Solubilization of Feather Keratin by a Copper-amine Complex

Chicken feather keratin was solubilized by cupri-ethylenediamine treatment and the solubilized products were separated into acidic and basic fractions by ion exchangers. In the solubilized products which had a molecular weight between 10,000 and 60,000, all the original cystine residues disappeared and cysteic acid residues were recovered instead of them but partly. The cupri-ethylenediamine reagent which catalyzed air-oxidation of cystine residues in keratin was removable mostly from the products by dialysis against water. The common copper-amine complexes were ineffective to solubilize feather keratin except for Schweitzer’s reagent. One strongly basic, unusual amino acid was detected in the basic solubilized fraction. This amino acid was eluted after arginine by usual column chromatography.     

Studies on the Structure of Feather Keratin: II. A β-Helix Model for the Structure of Feather Keratin

The assumption that the proline residues in feather keratin, which comprise 12 per cent of the total, are periodically located along the polypeptide chain is shown to lead to an essentially unique structure for this fibrous protein. The structure is based on a β-helix; i.e., an extended chain which coils slowly to form a helix of relatively large pitch. Such helices tend to aggregate by hydrogen bonding to form cylindrical units, which in turn can aggregate further into cable-like structures. This model has been tested with respect to its predictions concerning the x-ray diffraction pattern, infrared spectrum, mechanical properties, and chemical behavior of feather keratin. Preliminary results indicate that it is better capable of accounting for the data than previously proposed structures.     

嗜热脂肪芽孢杆菌高温蛋白酶分解毛发角蛋白的研究

本文研究了嗜热脂肪芽孢杆菌 (Bacillusstearothermophilus)WF - 1 4 6高温蛋白酶对毛发角蛋白的水解作用。结果表明 ,在体积分数为 2 %的巯基乙醇存在的条件下 ,WF - 1 4 6高温蛋白酶对毛发角蛋白有明显的水解作用。对酶解液氨基酸分析表明 ,酶解液中含有对照液中没有的游离蛋氨酸和亮氨酸 ,且游离氨基酸总量是对照样品的 2 .4倍 ,达 1 5 8mg·L-1 。此外 ,酶解液中含有大量的肽     

Hydrolysis of Heparin by the Immobilized Enzymatic Complex from Streptomyces kurssanovii

The possibility of obtaining low-molecular-weight heparins using a chitinolytic enzymatic complex immobilized on Silochrom has been demonstrated. The optimal conditions of this process (sodium acetate buffer, pH 7.0–7.5; temperature, 40–45°C; and duration of hydrolysis, 3 h) were determined. Depending on the ratio between heparin and the immobilized enzymatic complex, samples with molecular weights varying from 1.7 to 4.7 kDa, were obtained. These complexes inhibited factor Xa 2.0–3.7 times more effectively than the original heparin.     

降解鸡毛真菌板蜡蚧的鉴定及产酶培养优化

鉴定降解鸡毛真菌并通过单因素和正交实验优化其产角蛋白酶发酵培养条件.从加入鸡毛粉钓饵的医院花坛土中分离筛选获得3株角蛋白高效降解真菌,利用形态学和分子系统学鉴定均为板蜡蚧(Lecani-cillium testudineum).单因素实验表明,对优选菌株1Y2-12产酶能力具促进作用的碳源为乳糖,氮源为酵母膏,无机离子...     

Studies on the Structure of Feather Keratin: I. X-Ray Diffraction Studies and Other Experimental Data

Previous data as well as new results are examined with a view to determining the boundary conditions which present experimental information places on a satisfactory polypeptide chain model for the structure of feather keratin. Our studies indicate these conditions to include the following: (1) A 189 A identity period, with a pseudoidentity of 94.5 A; (2) characteristic fiber axis periodicities of 23.6₄, A and 18.9 A; (3) a meridian reflection of 2.96 A, but none in the 1.0 A region; (4) a strong, but sensitive, equatorial reflection of about 33 A spacing, with a possible equatorial reflection near 50 A; (5) perpendicular infrared dichroism of v (NH) of at least 5:1; (6) a limited extensibility along the fiber axis direction; (7) the natural accommodation of about 12 per cent of proline residues in the structure; (8) the possibility of breaking down the structure into units of about 10,000 molecular weight. The implications of these conditions with respect to a satisfactory model are considered.     

Keratinase of Doratomyces microsporus

 The fungus Doratomyces microsporus produced an extracellular keratinase during submerged aerobic cultivation in a medium containing a protein inducer for enzyme synthesis. The keratinase was purified to homogeneity using hydrophobic interaction chromatography followed by gel chromatography. The molecular weight was estimated to be 33 kDa (from SDS-PAGE analysis) or 30 kDa (by gel chromatography), suggesting a monomeric structure. The isoelectric point of the enzyme was determined to be around 9. The optimal pH and temperature for keratinolytic activity were pH 8–9 and 50 °C, respectively. The serine protease inhibitor PMSF totally inhibited the keratinase. The enzyme was not glycosylated. It was capable of hydrolysing different keratinous materials as well as some non-keratinous proteins. Hydrolysis of some synthetic substrates, specific for known proteinases, suggested that the keratinase of D. microsporus is close to proteinase K. Received: 9 July 1999 / Received revision: 13 September 1999 / Accepted: 17 September 1999     

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.     

Keratinase of a streptomycete.

Keratinase produced from Streptomyces Sp.A11 decomposed human hair, chicken feather, wool, silk and pure keratin extracted from human epidermis. Purification of the enzyme by DEAE-cellulose column chromatography resulted in 7.5-fold increase in activity relative to the activity of the culture filtrate. The enzyme was inducible, extracellular, homogeneous with a molecular weight of 49,000. The enzyme activity was inhibited by reduced glutathione, phenylmethyl sulphonyl fluoride and 2-mercaptoethanol.     

固定化酶-离子交换组合系统进行青霉素G水解生产6-APA的模型化研究

采用固定化青霉素酰化酶(Penicillin acylase)在反应器中进行青霉素G水解生产6－APA,同时与离子交换柱相组合以连续地去除反应混合液中的苯乙酸。建立了离子变换柱的分格模型(Comparunent model)．在确定了青霉素G和苯乙酸沿柱高的浓度分布的基础上,与描述固定化酶反应器的状态方程相结合,得到了固定化酶－离子交换组合系统的数学模型。在将计算机模拟值与实验值进行验证后,探讨了组合系统中树脂量、循环流速和组合起始时间对青霉素G酶解过程的影响。     

Hydrolysis of Galactose from Glycolipids and Glycoprotein by Young Rat Brain β-Galactosidases Immobilized to Sepharose 4B

An extract of glycosidic enzymes from young rat brain was immobilized to cyanogen bromide-activated Sepharose 4B. Most glycosidases retained approximately 10-25% of their activities after immobilization. Immobilized β-galactosidases were used repeatedly without detectable loss of enzyme activity in the hydrolysis of p-nitrophenyl-β-d -galactopyranoside. In addition to the synthetic substrate, the immobilized rat brain β-galactosidases could also hydrolyze galactose from lactose, galactosylcerebroside, asialofetuin, and GM₁-ganglioside. The hydrolysis of GM₁- to GM₂-ganglioside was confirmed on TLC.     

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.     

Hydrolysis of Butteroil by Immobilized Lipase Using a Hollow-Fiber Reactor: Part VI. Multiresponse Analyses of Temperature and pH Effects

A lipase from Aspergillus niger, immobilized by physical adsorption on hydrophobic hollow fibers made of microporous polypropylene, was used to effect the hydrolysis of the glycerides of melted butterfat at 40, 50, 55, and 60°C (pH 7.0), and at pH 3.0, 4.0, 5.0, 7.0, 8.0, and 9.0 (40°C). McIlvane buffer and melted butterfat were pumped cocurrently through the hollow fiber reactor. The concentrations of ten different free fatty acids in the effluent oil stream were measured by HPLC. Multiresponse nonlinear regression methods were employed to fit the data to multisubstrate rate expressions derived from a Ping Pong Bi Bi mechanism in which the rate controlling step is deacylation of the enzyme. Thermal deactivation of the immobilized lipase was also included in the mathematical model of reactor performance. A postulated normal distribution of v_max with respect to the number of carbon atoms of the fatty acid residue (with an additive correction for the number of double bonds) was found to provide the best statistical fit of the data. The models developed can be used to independently predict the effects of either the pH or the temperature, as well as the reactor space time and the time elapsed after immobilization, on the free fatty acid profile of the lipolyzed butteroil product.     

固定化酵母乙醇发酵及固化小球微生态的研究

比较了SA-PVA-SiO₂固定化酿酒酵母和游离酿酒酵母的乙醇发酵能力,采用批次发酵试验研究固定化酿酒酵母的发酵稳定性。结果表明,SA-PVA-SiO₂固定化酿酒酵母的发酵速度比游离酿酒酵母快,发酵周期短;发酵稳定性很好,30℃,橡胶塞、90 r/min摇床培养24 h时,乙醇体积分数均在3%~3.5％之间,连续发酵14批次后,固化小球的形态依然完好,不发粘。通过扫描电镜对酿酒酵母包埋微生态环境进行了分析,图像表明固定化小球的内部环境非常有利于酵母细胞的厌氧发酵产乙醇,充分证明了SA-PVA-SiO₂固定化酿酒酵母乙醇发酵的优越性。     

Sedimentation Studies of Epidermal Keratins. Keratin A and Keratin B

Electrophoretically homogeneous keratin A and keratin B were studied in the ultracentrifuge. Both preparations revealed two fractions: one which sedimented rapidly and another which sedimented slowly. This indicated that both preparations are heterogeneous with respect to particle size.     

Feather degradation by Kocuria rosea in submerged culture

A strain of Kocuria rosea with keratinolytic activity was studied. In batch culture, the optimum temperature for feather degradation, bacterial growth and protease secretion was at 40 °C. A specific growth rate of 0.17 h⁻¹ was attained in basal medium with feathers as fermentation substrate. Under these conditions, after 36 h of incubation, biomass and caseinolytic activity reached 3.2 g/l and 0.15 U/ml, respectively. Extracellular protease secretion was associated with the exponential growth phase. In batch fermentation, feather degradation up to 51% in 72 h was obtained with a conversion yield in biomass of 0.32 g/g. No organic acids were detected in the fermentation broth in significant amount. This revised version was published online in July 2006 with corrections to the Cover Date.