• Microbiology and Biotechnology Letters
• /
• v.40 no.4
• /
• pp.303-309
• /
• 2012

Keratinases are of particular interest because of their action on insoluble keratins and generally on a broad range of protein substrates. Alkalophilic and neutrophilic actinomycete strains isolated from different soil samples, rich in keratinaceous substances were screened for keratinolytic activity. An alkalophilic isolate, TBG-S13A5, was found to possess good keratinolytic activity and was able to utilize feather as the sole carbon and nitrogen source. TBG-S13A5 exhibited an off-white aerial mass color with a rectus-flexibilis type of spore chain. The morphological, microscopical and biochemical characters were comparable with that of Streptomyces albidoflavus. Fatty acid methyl ester profiling (FAME) and 16S rDNA sequence analysis confirmed its identity as a strain of S. albidoflavus. Under submerged fermentation conditions, maximum protease production was recorded on the $5^{th}$ day of incubation at $30^{\circ}C$, using basal broth of pH 9.0 with 0.25% (w/v) white chicken feather. This strain could affect feather degradation when the initial pH was 8 and above and maximum protease production was recorded when the initial pH was around 10.5. The effectiveness of the crude enzyme in destaining and leather dehairing were also demonstrated.

• Mycobiology
• /
• v.35 no.4
• /
• pp.219-225
• /
• 2007

A keratinolytic enzyme secreted by Aspergillus flavus K-03 cultured in feather meal basal medium (FMBM) containing 2% (w/v) chicken feather was purified and characterized. Keratinolytic enzyme secretion was the maximal at day 16 of the incubation period at pH 8 and $28^{\circ}C$. No relationship was detected between enzyme yield and increase of fungal biomass. The fraction obtained at 80% ammonium sulfate saturation showed 2.39-fold purification and was further purified by gel filtration in Sephadex G-100 followed by ion exchange chromatography on DEAE-Sephadex A-50, yielding an active protein peak showing 11.53-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms indicated that the purified keratinase is a monomeric enzyme with 31 kDa molecular weight. The extracellular keratinase of A. flavus was active in a board range of pH ($7{\sim}10$) and temperature ($30^{\circ}C{\sim}70^{\circ}C$) profiles with the optimal for keratinase activity at pH 8 and $45^{\circ}C$. The keratinase activity was totally inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, and ethylenediaminetetraacetate (EDTA) while no reduction of activity by the addition of dithiothreitol (DTT) was observed. N-terminal amino acid sequences were up to 80% homologous with the fungal subtilisins produced by Fusarium culmorum. Therefore, on the basis of these characteristics, the keratinase of A. flavus K-03 is determined to be subtilisins-like.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.12
• /
• pp.2190-2198
• /
• 2017

Thermoactinomyces sp. strain YT06 was isolated from poultry compost and observed to degrade integral chicken feathers completely at $60^{\circ}C$, resulting in the formation of 3.24 mg/ml of free amino acids from 50 ml of culture containing 10 g/l chicken feathers. Strain YT06 could grow and secrete keratinase using feather as the only carbon and nitrogen sources without other supplement, but complementation of 10 g/l sucrose and 4 g/l $NaNO_3$ increased the production of the keratinolytic enzyme. The maximum protease activity obtained was 110 U/ml and for keratinase was 42 U/ml. The keratinase maintained active status over a broad pH (pH 8-11) and temperature ($60-75^{\circ}C$). It was inhibited by serine protease inhibitors and most metal ions; however, it could be stimulated by $Mn^{2+}$ and the surfactant Tween-20. A reductive agent (${\beta}$-mercaptoethanol) was observed to cleave the disulfide bond of keratin and improve the access of the enzyme to the keratinaceous substrate. Zymogram analysis showed that strain YT06 primarily secreted keratinase with a molecular mass of approximately 35 kDa. The active band was assessed by MALDI-TOF mass spectrometry and was observed to be completely identical to an alkaline serine protease from Thermoactinomyces sp. Gus2-1. Thermoactinomyces sp. strain YT06 shows great potential as a novel candidate in enzymatic processing of hard-to-degrade proteins into high-value products, such as keratinous wastes.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.6
• /
• pp.818-825
• /
• 2012

Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support ($q_m$) and dissociation constant ($K_d$) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at $65^{\circ}C$. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.

• Microbiology and Biotechnology Letters
• /
• v.49 no.1
• /
• pp.65-74
• /
• 2021

Serine proteases are the most versatile proteolytic enzymes with tremendous applications in various industrial processes. This study was designed to investigate the biochemical properties, critical residues, and the catalytic potential of alkaline serine protease using in-silico approaches. The primary sequence was analyzed using ProtParam, SignalP, and Phyre2 tools to investigate biochemical properties, signal peptide, and secondary structure, respectively. The three-dimensional structure of the enzyme was modeled using the MODELLER program present in Discovery Studio followed by Molecular Dynamics simulation using GROMACS 5.0.7 package with CHARMM36m force field. The proteolytic potential was measured by performing docking with casein- and keratin-enriched residues, while the effect of the inhibitor was studied using phenylmethylsulfonyl fluoride, (PMSF) applying GOLDv5.2.2. Molecular weight, instability index, aliphatic index, and isoelectric point for serine protease were 39.53 kDa, 27.79, 82.20 and 8.91, respectively. The best model was selected based on the lowest MOLPDF score (1382.82) and DOPE score (-29984.07). The analysis using ProSA-web revealed a Z-score of -9.7, whereas 88.86% of the residues occupied the most favored region in the Ramachandran plot. Ser327, Asp138, Asn261, and Thr326 were found as critical residues involved in ligand binding and execution of biocatalysis. Our findings suggest that bioengineering of these critical residues may enhance the catalytic potential of this enzyme.

• Proceedings of the Korean Environmental Sciences Society Conference
• /
• 2006.05a
• /
• pp.414-416
• /
• 2006

도축 부산물인 우모는 높은 영양적 가치를 가짐에도 불구하고 현재 사용하는 물리화학적 처리법의 여러 단점으로 효율적으로 이용되지 못하고 있어서 경제적 방법인 생물 공학적 처리에 대한 연구의 필요성이 커지고 있다. 본 연구에서는 그러한 방법 중 대표적인 방법으로 미생물이 생산하는 keratinase 를 이용한 생분해에 대해 연구를 하기 위해 퇴비화 볏짚에서 keratinolytic protease 생성능이 우수한 균주를 분리하였고 생화학적 동정법과 16S rDNA를 이용한 동정결과 B. pumilus로 동정되었다 본 실험에서 분리된 B. pumilus는 기존에 활발히 연구되어 있지 않는 균주로서 native feather 분해도가 기존에 알려진 Bacillus 속들이 3일 정도에서 분해가 완료되는 것과는 달리 36시간$\sim$48시간 내에 깃대까지 완전히 분해하였다.

• Proceedings of the Korean Environmental Sciences Society Conference
• /
• 2006.11a
• /
• pp.432-435
• /
• 2006

전 세계적으로 방대하게 배출되는 도축 부산물인 우모는 높은 영양적 가치를 가짐에도 불구하고 현재 사용하는 물리화학적 처리법의 여러 단점으로 효율적으로 이용되지 못하고 있는 실정이다. 그 결과 2차 오염을 발생시키지 않는 경제적 방법인 생물학적 처리에 대한 연구의 필요성이 커지고 있다. 본 연구에서는 그러한 방법 중 대표적인 방법으로 미생물이 생산하는 keratinase를 이용한 생분해에 대해 연구를 하기 위해 경남 일대 퇴비화 볏짚에서 keratinolytic protease 생성능이 우수한 균주인 RS7을 분리하였고 생화학적 동정법과 16S rDNA를 이용한 동정결과 B. pumilus로 동정되었다. 본 실험에서 분리된 B. pumilus는 기존에 활발히 연구되어 있지 않는 균주로써 native feather 분해도가 기존에 알려진 Bacillus 속들이 3일 정도에서 분해 완료되는 것과는 달리 36시간 ${\sim}$48시간 내에 깃대까지 완전히 분해하였다. 또한 분리 균주의 경제적인 분해능을 토대로 분해산물이 식물 생장에 주는 영향과 아미노산 함유량을 검토한 결과 기존의 화학적 분해에 의한 분해산물보다 아미노산 함유량이 4배가량 많았으며 식물 생장에 있어서도 생장율과 개화시점으로 미루어볼 때 비료로써의 역할을 수행할 수 있었다.