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。     

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     

角蛋白酶及其研究进展

角蛋白在一般情况下很难被降解,工业上用高温、高压的办法降解角蛋白。然而,角蛋白酶能在温和的条件下降解角蛋白。本文对角蛋白的理化性质、发酵条件和分子生物学进行了综述。     

微生物角蛋白酶的特性及其应用研究进展

角蛋白作为家禽加工和农业废弃物的主要成分,因其结构中富含能抵抗普通蛋白酶和化学催化剂降解的稳定交联二硫键而难以被利用,因此每年都在环境中大量积累,造成了严重的环境污染。微生物角蛋白酶可将角蛋白废弃物转化为可再次利用的产物,带来了经济的可行性及环境的可持续发展。本文主要综述了角蛋白酶的生物化学特性、角蛋白酶的基本结构及其表达特性,总结了其应用价值及角蛋白降解机制,最后展望了微生物角蛋白酶的进一步研究方向。     

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。     

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。     

角蛋白降解菌的筛选及其UV诱变选育

从长年堆积废羽毛的土壤,屠鸡场下水道、池塘底污泥中分离出一株角蛋白降解菌C-1,摇瓶发酵测得其角蛋白酶活达35.6U,经形态观察和生理生化鉴定该菌株属于芽孢杆菌属.对菌株进行Uv诱变,在照射时间为100 s下得到一株产角蛋白酶活较高突变株C-1-4,其酶活比原始出发菌株提高了122%,且遗传性状稳定.     

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。     

地衣芽胞杆菌来源角蛋白酶N端对活力及其热稳定性的影响

【目的】通过对一株地衣芽孢杆菌来源的角蛋白酶N端进行分子改造,研究其对角蛋白酶活力和热稳定性的影响,进而提高角蛋白酶的热稳定性。【方法】将角蛋白酶N端前5个氨基酸进行分段缺失,并通过序列比对将N端的前5个氨基酸替换为来源于Thermoactinomyces vulgaris的嗜热蛋白酶的N端,将野生型和突变体角蛋白酶基因在枯草芽孢杆菌WB600中进行表达,并对重组酶进行纯化与酶学性质研究。【结果】角蛋白酶N端不同长度的缺失大幅度地降低了角蛋白酶的活力,其中缺失前5个氨基酸完全丧失了酶活力。将角蛋白酶N端前5个氨基酸替换为嗜热蛋白酶N端前12个氨基酸,虽然降低了近70%的活力,但是却增加了角蛋白酶的热稳定性,60°C条件下的半衰期t1/2由原来的9 min提高到20 min。【结论】角蛋白酶的N端对其酶活力具有较大的影响,与嗜热蛋白酶来源的N端进行替换可以有效提高角蛋白酶的热稳定性。     

Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain

AIMS: Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. METHODS AND RESULTS: The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74.3 U ml(-1). The ker gene was further subcloned into E. coli-Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml(-1). Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166.2 U ml(-1) (specific activity, 33.25 U mg(-1)) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0.4 OD600 nm and xylose concentration of 0.75% w/v. CONCLUSIONS: Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three-fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.     

Synthesis and regulation of extracellular keratinase in three fungi isolated from the grounds of a gelatin factory,Jabalpur, India

Chrysosporium queenslandicum, Graphium penicilloideus andScopulariopsis brevicaulis were grown on various supplemented basal salts media to compare keratinase induction, activity and repression. All three fungi can utilize keratin as a sole source of carbon, nitrogen and sulfur. Total keratinase activity inC. queenslandicum andS. brevicaulis, was not repressed by supplementation of keratin-containing medium with glucose, ammonium or sulfate. The production of keratinase activity was not derepressible in keratin-free media. Keratin utilization commenced before the detection of significant extracellular keratinase activity which was always associated with mycelial growth.     

Functional analysis of the C-terminal propeptide of keratinase from Bacillus licheniformis BBE11-1 and its effect on the production of keratinase in Bacillus subtilis

The keratinase from Bacillus licheniformis BBE11-1 is a serine protease and expressed as a pre-pro-precursor. To produce a mature and active keratinase, the propeptide must be cleaved on the C-terminal via cis or trans. In this study, to enhance the production of keratinase in Bacillus subtilis, single amino acid substitutions, single residue deletions and linkers were introduced at the C-terminus of the propeptide. The results showed that optimizing the residue of cleavage site of propeptide will affect the cleavage efficiency of propeptide, and the mature enzyme yield of Leu(P1)Ala mutant increases 50% compared with the wild-type. In addition, inserting linkers and deleting individual residues at the C-terminal of the propeptide decreases the mature keratinase production. Our results indicated that the primary structure of the C-terminus of propeptide is crucial for the mature keratinase production. Propeptide engineering at C-terminus may be an effective approach to increase the yield of keratinase.     

Hydrolysis of Feather Keratin by Immobilized Keratinase

Keratinase isolated from Bacillus licheniformis PWD-1 was immobilized on controlled-pore glass beads. The immobilized keratinase demonstrated proteolytic activities against both insoluble feather keratin and soluble casein. It also displayed a higher level of heat stability and an increased tolerance toward acidic pHs compared with the free keratinase. During a continuous reaction at 50(deg)C, the immobilized keratinase retained 40% of the original enzyme activity after 7 days. The immobilized keratinase exhibits improved stability, thereby increasing its potential for use in numerous applications.     

Stability and Stabilisation of Doratomyces microsporus Keratinase

Thermal and pH stabilities of a new crude keratinase ( Doratomyces microsporus ) were investigated in the ranges of 20-40°C and pH 4-10, respectively. The stability test was followed by activity measurement on two different substrates: human stratum corneum and haemoglobin. Activity measurement lasted more than 100 h. The effect of calcium ions on enzyme stability was also studied. Crude keratinase was stabilised by crosslinking with glutaraldehyde (GA). The same characteristics were determined for Proteinase K, the commercial enzyme, for comparative purposes. Crude keratinase was most stable at pH 8 in Tris/HCl and borate buffers. The type of buffer used proved to have higher effect on crude keratinase stability than on Proteinase K. Both enzymes were most stable at 20°C. Keratinase stability rapidly decreased at 40°C while Proteinase K showed higher thermal stability. A 1 mM solution of Ca 2+ ions did not significantly influence enzyme stability, but 2.5% GA solution stabilised crude keratinase at 40°C reducing the k d value by about 50%. Crude and crosslinked crude keratinase were used for crude calf skin degradation. A mathematical model, based on Michaelis-Menten kinetics, was developed to describe the crude calf skin degradation in a batch reactor. Validation of the model showed that it could describe the process over a defined range of its conditions.     

Stability and Stabilisation of Doratomyces microsporus Keratinase

Thermal and pH stabilities of a new crude keratinase ( Doratomyces microsporus ) were investigated in the ranges of 20-40°C and pH 4-10, respectively. The stability test was followed by activity measurement on two different substrates: human stratum corneum and haemoglobin. Activity measurement lasted more than 100 h. The effect of calcium ions on enzyme stability was also studied. Crude keratinase was stabilised by crosslinking with glutaraldehyde (GA). The same characteristics were determined for Proteinase K, the commercial enzyme, for comparative purposes. Crude keratinase was most stable at pH 8 in Tris/HCl and borate buffers. The type of buffer used proved to have higher effect on crude keratinase stability than on Proteinase K. Both enzymes were most stable at 20°C. Keratinase stability rapidly decreased at 40°C while Proteinase K showed higher thermal stability. A 1 mM solution of Ca 2+ ions did not significantly influence enzyme stability, but 2.5% GA solution stabilised crude keratinase at 40°C reducing the k d value by about 50%. Crude and crosslinked crude keratinase were used for crude calf skin degradation. A mathematical model, based on Michaelis-Menten kinetics, was developed to describe the crude calf skin degradation in a batch reactor. Validation of the model showed that it could describe the process over a defined range of its conditions.     

Production and characterization of keratinase from<Emphasis Type="Italic">Paracoccus</Emphasis> sp. WJ-98

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K₂HPO₄, 0.04% KH₂PO₄, and 0.01% MgCl₂·6H₂O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn²⁺ and Hg²⁺. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.     

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.     

Phosphate-Mediated Alteration of the Microsporum gypseum Germination Protease Specificity for Substrate: Enhanced Keratinase Activity

Inorganic phosphate was found to decrease the caseinolytic and ethyl-esterase activities of the Microsporum gypseum germination protease. The germination protease possessed exokeratinase (beta-keratinase) activity immediately after release from the fungal spore. After phosphate treatment of the enzyme, the germination protease also possessed endo-keratinase (alpha-keratinase) activity. Phosphate altered the protease's pH optimum from 9.0 to 7.0 and decreased the molecular weight from 33,000 to 16,000. These values were identical to those found for the keratinase. Alpha- and beta-keratinase activities were stimulated in excess of 200-fold by disulfide reducing agents. Natural and suspected keratin degradation products also enhanced keratinase activity. Cell fractionation and in vitro conversion of the alkaline germination protease into a functional keratinase suggested that the subunits comprising the germination protease and the keratinase were of a common origin.