Research On Phytogeny And Keratinase Of An Anaerobic Keratinolytic Bacteria Strain KD-1

Keratins are insoluble structural proteins that are extensively cross-linked by disulfide bonds,hydrogen bonds, and hydrophobic interactions. Keratins are abundant in hair, feathers, and horns. Theα-helix and β-sheet configurations of keratins are characteristically resistant to common proteasesincluding trypsin, pepsin and papain. However, keratins can be degraded by keratinase secreted bykeratinolytic microorganisms. Reflecting the ubiquity of keratinolytic microorganisms in naturalenvironments, keratin accumulation does not naturally occur. Keratinolytic microorganisms includebacteria (mainly actinobacteria and bacteria in the genus Bacillus), fungi, and archaea. It wasenvironment unfriendly and economic unreasonable to deal with the million tons of feathers waste everyyear by traditional physical and chemical method. Above these problems，based on the anaerobic keratindegradation bacteria18D-TA which was isolated by our laboratory, using Hungate anaerobic technologyand the separation and purification technology, We purified strain KD-1and done the phylogeneticanalysis and functional evaluation. Optimized keratinase production conditions of strain KD-1by singlefactor and orthogonal experimental. Determined the volatile acid component and sulfur compounds offeather degradation products by gas chromatography and other determination methods.; Observed thephysical changes in the process of feather degradation by scanning electron microscopy and analysisedthe mechanism of feather degration combined with metabolite analysis. Separated and purified keratinsesecreted by strain KD-1by hollow fiber cross-flow system, ion exchange chromatography and sizeexclusion chromatography.and analysised the physical and chemical properties of this keratinse. Theresults are as follows:（1）Strain KD-1had a95.3%,93.8%16S rRNAgene sequence similarity to Clostridium ultunenseBST(=DSM10521T) and Tepidimicrobium xylanilyticum PML14T(=CGMCC1.5080T=JCM15035T),respectively. And the cell fatty acid was predominantly C15:0iso, C15:0iso dma, C16:0dma, C16:0,C14:0dma, C11:0dma, C14:0, C17:0iso dma. The G+C content was28mol%. No respiratory quinonewas detected. Based on morphological and physiological characterization, together with phylogeneticanalysis based on16S rRNA sequencing, identified KD-1as a new species of a novel genus ofClostridiaceae. We propose the name Keratinibaculum paraultunense gen. nov., sp. nov., with KD-1(=JCM18769T=DSM26752T) as the type strain.（2）The cell of strain KD-1adhered on the surface of feathers and secreted thermophilic alkalineserine keratinase during feather degradation, but there is no disulfide reductase. It also produced highconcentrations of ammonia, and sulfur compounds such as thiol, sulfite, sulfate, sulfur ions andhydrogen sulfide. It suggested that strain KD-1and sulfite played a key role in the feather degration as adisulfide bond reducing agent. Keratinase mainly exercised a protease function.（3）The optimum conditions of K. paraultunense KD-1for keratinase production was feathers10.0g/L, MgSO40.2g/L, urea2.0g/L, NaCl3.0g/L, K2HPO43.0g/L, KH2PO42.5g/L, vitamin14110.0ml, trace elements14110.0ml and the initial pH8.0, temperature55°C by single factor andorthogonal experiments. The keratinase activity was improved21.3%higher than the original condition at optimal culture conditions.（4）Separated and purified a keratinase of strain KD-1with hollow fiber column, desalting column,anion exchange chromatography and size exclusion chromatography. The keratinase was20kDa, andthe optimum keratinase activity was pH10.0, temperature80°C. This keratinase was thermally stablebelow80°C, and was able to maintain most of the activity at pH5-11. It was partially inhibited byEDTA, and completely inhibited by PMSF, which indicated it was a serine protease. The reaction ofreducing agent was positive and metal ions, detergents and organic solvent to the reaction was negative.