Xiang Lin

Bio: Xiang Lin is an academic researcher from North Carolina State University. The author has contributed to research in topics: Keratinase & Bacillus licheniformis. The author has an hindex of 4, co-authored 5 publications receiving 500 citations.

Purification and Characterization of a Keratinase from a Feather-Degrading Bacillus licheniformis Strain

Abstract: A keratinase was isolated from the culture medium of feather-degrading Bacillus licheniformis PWD-1 by use of an assay of the hydrolysis of azokeratin. Membrane ultrafiltration and carboxymethyl cellulose ion-exchange and Sephadex G-75 gel chromatographies were used to purify the enzyme. The specific activity of the purified keratinase relative to that in the original medium was approximately 70-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Sephadex G-75 chromatography indicated that the purified keratinase is monomeric and has a molecular mass of 33 kDa. The optimum pH and the pI were determined to be 7.5 and 7.25, respectively. Under standard assay conditions, the apparent temperature optimum was 50°C. The enzyme is stable when stored at −20°C. The purified keratinase hydrolyzes a broad range of substrates and displays higher proteolytic activity than most proteases. In practical applications, keratinase is a useful enzyme for promoting the hydrolysis of feather keratin and improving the digestibility of feather meal. Images

Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1

Abstract: Bacillus licheniformis PWD-1 (ATCC 53757) secretes keratinase, a proteolytic enzyme which is active on whole feathers. By amino acid sequence similarity and phenylmethylsulfonyl fluoride inhibition, the keratinase was demonstrated to be a serine protease. The entire nucleotide sequence of the coding and flanking regions of the keratinase structure gene, kerA, was determined. A fixed oligonucleotide primer derived from the N-terminal sequence of the purified enzyme and a second random oligonucleotide primer were used in a procedure called PCR walking, which was developed to amplify and sequence the upstream and downstream regions of kerA. Another method, PCR screening, was conducted with a lambda phage vector with inserted PWD-1 genomic DNA fragments as templates and with the known sequences of the vector arms and the N-terminal sequence of the enzyme as primers. PCR amplification and sequence analysis of the lambda library completed the entire kerA sequence and established a set of gene deletions. The kerA gene shares a 97% sequence identity with the gene encoding subtilisin Carlsberg from B. licheniformis NCIMB 6816. The putative promoters, ribosome binding sites, and transcriptional terminators are also similar in these two bacteria. The deduced amino acid sequences indicate only three amino acid differences between the two mature proteases. Northern (RNA) analysis demonstrates that transcriptional regulation controls kerA expression on different growth media.

Dna encoding the bacillus licheniformis pwd-1 keratinase

Abstract: An isolated DNA encoding a keratinase is disclosed. The isolated DNA may be any of (a) isolated DNA which encodes the Bacillus licheniformis PWD-1 keratinase enzyme of the figure, (b) isolated DNA which hybridizes to an oligonucleotide probe, which hybridizes to the DNA of (a) above, and does not hybridize to DNA encoding the Bacillus licheniformis NCIB 6816 subtilisin Carlsberg serine protease, and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encodes a keratinase enzyme.

Method for expressing and secreting keratinase

Abstract: The present invention provides a Bacillus subtilis host cell capable of expressing and secreting keratinase. The host cell contains a recombinant DNA molecule comprising vector DNA and DNA encoding Bacillus licheniformis PWD-1 keratinase enzyme operatively associated therewith. The present invention also provides a method for producing keratinase enzyme. The method includes the steps of (a) culturing a Bacillus subtilis host cell containing a recombinant DNA molecule comprising vector DNA and DNA encoding Bacillus licheniformis PWD-1 keratinase enzyme operatively associated therewith, and (b) collecting keratinase enzyme from the cell culture.

Adn codant la bacillus licheniformis pwd-1 keratinase

Abstract: Cette invention se rapporte a un ADN isole codant une keratinase. Cet ADN isole peut etre: soit (a) un ADN isole qui code l'enzyme Bacillus licheniformis PWD-1 keratinase, illustree dans la figure, soit (b) un ADN isole qui s'hybride a une sonde oligonucleotidique, laquelle s'hybride a l'ADN (a) mentionne ci-dessus et ne s'hybride pas a l'ADN codant l'enzyme Bacillus licheniformis NCIB 6816 subtilisine Carlsberg serine protease; soit (c) un ADN isole qui differe des ADN isoles (a) et (b) mentionnes ci-dessus dans une sequence nucleotidique en raison de la degenerescence du code genetique, et qui code une enzyme keratinase.

Microbial keratinases and their prospective applications: an overview.

Abstract: Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.

Biochemical features of microbial keratinases and their production and applications.

Abstract: Keratinases are exciting proteolytic enzymes that display the capability to degrade the insoluble protein keratin. These enzymes are produced by diverse microorganisms belonging to the Eucarya, Bacteria, and Archea domains. Keratinases display a great diversity in their biochemical and biophysical properties. Most keratinases are optimally active at neutral to alkaline pH and 40–60°C, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Several keratinases have been associated to the subtilisin family of serine-type proteases by analysis of their protein sequences. Studies with specific substrates and inhibitors indicated that keratinases are often serine or metalloproteases with preference for hydrophobic and aromatic residues at the P1 position. Keratinolytic enzymes have several current and potential applications in agroindustrial, pharmaceutical, and biomedical fields. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling.

Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530.

Abstract: A serine protease from the keratin-degrading Streptomyces pactum DSM 40530 was purified by casein agarose affinity chromatography. The enzyme had a molecular weight of 30,000 and an isoelectric point of 8.5. The proteinase was optimally active in the pH range from 7 to 10 and at temperatures from 40 to 75 degrees C. The enzyme was specific for arginine and lysine at the P1 site and for phenylalanine and arginine at the P1' site. It showed a high stereoselectivity and secondary specificity with different synthetic substrates. The keratinolytic activity of the purified proteinase was examined by incubation with the insoluble substrates keratin azure, feather meal, and native and autoclaved chicken feather downs. The S. pactum proteinase was significantly more active than the various commercially available proteinases. After incubation with the purified proteinase, a rapid disintegration of whole feathers was observed. But even after several days of incubation with repeated addition of enzymes, less than 10% of the native keratin substrate was solubilized. In the presence of dithiothreitol, degradation was more than 70%.