| Keratin is a refractory protein which widely exists as animal hairs, feathers and so on in nature. Keratin wastes such as animal hairs and feathers can be transformed into available peptides and amino acids, which have significant application values in many fields such as fodder, leather treatment, foods and pharmaceuticals industry. Compared with traditional physical and chemical methods, bio-degradation of keratin wastes also has obvious environmental significance. Because the structure of keratin becomes loose and easy to be degraded at high temperatures, thermophilic bacteria and keratinases have attracted extensive attention. Currently, known mesophilic keratin degradative bacteria mainly belong to Bacillus and Streptomyces, and their feather degradation mechanisms are different. Bacillus sp. WF146and Thermoactinomyces sp. CDF are two thermophilic bacteria isolated from soil. Our preivious studies revealed that they can produce extracellular proteases with keratinase activity. From the point of view of the cellular morphology, they can be taken as "thermophilic version" of Bacillus and Streptomyces. These two thermophilic bacteria are ideal targets for comparative study of feather-degrading characteristics and mechanisms at high temperature.Here, we first investigated cultural conditions of the two bacteria for producing of extracellular proteases. The results showed that:nutrition (carbon and nitrogen resource) could significantly affect growth and extracellular proteases producing capacity of the two bacteria. For Bacillus sp. WF146, growth of the bacteria could be significantly inhibited by reducing nutrition of the medium. Compared to cultivation in LB medium, the maximum quantity of the bacteria in the medium with reduced nutrition was reduced4-5orders of magnitude, and the bacterial morphology was changed. However, nutrition reduced culturing could improve extracellular proteases producing capacity of Bacillus sp. WF146. By use of nutrition reduced dLB2medium, enzyme producing process was accelerated, and extracellular protease activity at stationary stage increased by360%. Meanwhile, the main extracellular protease (WF146protease) existed as mature form. For Thermoactinomyces sp. CDF, due to generation of the mycelium structure, we monitored the growth by measuring the colony forming units. The results showed that nutritional reduced conditions could significantly affect its growth. Compared to cultivation in LB medium, the maximum quantity of viable cell in dLB2medium was reduced about1order of magnitudes, and extracellular proteolytic activity at stationary phase reduced by84%. This change was caused by decreasing of the quantity of viable cell and reducing of enzyme producing capacity of cells.Secondly, comparison of feather-degrading characteristics between the two thermophilic bacteria was performed. We found that the two thermophilic bacteria all had degradation capacity of keratin in feathers, and could grow by using feather as the main carbon and nitrogen resource. The additional feather of the medium could promote growth of the two bacteria, and has significant effect on their protease-producing ability. When growing in nutritional stress condition, Bacillus sp. WF146could produce extracellular proteases to degrade feather. Meanwhile, feather (4g/L) could promote the growth and enzyme producing ability of the bacteria. Compared to cultivation in dLB2medium, the maximum quantity of the bacteria in dLB2F medium with additional feathers increased1order of magnitude, and the extracellular proteolytic activity at stationary phase increased by85%. Similar to Bacillus sp. WF146, additional feather (4g/L) could also significantly promote the growth of Thermoactinomyces sp. CDF. Compared to cultivation in LB/dLB2medium, the maximum quantity of Thermoactinomyces sp. CDF in LBF/dLB2F medium with the additional feather (4g/L) increased by1order of magnitude. Meanwhile, the extracellular proteolytic activity in LBF/dLB2F was increased by31%and209%, respectively. This improvement was caused by the increase of the quantity of viable cell and change of cellular enzyme producing capacity. Effect of feather on extracellular proteases producing capacity of Thermoactinomyces sp. CDF in LB medium might be related to the degrading process.In order to study further about effect of feather on growth and enzyme producing capacity of the two bacteria, we have designed three oligotrophic media: TYNF, YNF and NF (feather content was0.5g/l). Bacillus sp. WF146in YNF medium could completely degrade feathers, and enzymes producing process began at logarithmic phase. However, Thermoactinomyces sp. CDF could degrade feathers in TYNF, YNF and NF media, with a lot of fine feather fragments remained. It was worth noting that when cultured in NF medium, feather was degraded but only little extracellular proteases were produced. It indicated that besides the mechanism of protease hydrolysis, other feather mechanisms may involve. Oligotrophic cultivation of the two bacteria showed that, although the two bacteria could grow with feather as the main carbon and nitrogen resource, feather degrading characteristics and mechanism of the two bacteria were significantly different. Finally, the mechanisms of the two bacteria in the degradation process offeathers were compared. Both the two bacteria could produce serine protease with keratin degradation capacity. Protease C2produced by Thermoactinomyces sp. CDF was more inclined to keratin substrates. Compared the feather degradation mechanisms of these two bacteria, Bacillus sp. WF146degrades feather by damaging disulfide bond of keratin with reducing power provided by the surface of cells. While Thermoactinomyces sp. CDF mycelium may not only provide reducing power, but also physics destroy the structure of feather. Therefore, different feather degrading mechanisms of the two bacteria led to their different feather-degrading characteristics. |
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