Isolation And Taxonomic Identification Of Marine Bacteria And Evaluation Of The Potential Of Marine Bacterial Protease In Tissue Engineering

The ocean is one of the most important ecosystems on the earth that contains various and aboudant organisms,offering a great potential for the discovery of new resources.In order to adapt to marine environments(i.e.low temperature,high salt and high pressure),marine bacteria have evolved a variety of physiological mechanisms.Novel enzymes with wide range of enzymatic activities have been discovered from marine bacteria.Many marine-derived active proteases can significantly clear necrotic tissues,blood crust and accelerate wound healing when used as pharmaceutical components.Therefore,the marine proteases can offer novel biocatalysts for biotechnology and great potentials for medical application.Tissue engineering and regenerative medicine is an exciting research area that aims at regenerating alternatives for transplantation.Biomaterials can be used as scaffolds to provide three-dimensional(3D)templates and synthetic extracellular matrix environments for tissue regeneration.Compared with synthetic and biomimetic materials,biologically produced materials are usually considered as the first choice for tissue scaffolds owing to their low toxicity and high cell compatibility.Collagen is the most abundant protein within the mammalian extracellular matrix(ECM).The ECM as a native scaffold prepared by decellularization of the natural tissue are mostly close to the natural tissue in composition and texture,so the ECM scaffold is more preferably used as a regeneration template for damaged tissue.Chemical,physical,and enzymatic approaches have been employed for decellularization of tissue and organ.Enzymes used in tissue decellularization include trypsin,dispase and nucleases.However,trypsin and dispase can disrupt ECM ultrastructure and remove ECM constituents such as collagen and fibronectin,and nucleases may hardly difficult to remove cells from the tissue and invoke immune respones.Therefore,the development of novel proteases that can be applied to tissue decellularization is important for the preparation of ECM scaffolds with better performance.Tissue regeneration requires transfer intercellular and intracellular communications between different cell populations and organ systems,a process that growth factors play a crucial role.Growth factors are also known as intercellular signaling molecules,which promote cell proliferation,migration and differentiation and stimulate matrix formation and reconstruction of damaged tissue.Recently,synthetic,biomimetic and chemical schemes are being developed to enable in situ cross-linking and release of incorporated growth factors and thus enhance properties of ECM-growth factor composite scaffolds.Therefore,the importance of exogenous delivery of these molecules in tissue engineering is unsurprising,considering their importance for tissue engineering and regenerative medicine.In order to obtain new proteases,protease-producing marine bacteria was first isolated from sea samples and identified in this study.Then,we focus on two marine protease-producing bacteria isolated from deep sea sediments,Myroides profundi D25 and Pseudoalteromonas sp.SM9913.The strain D25 produced a novel elastinolytic protease myroilysin,which could not hydrolysis collagen but had strong collagen swelling ability.Strain SM9913 produced a collagenolytic protease MCP-01.MCP-01 was a multi-domain protein composed of a catalytic domain(CATD),a linker,a P-proprotein domain and a ploycystic kidney disease(PKD)domain.Our previous study demonstrated that the PKD domain at its C-terminus could bind collagen.In this study,we explored the potentials of myroilysin and PKD domain in tissue engineering.Take advantage of the efficient collagen swelling ability of myroilysin,we prepared ECM by using myroilysin.On the other hand,based on the collagen binding ability of PKD domain,a novel composite scaffold was prepared where collagen matrices bioactivated with vascular endothelial growth factor(VEGF)and transforming growth factor(TGF).The ECM scaffolds bearing with growth factors were characterized and its application potential was assessed in animal models.1.Isolation and taxonomic identification of marine bacteriaIn total 142 marine bacterial strains belonging to 73 species were isolated from sea water and sea sediment samples,leading to the discovery of 6 potential novel species.Three marine protease-producing bacteria were identified by polyphasic taxonomic analysis.Base on the polyphasic data,strain SM1211T was considered as a representive of a novel specie in a new genus within the family Rhodobacteraceae,for which the name Puniceibacterium antarcticum gen.nov.,sp.nov.was proposed.Strain SM1212T was represented a novel species in a new genus in the family Flavobacteriaceae,for which the name Arcticiflavibacter luteus gen.nov.,sp.nov.was proposed.Strain SM1216T was represented a novel species in the genus Algimonas,for which the name Algimonas arctica sp.nov.was proposed.Isolation of these strains laid the foundation for their further research and potential application.2.Preparation of porcine acellular dermal matrix(PADM)by using marine bacterial protease myroilysin and its evaluationStudies on the mechanism underlying collagen swelling by myroilysin treatment indicated that myroilysin did not destroy the basic structure of collagen but cleaved the cross-linking between collagen fibers by degrading proteoglycan.The native ECM scaffold of PADM was prepared by myroilysin treatment.Compared with the PADM treated with SDS and trypsin,the PADM prepared by myroilysin had higher porosity,higher water absorption,higher in vitro degradation rate,and maintained moderate mechanical strength.In addition,the residual cells and DNA were removed more thoroughly than those treated with SDS and trypsin.There were less immunogenic molecular,but more bioactive cytokines,in myroilysin-treated PADM.Moreover,comprehensive evaluation indicated that the PADM had the characteristics of easy sterilization,nontoxicity and good biocompatibility.These results suggest that the PADM prepared by myroilysin treatment meet the requirement of ECM scaffolds in tissue engineering and regenerative medicine.3.Preparation and evaluation of recombinant growth factor fusion proteins with PKD domain in marine bacterial protease MCP-01 and structural basis for collagen binding of PKD domainPrevious studies indicated that the PKD domain of MCP-01 had the ability of collagen binding without destroying the triple-helix structure of collagen.Two fusion proteins,PKD-VEGF and PKD-TGF,with fusion of the PKD domain and growth factors were constructed and expressed in the insect cell expression system sf9.The fusion proteins were investigated for their stability,collagen binding ability and biological activity.The results indicated that the fusion protein had low degradation rate and capability of binding collagen.Moreover,both PKD-VEGF and PKD-TGF could induce the proliferation and differentiation of human umbilical vein endothelial cells(HUVECs),suggesting that the enhanced ECM-growth factor composite represents a promising growth factor delivery strategy for tissue regeneration.In order to investigate how the fusion proteins PKD-VEGF and PKD-TGF bound on the surface of collagen scaffolds,the crystal structure of PKD domain was analyzed.The mechanism of PKD domain binding was deduced by molecular docking experiment.The results demonstrated that the ?-sandwich structure of the PDK domain is divided into two sheets of three ?-strands each.The collagen binding ability of PKD domain was mainly relied on the electrostatic interaction between PKD domain and collagen.PKD-VEGF and PKD-TGF exhibited a considerable stability when exposed to the microenvironment of scaffolds,which provides an idea for the fixation of the active factor on the surface of the scaffold.4.Preparation of full-thickness wound animal model and evaluation of composite scaffolds binding with growth factorA novel composite scaffold,PADM comprised of PKD-VEGF and/or PKD-TGF,was prepared.In order to evaluate its clinical potentiality,in vitro and in vivo experiments were carried out on the new composite scaffolds.In vitro assays indicated that composite scaffolds not only provided 3D space for cell growth,but also induced the activity of cell proliferation and differentiation.Further,full-thickness dermis defects of 2 cm in diameter were created on the dorsal part of rats and the composite scaffolds were implanted in the dermis defect sites.The wounds were inspected on their morphological and histological changes.The studies indicated that the composite scaffold could accelerate reconstruction and reparation of wound tissue and promote angiogenesis.Taken together,our study indicates that the composite scaffold,PADM binding with PKD-VEGF and/or PKD-TGF,can potentially be used in diverse tissue engineering,regenerative medicine,and drug delivery systems.In this study,marine bacterial strains were isolated from sea water and sea sediment samples and three marine protease-producing bacteria were identified by polyphasic taxonomic analysis.The PADM prepared by myroilysin treatment meet the requirement of ECM scaffolds in tissue engineering and regenerative medicine.The collagen binding ability of PKD domain in marine bacterial protease MCP-01 was used to prepare the fusion proteins of PKD-VEGF and PKD-TGF.Then the the composite scaffold,PADM binding with PKD-VEGF and/or PKD-TGF,can potentially be used in clinical application.From the above,the marine contains various and aboudant organisms,offering a great potential for the discovery of new resources,can support novel biocatalysts for biotechnology and great potentials for medical application.