily bound in the groove of CD without structural clash. The structure and amno acid compositions of the substrate-binding groove of the CD and Carlsberg were analyzed. It was shown that three loops are composed of the substrate-binding pocket of the enzymes and may have direct function in the binding of collagens. The comparison showed that some amino acid residues in these loops of the CD and Carlsberg were different. Acidic and aromatic residues frequently appear on the loops of CD, which are insteaded by aliphatic residues in Carlsberg. Moreover, these acidic and aromatic residues are conserved in the loops of serine collagenolytic proteases. Therefore, these acidic and aromatic residues may be the important sites in the CD in collagen binding and degradation. Site-directed mutagenesis of the residues was introduced, and the result showed that mutation of D140, D202, P210, P256, F177 and E102 led to significant reduce of the collagenolytic activity of CD, which indicates that these residues are vital sites for the interaction of the CD with collgens.The above results give an insight into the collagenolytic mechanism of MCP-01 and provide an indication for the functional evolution in collagen degradation of serine proteases.All the above results give an insight into the collagenolytic mechanism of MCP-01. MCP-01 is a represent of deseasins that may be widely present in deep-sea sediment. Collagen is an important component of deep-sea PON. So the results also give an indication of the function of deseains in the degradation of deep-sea HMWON, and laid a foundation for the development of new proteases.5. Meat tenderization ability of MCP-01 and the involved mechanism.Collagen occupies 80% of the connective tissue and is considered to be a significant factor for meat texture. The above results showed that MCP-01 is a cold-adapted collagenolytic protease and is capable of digesting various collagens under low and room temperatures, suggesting that it may be useful in meat tenderization. Taking beaf meat as a sample, we evaluated the meat-tenderizing ability of MCP-01 and the involved mechanism in comparison with the commercially used meat tenderizers, Papain and Bromelain. It was shown that MCP-01 treatment could significantly reduce the shear force of meat at 4?. After MCP-01 treatment, the meat kept its fresh color and shape, and a low water loss. In comparision, the meat treated with Papain or Bromealain showed a darker color, a changed shape and a high water loss. Further research showed that MCP-01 had a stronger activity than Papain and Bromelain in selectively degrading collagen at 4?, suggesting that MCP-01 can destruct collagens in the connective tissue of meat during tenderization. The digestion pattern of MCP-01 on meat myofibrillar proteins was also different from that of Papain or Bromelain. MCP-01 only degraded some proteins including myosin heavy chain, and could not degrade the actin. In comparision, Papain and Bromelain unselectively degraded all the myofibrillar proteins into peptides lower than 30 kDa. SEM observation showed that the surface of the meat treated with MCP-01 was quite different from that treated with Papain or Bromelain, which may result from their different tenderization mechanisms. The above results indicate the promising use of MCP-01 as a new meat tenderizer with a distinctive tenderization mechanism from commercially used tenderizer.