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Purification and characterization of keratinase from a new Bacillus subtilis strain

Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2008年第9期9卷 713-720页

作者： Cheng-gang CAI1,2, Ji-shuang CHEN3, Jiong-jiong QI2, Yun YIN2, Xiao-dong ZHENG2 (1College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China) (2College of Food Science and Biosystem Engineering, Zhejiang University, Hangzhou 310029, China) (3Institute of Bioengineering, Zhejiang Sci-Tech University, Hongzhou 310018, China) College of Biology and Environmental Engineering Zhejiang Shuren University Hangzhou 310015 China College of Food Science and Biosystem Engineering Zhejiang University Hangzhou 310029 China.

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 °C was 8.5 and the optimum temperature at pH 8.5 was 55 °C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.

关键词： Ammonium sulfatate Bacillus subtilis Characterization Feather Keratin keratinase Purification Reducing agents

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Recent advances in keratinase production via protein engineering,breeding,and fermentation

Advanced Agrochem

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Advanced Agrochem 2024年第3期3卷 188-196页

作者： Ali Raza Ishaq Zheng Zhang Penghui He Min Xiong Shouwen Chen State Key Laboratory of Biocatalysis and Enzyme Engineering Environmental Microbial Technology Center of Hubei ProvinceCollege of Life SciencesHubei UniversityWuhan430062China

The gene editing and synthetic biological tools have led to the implementation of diverse metabolic engineering approaches to enhance the production of specific *** keratinases can convert keratin wastes into valuable compounds for *** the market for keratinases cannot be satisfied by production from wild hosts,it is obligatory to develop hosts with high keratinase *** intention of this review is to evaluate microbial keratinase advancement through protein engineering,breeding techniques,and fermentation *** main aim of protein engineering is to improve the heat resistance ability and catalytic activity of keratinases by employing mutagenesis ***,modifying the expression elements and host engineering are also two unique ways to augment the keratinase *** to accelerate the production of modified keratinase,this review attempts to highlight the optimization of expression elements,such as promoter engineering,UTR,signal peptide,and codon ***,the approaches of host engineering including strengthening precursor supply,membrane surface engineering,and optimization of secretion pathways were also explained ***,it is also essential to optimize the medium composition and fermentation condition for high keratinase *** review also addressed the present advancements,difficulties,and tendencies in the field of microbial keratinase production,along with its potential.

关键词： keratinase Protein engineering Expression elements Breeding Fermentation

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Applying the mutation of <i>Bacillus subtilis</i>and the optimization of feather fermentation medium to improve <i>keratinase</i>activity

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Advances in Biological Chemistry 2012年第1期2卷 64-69页

作者： Xin Zhang Jilin Business and Technology College Changchun China

Energetic Bacillus subtilis was preliminarily isolated from feather meal selection medium experiment, which could be used to produce keratinase. Through using the ultraviolet ray produced by ultraviolet light and the compound mutation of sodium nitrite solution, a mutated strain was produced which can yield Kerati-nase with a high activity. The activity of keratinase was 75.9% higher than that before the compound mutation. This was achieved by optimizing the fermentation medium of mutated strain. The optimum fermentation medium had the parameters as feather meal 5.5%, maize silage 0.8%, K+ 0.018 mol/L, Mg2+ 0.065 mol/L, Ca2+ 0.072 mol/L, Fe2+ 0.010 mol/L, and Na+ 0.088 mol/L. Based on the optimized fermentation medium, the highest yielding rate for producing enzyme was 1.117 U/mL, 25.22% higher than that before the optimization. The amino acid in the fermentation medium fluid reached 22.66 mg/mL. This paper presents a simple, and low cost way to produce high quality bio-fermentation feather meal.

关键词： keratinase Activity Bacillus subtilis Mutation Optimization

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Nutritional Applications of keratinase

Nutritional Applications of Keratinase

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第二届全国家禽营养与饲料科技研讨会

作者： Jason C.H.Shih Jeng-Jie Wang J.D.Garlich North Carolina State University RaleighNC 27695-7608USA BioResource International Inc.MorrisvilleNC 27560USA

A feather-degrading bacterium,its keratinase,and the gene encoding this enzyme were discovered and isolated. Over expression of keratinase by genetic manipulation and fermentation in large scales made mass production of the enzyme *** proteolytically active on all kinds animal and plant proteins tested,keratinase has been tested for nutritional *** processing feathers,the enzyme was proved to be able to hydrolyze feathers into a more digestible feed pro- *** of the enzymatic process for feather meal is in *** the other hand,the enzyme was tested posi- tively as a feed *** the last seven years,numerous experiments have been carried out with broilers chickens. These studies included cage tests of young chicks to 3～4wk of age,pen trials on research farms to 6～7wk of market age and commercial farm tests conducted by private companies.A large poultry integrator in the *** using the commercial product of keratinase,namely,Versazyme,for broiler production in a large *** summary,Versazyme additive in- creases body weight gain at market age and decreases feed conversion ratio (FCR).It also improves meat yield of the broil- ers when moderate and high protein diets are *** intestinal keratinolytic activity and increased amino acid ileal digestibility were found in the birds on the diets supplemented with *** a study at CAU,it was found that cot- tonseed meal could replace 50% soybean meal in feed without depressing the broiler growth when Versazyme additive was *** summary,a new enzyme additive called Versazyme that improves protein digestibility has been discovered and introduced to the feed and animal *** the original laboratory discovery made 20 years ago to a new product in the market place,it is an exemplary case of the development of knowledge-based agricultural technology and business.

关键词： Feather degradation keratinase VersazymeTM Feed enzyme Protein nutrition Broiler chicken

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Screening for a new Streptomyces strain capable of efficient keratin degradation

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Journal of Environmental Sciences 2007年第9期19卷 1125-1128页

作者： CHAO Ya-peng XIE Fu-hong YANG Jing LU Jing-hua QIAN Shi-jun State Key Laboratories of Transducer Technology Institute of Microbiology Chinese Academy of Sciences Beijing 100080 China.

Keratinous wastes could be degraded by some microorganisms in nature. Native human foot skin （NHFS） was used as sole nitrogen source to screen microorganisms with keratin-degrading capability. From approximately 200 strains, a strain of Streptomyces sp. strain No. 16 was found to possess the strongest keratinolytic activity, and the total activity in the culture was 110 KU/ml with specific activity of 2870 KU/mg protein （KU： keratinase unit）. Substrate specificity test indicated that the crude keratinase could degrade keratin azure, human hair, cock feathers and collagen. The optimal pH of the crude keratinase ranged from 7.5 to 10 and the temperature ranged from 40℃ to 55℃. Metal chelating agent ethylenediamine tetraacetic acid obviously stimulated the keratinolytic activity but suppressed the proteolytic activity. To our knowledge, this is the first report on specific induction of keratinases by NHFS from an actinomycete. Moreover, excellent characteristics of its crude keratinase may lead to the potential application in waste treatment and recovery, poultry and leather industry, medicine, and cosmetic development.

关键词： keratinolytic capability Streptomyces sp. native human foot skin (NHFS) keratinase substrate specificity

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Distribution and Prevalence of Dermatophytes in Semi-Arid Region of India

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Advances in Microbiology 2015年第2期5卷 93-106页

作者： Vishnu Sharma Tarun Kumar Kumawat Anima Sharma Ruchi Seth Subhash Chandra Department of Biotechnology JECRC University Jaipur India Vardhaman Mahaveer Open University Kota India

Earth has been documented as a natural territory for fungi which cover individual kingdom with evolution. In subsequently vertebrates developed keratin which was a part of life as a structural aspect. Few moulds have skilled to digest keratin and crop up from soil and wastewater habitats. They take part as a keratinolytic agent in the purification of α-keratins with an incidence of disulphide and hydrogen bonds which are improperly biodegradable. The best moulds genera to decay of keratin are Microsporum, Trichophyton and Epidermophyton. The presences of these genera are open health issues in developing countries where they cause the mortal mycotic contagion. The reason behind this is perceived to be the poor hygienic environment and socioeconomic behaviour among people. The present review is a compilation of updated information concerning the nature of these keratinolytic moulds and abundances of most contributed developing countries including India.

关键词： Moulds Keratinophytes Dermatophytes keratinase Mycoses

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Isolation and identification of two feather-degrading strains of bacterium from spider gut and the purification and analysis of related key microbial enzymes

Isolation and identification of two feather-degrading strain...

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中国生物化学与分子生物学会第十二届全国会员代表大会暨2018年全国学术会议

作者： Fang Qu Jinjun Chen College of Bioscience and Biotechnology Hunan Agricultural University

Microbial enzymes play an increasingly important role in industrial production,agriculture and daily life ***,some of these enzymes are derived from human-safe *** our study,two novel feather-degrading bacteria,named CA-1 and CH-1 respectively,were isolated from the gut of the spider Chilobrachys guangxiensis,and were identified by sequence analysis of 16 S rRNA genes and phenotypic characteristics,which can degrade native whole chicken *** on morphologic and molecular analysis,the CA-1 was confirmed to belong to Stenotrophomonas maltophilia,CH-1 as a Bacillus ***,the purified enzymes under the optimal and suppressive conditions were analyzed respectively by SDS-PAGE and LC-MS/MS,and proteomic analysis of protein bands *** enzymes,namely alkaline serine protease(29.1 kDa),ABC transporter permease(27.5 kDa),and alkaline phosphatase(40.8 kDa),were purificated and identified from the supernatant of the CA-1 optimal culture and were considered to play principal *** kinds of enzymes which include extracellular protease Vpr(68 kDa),g-glutamyl transpeptidase(64 kDa),peptidase T(45 kDa) and glyoxalmethylglyoxal reductase(32 kDa) derived from *** study provides a new angle for discovering featherdegrading *** purification and analysis of the enzymes shines a light on further understanding the molecular mechanism of feather degrading by *** are also potential applications in cost-effectively degrading feathers into feeds and fertilizers through careful optimization and engineering of the newly identified enzymes.

关键词： Stenotrophomonas maltophilia Bacillus subtilis feather degradation keratinase spider gut synergic enzymes symbiotic microorganism

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