Isolation And Identification Of Actinomycetes Degrading Silk Fibroin, The Optimization Of Fermentation Technology And The Degradation Mechanism Of Fibroin

As an excellent natural protein fibre, Bombyx mori silk is widely used in the field of textile industry. With the development of high and new-technology industries in recent years, the special functions of silk fibroin are paid more and more attention to due to the crossing and recombination of different subjects. Silk fibroin owns considerable application value especially in the area such as biomaterial, medicine, food science and chemical industry because silk fibroin is an ideal biomacromolecule with perfect biology compatibility. At the same time, silk fibroin is difficult to degrade for its unique structure as a macromolecule. The classical methods to degrade silk fibroin are salt degradation, acid degradation and alkali degradation. As a result, only macromolecular regenerated fibroin powder could be obtained by salt degradation, and many amino acids are destroyed by acid degradation and alkali degradation. They all need the complicated process of desalinization and deacidification, and meanwhile, cause serious environmental problems due to their high energy consumption as well as heavy pollution. Therefore, enzyme degradation is the ultimate way to solve the problem if the obstacle from silk fibroin structure, the typicalβ-sheet form, which is so steady that it can not be degraded by common proteinase. It is a good route to develop the microbe resources and find the exact enzyme degrading silk fibroin. Based on these understanding, the 52 actinomycetes were successfully isolated from soil which all carried the characteristics of degrading silk fibroin in this paper. Out of them, Amycolatopsis decaplanina SA12 was screened out due to its excellent performance in fibroin degradation. After a series of single-factor experiments, the rotation-orthogonal composite design was utilized to obtain the optimal fermentation technology of fibroin degradation. Furthermore, the fibroin degradation mechanism was probed by the way of Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Gravitational Thermal Analysis (GTA), mechanical drawing test, amino acid analysis, etc. the results were as follows.1. The soil samples were collected from different region. After plenty of pretreatments of these samples, the enrichment culture, gradient dilution as well as lineation separation were adopted to obtain the actinomycetes which can grow normally on the inorganic salt culture using silk fibroin as the sole carbon resources and nitrogen resources. 52 actinomycetes were gained and among them, 18 strains were especially screened out due to their excellent ability of silk fibroin degradation. The 18 actinomycetes were all identified into the genus level after morphological observation, the physiological and biochemistry indexes mensuration, 16SrDNA sequencing and phylogenetic tree construction.2. The actinomycete strain A. decaplanina SA12, which carries the perfect ability to degrade silk fibroin, was optimized in order to achieve its ultimate fermentation technology. The single-factor experiments were consists of the optimal fermentation temperature, the optimal initial fermentation pH, the optimal carbon resources as well as the optimal concentration of the obtained carbon resources, the optimal nitrogen resources as well as the optimal concentration of the obtained nitrogen resources, the effects of metal ions. Based on these results, the rotation-orthogonal composite design was utilized and the optimal fermentation technology of A. decaplanina SA12 was finally obtained: 32.5℃, pH8.0, KNO3 concentration 1.25%, corn powder concentration 1.25%. At the same time, Mg²⁺,Zn²⁺,Fe²⁺,Ca²⁺ could enhance the fibroinase activity while Hg⁺,Cu²⁺,Fe³⁺ inhibit the fibroinase activity.3. The fibroinase from A. decaplanina SA12 was extracellular enzyme and inducible enzyme. It owned a relatively wide substrate scope, degrading silk fibroin protein, feather keratin, wool keratin and even the human hair keratin, high-temperature-treated or non-high-temperature-treated. Meanwhile, it could also degrade casein. The wide substrate scope meant a far application future of this unique silk fibroin protein from actinomycete. The fibroinase from A. decaplanina SA12 degraded all the substrates above after high-temperature-treatment at a high level than those without the high-temperature-treatment, which suggested that the protein denaturalization from high-temperature-treatment played an important role in the protein degradation progress.4. The SEM morphological observation showed that there was a unique pellicle formed in the early state of degradation. The pellicle was compact and covered the silk fibre and it was where the actinomycete colony appeared on. It was presumed that there was something special inside the pellicle and it could destroy the disulfide bond in the fibroin molecule and among the fibroin molecule, which was the just obstacle of the anti-degradation structure for the infusible silk fibroin. In addition, many kinds of fractures were observed while the hole and crack were not found in the surface of silk fibre in the experiment of SEM. 5. The most obvious changing for the structure of silk fibroin in the process of degradation was the sharp fall of silk mechanical properties, especially the indexes of tensile strength, tensile modulus and extensibility, which indicated the strength and the flexibility of silk fibre. All the three indexes above dropped to a collapse level from second day to fourth day in the degradation process. The tensile strength declined from 25.23±5.7 MPa to 2.11±0.51 MPa, and the tensile modulus from 175.55±28.34 GPa to 18.86±7.49 GPa during this period.6. The secondary structure of degrading silk fibroin due to A. decaplanina SA12 was quantificationally described by the technique of FTIR and X-ray diffraction. In the process of degradation, the content of steady secondary structure,β-sheet conformation as well as the silkâ…¡crystal form, were all showed a decrease trend in a whole, with a little undulation. It suggested that the fibroin degradation was a dynamic process, influenced by many factors such as the inorganic salt ion in the culture solution, the shearing force from the shake cultivation in the fermentation, the break and recombination of hydrogen bond in the complicated degradation solution. It was a normal phenomenon in the degradation process whenβ-sheet conformation dynamically transformed into random coil, or silkâ…¡crystal form transformed into silkâ… and amorphism.7. The free amino acids occurred in the degradation solution in the sixth day. Glycin and alanine, with small side chain amino acid residue, emergenced in a large scale at the earliest time, while the other important component, serine, with the same characteristics of small side chain, was appeared in a far low level than that in the fibroin compose. It is presumed that the fibroinase from A. decaplanina SA12 might cut the site just between glycin and alanine. Meanwhile, aspartic acid, as a symbol of protein hydrolysis, was also occurred in the early stage of the degradation.8. It is an effective way for the protein degradation mechanism to investigate the changing of sulfur compound in the protein degradation solution as well as the influence of additive sulfur compound to the protein degradation process. In this paper, the content of sulphate reached the maximum on the first day of degradation in the culture solution of A. decaplanina SA12. After the comprehensive consideration of the results of the fibroinase activity changing as well as the silk fibroin loss rate, it was found that the silk fibroin loss and soluble protein in the degradation solution all occurred before the fibroinase activity could be detected in the solution, which suggested that there existed a non-enzyme-degradation-mechanism. It was presumed that there was a close relationship between the maximum content of sulfate in the first day of degradation and the pellicle occurred in the early time before the actinomycete colony formed in the photos of SEM. The conclusion could be drawn that the process of silk fibroin degradation was synergetic effect of non-enzyme-degradation-mechanism, which happened at the early stage, and followed by the proteinase-degradation thereafter. It was the non-enzyme-degradation that broke the disulfide bond inhibited the proteinase hydrolysis and so the non-enzyme-degradation was the key step for the fibroin degradation from A. decaplanina SA12.