Maturation process and characterization of a novel thermostable and halotolerant subtilisin-like protease with high collagenolytic but low gelatinolytic activity

2021 ◽  
Author(s):  
Kui Zhang ◽  
Qianqian Huang ◽  
Yu Li ◽  
Lanhua Liu ◽  
Xiao-Feng Tang ◽  
...  
Keyword(s):  
High Temperatures ◽  
Type I Collagen ◽  
Elevated Temperatures ◽  
Catalytic Domain ◽  
Ion Pairs ◽  
Gelatinolytic Activity ◽  
Type I ◽  
Fish Scale ◽  
Collagenolytic Proteases ◽  
Collagenolytic Protease

Enzymatic degradation of collagen is of great industrial and environmental significance; however, little is known about thermophile-derived collagenolytic proteases. Here, we report a novel collagenolytic protease (TSS) from thermophilic Brevibacillus sp. WF146. The TSS precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain, a β-jelly roll (βJR) domain, and a prepeptidase C-terminal (PPC) domain. The maturation of TSS involves a stepwise autoprocessing of the N-terminal propeptide and the PPC domain, and the βJR rather than the PPC domain is necessary for correct folding of the enzyme. Purified mature TSS displayed optimal activity at 70°C and pH 9.0, a half-life of 1.5 h at 75°C, and an increased thermostability with rising salinity up to 4 M. TSS possesses an increased number of surface acidic residues and ion pairs, as well as four Ca 2+ -binding sites, which contribute to its high thermostability and halotolerance. At high temperatures, TSS exhibited high activity toward insoluble type I collagen and azocoll, but showed a low gelatinolytic activity, with a strong preference for Arg and Gly at the P1 and P1’ positions, respectively. Both the βJR and PPC domains could bind but not swell collagen, and thus facilitate TSS-mediated collagenolysis via improving the accessibility of the enzyme to the substrate. Additionally, TSS has the ability to efficiently degrade fish scale collagen at high temperatures. IMPORTANCE Proteolytic degradation of collagen at high temperatures has the advantages of increasing degradation efficiency and minimizing the risk of microbial contamination. Reports on thermostable collagenolytic proteases are limited, and their maturation and catalytic mechanisms remain to be elucidated. Our results demonstrate that the thermophile-derived TSS matures in an autocatalytic manner, and represents one of the most thermostable collagenolytic proteases reported so far. At elevated temperatures, TSS prefers hydrolyzing insoluble heat-denatured collagen rather than gelatin, providing new insight into the mechanism of collagen degradation by thermostable collagenolytic proteases. Moreover, TSS has the potential to be used in recycling collagen-rich wastes such as fish scales.

Curcumin loaded nano graphene oxide reinforced fish scale collagen – a 3D scaffold biomaterial for wound healing applications

RSC Advances ◽  
2015 ◽  
Vol 5 (119) ◽  
pp. 98653-98665 ◽  
Author(s):  
Tapas Mitra ◽  
Piyali Jana Manna ◽  
S. T. K. Raja ◽  
A. Gnanamani ◽  
P. P. Kundu
Keyword(s):  
Tissue Engineering ◽  
Wound Healing ◽  
Graphene Oxide ◽  
Type I Collagen ◽  
Type I ◽  
Fish Scale ◽  
3D Scaffold ◽  
Engineering Research ◽  
Nano Graphene ◽  
Tissue Engineering Research

We prepare a highly stabilized nano graphene oxide functionalized with type I collagen to make a 3D scaffold as a novel platform for better tissue engineering research..

scholarly journals Effect Collagen Concentration Tilapia Scales on the Quality of Burn Ointment

2021 ◽  
pp. 87-95
Author(s):  
Muhammad Firham Ramadhan ◽  
Junianto . ◽  
Rusky Intan Pratama ◽  
Iis Rostini
Keyword(s):  
Type I Collagen ◽  
Ph Value ◽  
Quality Standard ◽  
National Standard ◽  
Control Treatment ◽  
Type I ◽  
Fish Scale ◽  
Collagen Concentration ◽  
Biomedical Material ◽  
Skin Collagen

Collagen is one of the main connective tissue animal proteins and has been widely used as a biomedical material. Collagen is divided into XIX types. Type I collagen, among others, is obtained from bone, scales and skin. Collagen derived from type I can repair tissue or accelerate tissue regeneration to heal burns. The purpose of this research was to determine the addition of fish scale collagen extract to the characteristics of the burn ointment preparation in accordance with the Indonesian National Standard (SNI) and the best quality. The method used in this research is an experimental method of Completely Randomized Design (CRD) consisting of 4 collagen addition treatments: 0%, 2%, 4% and 6% repeated 5 times. Parameters in this method include physical-chemical parameters (pH, spreadability, shelf life and homogeneity) and organoleptic parameters (appearance, aroma, texture and color). Bayes test results, the concentration of the addition of tilapia scale collagen in the ointment preparation of 4% resulted in a value close to the control treatment. The addition of 4% collagen was the best treatment compared to 2% and 6% with a pH value of 6.12, dispersion of 3.22 cm, safe ointment preparation did not change at all during 28 days of storage. Based on the results of the organoleptic test parameters, the ointment at this concentration had a homogeneous appearance, slightly yellowish white color, a distinctive smell of collagen and a semi-solid texture, this was in accordance with the quality standard of the ointment and had the best quality characteristics.

Application of bacterial collagenolytic proteases for the extraction of type I collagen from the skin of bigeye tuna (Thunnus obesus)

LWT ◽  
2018 ◽  
Vol 89 ◽  
pp. 44-51 ◽  
Author(s):  
Raju Ahmed ◽  
Adane Tilahun Getachew ◽  
Yeon-Jin Cho ◽  
Byung-Soo Chun
Keyword(s):  
Type I Collagen ◽  
Type I ◽  
Bigeye Tuna ◽  
Collagenolytic Proteases ◽  
Thunnus Obesus

Fish Scale Collagen Functionalized Thermo-Responsive Nanofibres

2020 ◽  
Vol 846 ◽  
pp. 189-194
Author(s):  
Kim Yeow Tshai ◽  
Mei Hua Chin ◽  
Siew Shee Lim ◽  
Hwei San Loh ◽  
Ernest Hsin Nam Yong ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Fibre Diameter ◽  
High Viscosity ◽  
Measured Quantity ◽  
Type I ◽  
Fish Scale ◽  
Electrospinning Technique ◽  
Sem Observation ◽  
Fibrous Protein ◽  
Average Fibre Diameter

Smart thermosensitive polymer such as poly (N-isopropyl acrylamide) (PNIPAM) and dominant fibrous protein of connective tissue such as collagen (CLG) possess great potential in biomedical and tissue engineering applications. The objectives of current work aim to explore potential of PNIPAM and collagen by (i) establish a stable procedure to extract collagen from fresh water Tilapia fish scale (TFS) and (ii) fabricate PNIPAM and hybrid PNIPAM-CLG nanofibrous scaffolds through electrospinning technique and investigate their material-process-structure behaviour. Type I collagen was derived through acid hydrolysis of TFS. Electrospinning of PNIPAM was carried out with 16, 18 and 20 wt% PNIPAM concentration in methanol (MeOH) while PNIPAM-CLG was prepared through blending measured quantity of PNIPAM dissolved in water with collagen dissolved in acetic acid. Material properties, viscosity, morphology and thermo-physical behaviors of the derived collagen, electrospun PNIPAM and PNIPAM-CLG scaffolds were characterized. Results from SDS-PAGE and FTIR confirmed that the isolated TFS collagen is of type I. EDX revealed that demineralization eliminated the aluminium, magnesium, silicon and phosphorus while significantly reduced the sulfur elements from raw TFS. SEM observation of the collagen morphology shown a fluffy and fibrillary lamellae structure. Electrospun scaffolds were successfully fabricated with 16 and 18 wt% PNIPAM in MeOH. Both homogeneity and average fibre diameter (Davg) were greater in the 18 wt% PNIPAM scaffold, in which the Davg for 16 and 18 wt% were ~110 and ~131.7 nm respectively. However, PNIPAM at 20 wt% failed to be electrospun owing to its excessively high viscosity. On the other hand, SEM observation revealed that the electrospun hybrid PNIPAM-CLG scaffold has Davg of ~105.5 nm amid the presence of numerous elongated beads.

scholarly journals Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Xiaowei Gao ◽  
Jing Zeng ◽  
Huawei Yi ◽  
Fang Zhang ◽  
Bing Tang ◽  
...  
Keyword(s):  
High Temperatures ◽  
Calcium Binding ◽  
Catalytic Domain ◽  
Pyrococcus Furiosus ◽  
Ion Pairs ◽  
Structure Stability ◽  
Single Mutation ◽  
Structural Elements ◽  
Content Type ◽  
Enzyme Thermostability

ABSTRACT Pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus is the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily (subtilases). It contains four inserts (IS147, IS29, IS27, and IS8) of unknown function in the catalytic domain. We performed domain deletions and showed that three inserts are either essential (IS147 and IS27) or important (IS8) for efficient maturation of pyrolysin at high temperatures, whereas IS29 is dispensable. The large insert IS147 contains Ca3 and Ca4, two calcium-binding Dx[DN]xDG motifs that are conserved in many pyrolysin-like proteases. Mutagenesis revealed that the Ca3 site contributes to enzyme thermostability and the Ca4 site is necessary for pyrolysin to fold into a maturation-competent conformation. Mature insert-deletion variants were characterized and showed that IS29 and IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites: one within IS29 and the other in IS27. Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Interestingly, autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation. Additionally, a single mutation in IS29 increases resistance to salt-induced autocleavage and further increases enzyme thermostability. Our results suggest that these extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments. IMPORTANCE Pyrolysin-like proteases belong to the subtilase superfamily and are characterized by large inserts and long C-terminal extensions; however, the role of the inserts in enzyme function is unclear. Our results demonstrate that four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease.

scholarly journals Molecular Dissection of the Structural Machinery Underlying the Tissue-invasive Activity of Membrane Type-1 Matrix Metalloproteinase

2008 ◽  
Vol 19 (8) ◽  
pp. 3221-3233 ◽  
Author(s):  
Xiao-Yan Li ◽  
Ichiro Ota ◽  
Ikuo Yana ◽  
Farideh Sabeh ◽  
Stephen J. Weiss
Keyword(s):  
Matrix Metalloproteinase ◽  
Type I Collagen ◽  
Cell Function ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Type I ◽  
Cell Trafficking ◽  
Membrane Type

Membrane type-1 matrix metalloproteinase (MT1-MMP) drives cell invasion through three-dimensional (3-D) extracellular matrix (ECM) barriers dominated by type I collagen or fibrin. Based largely on analyses of its impact on cell function under two-dimensional culture conditions, MT1-MMP is categorized as a multifunctional molecule with 1) a structurally distinct, N-terminal catalytic domain; 2) a C-terminal hemopexin domain that regulates substrate recognition as well as conformation; and 3) a type I transmembrane domain whose cytosolic tail controls protease trafficking and signaling cascades. The MT1-MMP domains that subserve cell trafficking through 3-D ECM barriers in vitro or in vivo, however, remain largely undefined. Herein, we demonstrate that collagen-invasive activity is not confined strictly to the catalytic, hemopexin, transmembrane, or cytosolic domain sequences of MT1-MMP. Indeed, even a secreted collagenase supports invasion when tethered to the cell surface in the absence of the MT1-MMP hemopexin, transmembrane, and cytosolic tail domains. By contrast, the ability of MT1-MMP to support fibrin-invasive activity diverges from collagenolytic potential, and alternatively, it requires the specific participation of MT-MMP catalytic and hemopexin domains. Hence, the tissue-invasive properties of MT1-MMP are unexpectedly embedded within distinct, but parsimonious, sequences that serve to tether the requisite matrix-degradative activity to the surface of migrating cells.

Elemental distribution analysis of type I collagen fibrils in tilapia fish scale with energy-filtered transmission electron microscope

Micron ◽  
2009 ◽  
Vol 40 (5-6) ◽  
pp. 665-668 ◽  
Author(s):  
Mitsuhiro Okuda ◽  
Masaki Takeguchi ◽  
Motohiro Tagaya ◽  
Toru Tonegawa ◽  
Ayako Hashimoto ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Transmission Electron Microscope ◽  
Type I Collagen ◽  
Collagen Fibrils ◽  
Elemental Distribution ◽  
Type I ◽  
Distribution Analysis ◽  
Fish Scale ◽  
Transmission Electron

scholarly journals In Situ Localization of Two Fibrillar Collagens in Two Compact Connective Tissues by Immunoelectron Microscopy After Cryotechnical Processing

1997 ◽  
Vol 45 (1) ◽  
pp. 119-128 ◽  
Author(s):  
G. Nicolas ◽  
F. Gaill ◽  
L. Zylberberg
Keyword(s):  
Osmium Tetroxide ◽  
Type I Collagen ◽  
Freeze Substitution ◽  
Immunogold Electron Microscopy ◽  
Type I ◽  
Fish Scale ◽  
Electron Microscopic ◽  
Lr White ◽  
Fibrillar Collagens ◽  
Freeze Fixation

Two fibrillar collagens, the worm cuticular collagen and the vertebrate Type I fish scale collagen, both organized in a compact tissue, were localized by immunogold electron microscopy in resin sections after freeze-fixation and freeze-substitution. Identification of these two fibrillar collagens failed with the-use of postembedding labeling after conventional electron microscopic processing. Positive labeling of the Type I collagen was observed in sections of fish scales freeze-fixed by either slam-freezing or high-pressure freezing, freeze-substituted in acetone with or without osmium tetroxide, and embedded in LR White. The worm cuticular collagen was detected in sections of cuticle that were freeze-fixed, freeze-substituted (necessarily with osmium tetroxide added to acetone), and embedded in either LR White or Epon. It was also detected in specimens pre-fixed by aldehydes before freeze-fixation. The Type I fish scale collagen appears to be more sensitive than the fibrillar cuticular collagen of worms to the procedures employed for postembedding immunoelectron microcopy. Our results have shown that freeze-fixation and freeze-substitution preserved the antigenicity of the fibrillar collagens organized in a compact three-dimensional network, whereas immunolabeling failed after conventional electron microscopic procedures. These cryostabilization techniques appear to be of value to improve the immunolocalization of collagens.

scholarly journals Synthesis and Characterization of Wound Dressing Material from Bio-Wastes Impregnated with the Spider Web and the Ethanolic Weaves Extract of Mangifera indica (L.)

2021 ◽  
Vol 12 (2) ◽  
pp. 1998-2012
Keyword(s):  
Cost Effectiveness ◽  
Wound Dressing ◽  
Type I Collagen ◽  
Mangifera Indica ◽  
Biological Evaluation ◽  
Spectroscopic Techniques ◽  
Type I ◽  
Fish Scale ◽  
Good Efficacy ◽  
Spider Web

Substantial progress in wound therapy has not snuffed out the passion in search of innovative wound dressing materials. This work is to analyze the physiochemical characterizations and biological evaluation of a wound dressing material. A wound dressing material had been synthesized from Physiologically Clotted Fibrin (PCF), Fish Scale Collagen (FSC). Also, the wound dressing material had impregnated with the folklore medicinal impact of the Spider Web (SW) and the Ethanolic Extract of Mangifera indica (L.)(EEMI). Infrared spectroscopic techniques confirmed the presence of Type I collagen. Surface morphology established the smooth, uniform, porous, biocompatible surface of the material. Water absorption studies, porosity measurements showed the required characteristics, the antibacterial activity favored the resistance, and the ash test supported the eco-friendly environment of the wound dressing material. Human erythrocytes had reviewed biocompatibility. The supernatant of the wound dressing material at different concentrations and incubation times had determined for percent hemolysis. Plots between percent hemolysis and concentration showed the non-hemolytic behavior of the wound dressing material. The synthesized biomaterial could propose as a wound dressing material with good efficacy, cost-effectiveness, and eco-friendly. The synthesized biomaterial could have been a better wound dressing material with good efficacy, cost-effectiveness, and eco-friendly.

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