角蛋白酶的研究与应用前景

角蛋白酶(keratinase) 是一种可以特异性降解角蛋白的酶类,其来源广泛,多种微生物在羽毛降解过程中均可产生角蛋白酶。不同菌种来源的角蛋白酶,其结构、理化性质、活性和底物也不同。其在饲料行业、制革工业和环境废弃物处理等多个方面具有广泛的应用前景,能够产生巨大的社会效益和经济效益。本文系统总结了角蛋白酶的来源、分类、理化性质、作用机理及其在基因工程研究等方面的一些最新进展,简要介绍了其应用研究现状,并展望了角蛋白酶的应用前景。     

角蛋白酶研究进展

角蛋白化学结构稳定,不易被一般的蛋白酶降解;但作为角蛋白酶的专一性底物,角蛋白可被角蛋白酶降解.本就角蛋白酶的生产菌、降解角蛋白的生化机制、角蛋白酶的生化及分子生物学特性等方面的最新研究进展作一综述.     

链霉菌降解角蛋白的生化机制研究

对弗氏链霉菌S-221变种降解角蛋白的生化机制进行了初步研究。该菌在角蛋白底物作用下诱导产生角蛋白酶。它是一种复合蛋白酶,含有二硫键还原酶和多肽水解酶等多种酶活性组分。硫酸钠、亚硫酸钠和巯基乙醇对角蛋白酶具有强烈的激活作用,其主要表现作用于角蛋白酶中的二硫键还原酶。亚硫酸钠在0．01mol／L浓度下不仅作用于二硫键还原酶,而且还作用于多肽水解酶。硫代硫酸钠对二硫键还原酶有强烈的抑制作用。角蛋白酶降解羽毛角蛋白首先是角蛋白酶中的二硫键还原酶使角蛋白中二硫键裂解产生变性角蛋白,然后变性角蛋白在多肽水解酶的共同作用下逐步水解成多肽、寡肽和游离氨基酸,使角蛋白彻底降解。在角蛋白降解过程中,角蛋白中的硫也随之转化成巯基化合物,H2S和硫酸盐3种含硫化合物存在于降解产物中。     

Stenotrophomonas maltophilia产角蛋白酶对羊毛的作用机理探讨

采用Stenotrophomonas maltophilia产角蛋白酶降解羊毛角朊蛋白,通过测定反应前后残液中巯基和肽键的变化探讨角蛋白酶作用机理。结果表明,角蛋白酶主要作用是断裂角蛋白中的二硫键,也能降解大分子蛋白质,但作用效果不强。羊毛胱氨酸分析结果进一步证明角蛋白酶能断裂羊毛鳞片层中胱氨酸二硫键。SEM结果显示单独使用角蛋白酶对羊毛鳞片去除效果不佳,但角蛋白酶和蛋白酶二浴法工艺能有效降解、剥离羊毛鳞片。     

角蛋白酶研究进展

角蛋白酶（keratinase）是分解角蛋白的一类蛋白酶,应用广泛,如分解含角蛋白的毛发、羽毛、角蹄、鳞等等,而角蛋白的化学结构稳定,不溶于水,一般的蛋白酶对它们降解不起什么作用。只有角蛋白酶对角蛋白有分解活性,所以称这种酶为角蛋白酶。     

链霉菌降解角蛋白的生化机制研究*

对弗氏链霉菌S-221变种降解角蛋白的生化机制进行了初步研究。该菌在角蛋白底物作用下诱导产生角蛋白酶。它是一种复合蛋白酶,含有二硫键还原酶和多肽水解酶等多种酶活性组分。硫酸钠、亚硫酸钠和巯基乙醇对角蛋白酶具有强烈的激活作用,其主要表现作用于角蛋白酶中的二硫键还原酶。亚硫酸钠在0·01mol/L浓度下不仅作用于二硫键还原酶,而且还作用于多肽水解酶。硫代硫酸钠对二硫键还原酶有强烈的抑制作用。角蛋白酶降解羽毛角蛋白首先是角蛋白酶中的二硫键还原酶使角蛋白中二硫键裂解产生变性角蛋白,然后变性角蛋白在多肽水解酶的共同     

角蛋白单体获得及诱导S.maltophilia DHHJ产角蛋白酶研究

可降解羽毛微生物能以羽毛粉为唯一营养源生长,而羽毛粉是一种大颗粒的不溶性底物,不能直接进入细胞内作为营养源同时作为一级信使来诱导酶基因表达.本文通过化学还原法水解羽毛得到可溶性羽毛角蛋白溶液,利用电泳和质谱验证其分子量约10 kD,为角蛋白单体.分别以该可溶性羽毛角蛋白及角蛋白单体酶解片段为诱导源,在无诱导源、羽毛粉为对照的情况下,测定72 h内嗜麦芽窄食单胞菌(S.maltophilia)DHHJ产生的角蛋白酶的酶活.在无诱导源时,角蛋白酶基因表现本底表达(0.5 U/mL);培养基中添加羽毛粉及角蛋白单体时,角蛋白酶表达量高,分别可达15 U/mL和20 U/mL.并且角蛋白单体酶解片段诱导酶表达较低(2.3 U/mL).该结果初步表明,水溶性角蛋白单体作为信号源与细菌细胞接触或进入细胞内,控制角蛋白酶基因表达.该结果为细菌降解角蛋白分子机制研究奠定基础.     

角蛋白酶的结构、功能及应用

角蛋白是广泛存在于羽毛及毛发中的难溶性蛋白质。角蛋白酶可以催化角蛋白的降解，在畜牧、皮革加工、医疗等领域中具有较大的应用价值与潜力。近年来，研究人员对角蛋白酶的来源、分类、结构、功能优化等方面进行了大量的研究，并取得了很多成果，为角蛋白酶的商业化应用提供了很好的基础。本文综述了角蛋白酶结构、功能、应用等方面的研究进展，并提出了今后的研究方向。     

科技信息

角蛋白酶研究进展角蛋白酶(keratinase)是分解角蛋白的一类蛋白酶,应用广泛,如分解含角蛋白的毛发、羽毛、角蹄、鳞等等,而角蛋白的化学结构稳定,不溶于水,一般的蛋白酶对它们降解不起什么作用。只有角蛋白酶对角蛋白有分解活性,所以称这种酶为角蛋白酶。除某些真菌、放线菌具有产生角蛋白酶能力之外,在细菌中某些细菌菌种具有分解角蛋白的活力,如黄杆菌属Chryseobacterium、Bacillus等属的某些种产生角蛋白酶均有分解角蛋白酶能力,其中有两种细菌值得注意:一是地衣芽孢杆菌(Bac.lincheniformis),它似乎以羽毛为唯一有机底物生长繁殖,从分…     

角蛋白酶kerC基因的克隆及序列分析

为克隆分析角蛋白酶ker C基因,本研究以羽毛为底物从11株实验室保存的芽孢杆菌中筛选角蛋白酶产生菌。得到了一株具有高效降解羽毛能力的枯草芽孢杆菌BS10,该菌株3 d即可降解一根完整的羽毛,以羽毛粉、天青角蛋白为底物测定其酶活力,分别达(1.88±0.10)U/mL、(1.79±0.49)U/mL。以同源克隆的方法克隆ker C基因,获得了一条全长1 149 bp的kerC基因(Gen Bank登录号:KX108888),编码383个氨基酸。利用BLAST、ProtParm、SOPMA和MEGA等生物信息学工具对其理化性质、二级结构及系统进化树等进行分析,发现其与NCBI中的角蛋白酶基因相似性达到85%;相对分子质量为39.095 kD,等电点为9.23;该基因蛋白为亲水性蛋白;系统进化树分析结果与菌株信息相吻合。羽毛降解菌的获得可促进废弃羽毛资源化利用;角蛋白酶基因的获得及其分子特征的分析,为进一步通过基因工程手段提高角蛋白酶活性提供了一定的理论依据。     

微生物角蛋白酶的分子生物学研究进展

角蛋白酶具有分解角蛋白的活性,在饲料,食品加工,环境废物处理等方面,具有广泛的应用前景。本文综述了微生物角蛋白酶的来源,角蛋白酶的理化性质,作用机理,基因分离和表达等分子生物学和基因工程研究进展,并对其应用前景进行了展望,对其今后的发展趋势进行了讨论。     

Characterization of an alkaline active-thiol forming extracellular serine keratinase by the newly isolated Bacillus pumilus

Aims: The aim of the study was to optimize microbial degradation of keratinous waste and to characterize the alkaline active keratinase showing its biotechnological importance. Method and Results: An extracellular keratinase enzyme was purified from the culture medium of a bacterial isolate and the conditions were optimized. The molecular weight of DEAE‐Sepharose‐purified keratinase was determined by SDS‐PAGE. Instrumental analyses were investigated to study the mechanism of bovine hair hydrolysis. Isolate was identified as Bacillus pumilus based on phenotypic characteristics and 16S rDNA sequence. The optimized condition for its growth was pH 8 and 35°C. The molecular weight of the keratinase was estimated as 65 kDa. Activity inhibition by phenyl methyl sulphonyl fluoride confirmed keratinase as serine protease type. Instrumental analysis revealed the sulphitolysis and proteolysis involved mechanism in bovine hair hydrolysis. Conclusion: This study indicates that the isolated keratinase is an alkaline active serine protease with a high degree of activity towards bovine hair. Significance and Impact of the Study: This study examines a serine protease with high keratinolytic activity and degradation mechanism for bovine hair. The keratinolytic activity of the isolated strain and its reaction mechanism on bovine hair could show biotechnological potential in the leather industry.     

Microbial production and industrial applications of keratinases: an overview

Massive production of keratinaceous byproducts in the form of agricultural and industrial wastes throughout the world necessitates its justified utilization. Chemical treatment of keratin waste is proclaimed as an eco-destructive approach by various researchers since it generates secondary pollutants. Microbial degradation of keratin waste is an emerging and eco-friendly approach and offers dual benefits, i.e., treatment of recalcitrant pollutant (keratin) and procurement of a commercially important enzyme (keratinase). This review summarizes the potential utility of some bacterial and fungal species for the production of keratinase using a variety of keratinaceous wastes as growth substrates. The application of microbial keratinases in waste management; animal feed, detergent, and fertilizer manufacturing; and leather, cosmetic, and pharmaceutical industries is also abridged in this review.     

Azo dying of α-keratin material improves microbial keratinase screening and standardization

Microbial conversion through enzymatic reactions has received a lot of attention as a cost-effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current available methods lack sensitivity and standardization. Here, we suggest an optimized Azokeratin assay, with substrate generated directly from azo-dyed raw keratin material. We introduced supernatant filtration in the protocol for optimal stopping of keratinase reactions instead of the widely used trichloroacetic acid (TCA), as it generated biases and impacted the sensitivity. We furthermore suggest a method for standardization of keratinase activity signals using proteinase K, a well-known keratinase, as a reference enabling reproducibility between studies. Lastly, we evaluated our developed method with several bacterial isolates through benchmarking against a commercial assay (Keratin Azure). Under different setups, the Azokeratin method was more sensitive than commonly used Keratin Azure-based assays (3-fold). We argue that this method could be applied with any type of keratin substrate, enabling more robust and sensitive results which can be used for further comparison with other studies, thus representing an important progress within the field of microbial keratin degradation.     

Viable airborne microbial counts from air-cooling units with and without complaints of urine and body odors

Viable airborne microbial counts are commonly used in indoor air quality (IAQ) assessment, but studies linking the microbial counts to a specific type of indoor microbial contamination are limited. We hypothesize that the airborne microbial counts can differentiate air-cooling units with and without complaints of urine and body odors. The keratinolytic property of some isolated bacteria prompts to the hypothesis that keratinase is present in the units to break down keratins, structural proteins that form human skin scales, as sources of amino acids and ammonium to produce the odors. Seven bacterial species and four fungal species were identified in the units and room air. Airborne Staphylococcus haemolyticus and Methylobacterium organophilum counts contributed the most to the microbial dissimilarities of units with and without odor complaints. Keratinolytic bacteria and a methylotrophic bacterium were abundant in the units. All the units contained ammonium, and keratinase activity was higher in the units with odor complaints. Extracellular keratinase activity was more effective at 20 °C than at 30 or 4 °C. Keratinolytic bacteria produced high levels of ammonium in the culture with skin cells. Viable airborne microbial counts can help IAQ inspectors to identify potential odor-causing air-cooling units. Keratins may be broken down in the units and associated with the odor complaints.     

Versatility and commercial status of microbial keratinases: a review

The world’s increasing population and shortage of food and feed is creating an urgently for us to look for new protein sources from waste products like keratinous waste. Poor management of these wastes has made them one of the major recalcitrant pollutants in nature. Microbial keratinases offers an economic and eco-friendly alternative for degrading and recycling keratinous waste into valuable byproducts. Diverse groups of microorganisms viz., bacteria, fungi and actinomycetes have the ability to degrade recalcitrant keratin by producing keratinase enzyme. Microbial keratinases exhibits great diversity in its biochemical properties with respect to activity and stability in various pH and temperature ranges as well as in the range of recalcitrant proteins it degrades like those present in feathers, hairs, nails, hooves etc. Owing to diverse properties and multifarious biotechnological implications, keratinases can be considered as promising biocatalysts for preparation of animal nutrients, protein supplements, leather processing, fiber modification, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical, cosmetic and biomedical industries, and waste management. This review article presents an overview of keratin structure and composition, mechanism of microbial keratinolysis, diversity of keratinolytic microorganisms, and their potential applications in various fields.     

Keratinase Production by Newly Isolated Antarctic Actinomycete Strains

Summary The ability of actinomycete strains newly isolated from Antarctic soils to produce keratinolytic enzymes during growth on sheep wool waste was investigated. The strains which displayed highest keratinase activity and identified as Streptomyces flavis 2BG (mesophilic) and Microbispora aerata IMBAS-11A (thermophilic) were selected for a more detailed analysis. The addition of starch to the growth medium affected keratinase secretion by both strains. After 5 days of cultivation, a 6-fold increase in keratinase activity of strain 11A was observed in the presence of 11 g starch/l and a 9-fold increase in keratinase activity of the strain 2BG in the presence of 5 g starch/l. The results obtained showed that both newly isolated strains are very promising for effective processing of native keratinous wastes. To our knowledge, this is the first report of Antarctic actinomycete strains that were able to grow on keratin-containing wastes by producing keratinolytic enzymes.     

Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1

Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2?U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2?U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65?°C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50–60?°C for almost 90?min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.