Enzyme Bioprospection of Marine-Derived Actinobacteria from the Chilean Coast and New Insight in the Mechanism of Keratin Degradation in Streptomyces sp. G11C

Marine actinobacteria are viewed as a promising source of enzymes with potential technological applications. They contribute to the turnover of complex biopolymers, such as pectin, lignocellulose, chitin, and keratin, being able to secrete a wide variety of extracellular enzymes. Among these, keratinases are a valuable alternative for recycling keratin-rich waste, which is generated in large quantities by the poultry industry. In this work, we explored the biocatalytic potential of 75 marine-derived actinobacterial strains, focusing mainly on the search for keratinases. A major part of the strains secreted industrially important enzymes, such as proteases, lipases, cellulases, amylases, and keratinases. Among these, we identified two streptomycete strains that presented great potential for recycling keratin wastes—Streptomyces sp. CHA1 and Streptomyces sp. G11C. Substrate concentration, incubation temperature, and, to a lesser extent, inoculum size were found to be important parameters that influenced the production of keratinolytic enzymes in both strains. In addition, proteomic analysis of culture broths from Streptomyces sp. G11C on turkey feathers showed a high abundance and diversity of peptidases, belonging mainly to the serine and metallo-superfamilies. Two proteases from families S08 and M06 were highly expressed. These results contributed to elucidate the mechanism of keratin degradation mediated by streptomycetes.     

Epidermal proteases of the Japanese eel

A fluorescent-sensitive assay was used to demonstrate the protease activity in the dorsal skin of Japanese eel (Anguilla japonica). Two distinct extracts were separately prepared from skin mucus and epidermal cell layers, with no mutual contamination. The epidermal extract was sensitive to various substrates, whereas there was no, or only marginal, susceptibility to the same substrates for the mucous extract. Optimum hydrolysis pHs of the epidermal extract was variable and below pH 7.0, and the optimum hydrolysis temperatures were between 40 and 50 °C. In addition, Tos-Phe-Ch₂Cl, chymostatin, CdCl₂, CuCl₂, HgCl₂ and ZnCl₂ inhibited protease activities to different extents. Several other reagents specifically affected the protease activities, and their induced effects were useful for the identification of epidermal proteases. The findings indicate that a proteolytic factor, exhibiting various enzymological specificities, is retained within epidermal cell layers of Japanese eel. This factor is composed of 4 distinct proteases, such as cathepsins L and B-like proteases, a serine protease and an aminopeptidase.     

Proteases of Clostridium botulinum. V. Studies on the serological relationship between proteases from Clostridium botulinum and other spore-forming bacteria

By the use of the electrophoretic casein precipitating inhibition test (CPI-test) the serological relationship between proteolytic enzymes produced by different species within the genera Clostridium and Bacillus has been tested. The proteases produced by Clostridium botulinum types A, B, C, D and F cross-reacted with each other. Clostridium botulinum strain 84 was inhibited by antiproteases produced against Clostridium sporogenes, Clostridium botulinum types C and F (protease F I and F II), but not by antiproteases against Clostridium botulinum types B and F (protease II), Clostridium bifermentans and Clostridium perfringens. The protease of the newly described Clostridium botulinum strain 89 (type G) was inhibited by Clostridium sporogenes antiprotease, but not by any of the other antiproteases. It is not possible to differentiate between Clostridium botulinum, Clostridium sporogenes and Clostridium perfringens by use of serological differentiation of their proteolytic enzymes. The protease of Clostridium bifermentans is not serologically related to any of the species tested in this investigation. Proteases produced by different Bacilli were not inhibited by antiproteases from Clostridium botulinum types B, C and F, Clostridium sporogenes, Clostridium bifermentans, and the two strains of Clostridium perfringens tested. This investigation indicates a serological relationship between proteases from different Clostridium species, but not a serological relationship between proteases produced by the Clostridium species and Bacillus species tested.     

腐蹄病节瘤拟杆菌的分子致病机制的研究进展

腐蹄病是反刍动物绵羊、山羊、鹿和奶牛等常见的一种高度接触性传染病。节瘤拟杆菌是致病作用的主要病菌之一,它是通过IV型纤毛和细胞外蛋白酶而产生作用。但从腐蹄病发病症状看,节瘤拟杆菌所致疾病的严重程度不是相同的,据此节瘤拟杆菌被分为毒性、弱毒性和良性菌,这种分类通过对该菌基因组毒性关联蛋白Vap(Virulence-associated protein)区域和毒性相关位点vrl(Virulence Related Locus)区域的研究,发现其致病特点与基因的顺序有很大联系。     

Proteolytic activity in soil: A review

The aim of this work is to review current knowledge on inputs, sources and regulation of protease activities in soils from different ecosystems, while exploring limitations to proteolysis and N mineralisation. Extracellular proteases enter the soil via microbial production and other sources, including plant root exudates, animal excrements, decomposition processes and leaching from agro-industrial fertilisers. The synthesis and activities of proteases in soil are regulated by many factors, including climate, soil properties and the presence of organic compounds of plant and microbial origin. Two particularly important areas for future research are the regulation of proteolysis by low-molecular-weight organic compounds, including amino acids, sugars, flavonoids, plant hormones and siderophores, as well as the identification and characterisation of proteinaceous protease inhibitors of plant and microbial origin in the soil. Despite all the work that has been performed on soil proteases, our understanding of the roles of extracellular plant root proteases in N nutrition is weak. Furthermore, the regulation of soil proteolytic activities of different ecosystems, especially in terms of pollutant inputs and the impact of climate change, requires investigation. Other areas that pose important questions for the future include assessments of protease inhibitor inputs to the soil, regulation of these inhibitors via naturally occurring soil organic compounds and the interactions between soil organisms.     

Development of Digestive Enzymes in California Halibut <Emphasis Type="Italic">Paralichthys californicus</Emphasis> Larvae

California halibut Paralichthys californicus is an important commercial species with high aquaculture potential in Baja California Sur, México. To optimize the feeding process using live prey and/or inert diets, we evaluated alkaline proteases, pepsin, trypsin, chymotrypsin, leucine aminopeptidase, lipase, α-amylase, and acid and alkaline phosphatase activities on starved larvae and larvae fed live prey. Highest activities were observed for alkaline protease, trypsin, chymotrypsin, leucine aminopeptidase, and alkaline phosphatase in feeding larvae than starved larvae on day 4 after hatching. At day 5, a sizeable increase in all enzymatic activities was detected in feeding larvae. Alkaline protease, trypsin, chymotrypsin, and alkaline phosphatase decreases progressively from day 5 until day 18. At day 18, a slight pepsin activity was observed. This was considered an indicator of the start of digestive system maturation. We concluded that total enzymatic equipment for this species is complete between day 18 and 30 after hatching. Based on this evidence, early weaning from live prey to inert feed would be possible at this time.     

Fractionation of hydrolase-humus complexes by gel chromatography

Summary Purification of soil phosphatase-, urease-, casein- and benzoylarginamide-hydrolysing proteases was obtained by exhaustively ultrafiltering a soil extract using 0.1M pyrophosphate solution at pH 7.1, separating the retained material into fractions of molecular weight higher (A_I) and lower (A_II) than 10⁵ and eluting the fractions on gel chromatography.Three peaks of phosphatase and urease activity were obtained after gel chromatography of fraction A_I on Sephadex G200 using 0.1M pyrophosphate solution as eluant. Only one distinct activity peak was observed when casein- and benzoylarginamide-hydrolysing proteases were assayed in the eluted fractions. Elution diagrams obtained by gel chromatography of fraction A_II on Sephadex G100, using a water as eluant, were characterized by one peak each of phosphatase-, casein- and benzoylarginamide-hydrolysing activity and by two peaks of urease activity.Gel chromatography of both A_I and A_II, generally, but not always, increased specific activity on a C and N basis of derivative fractions. Both proteases showed the highest increase in specific activity due to a marked decrease in organic C and N and an increase in total activity.     

Efficient Screening of Marine Extracts for Protease Inhibitors by Combining FRET Based Activity Assays and Surface Plasmon Resonance Spectroscopy Based Binding Assays

The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose.     

新疆阿勒泰和温泉县冷水鱼肠道产低温蛋白酶菌株的筛选及其酶学性质的研究

[目的]从50株冷水鱼肠道产蛋白酶菌株中筛选优良产酶菌株,并研究其酶学性质,为高效低温蛋白酶技术的研发奠定基础。[方法]对比脱脂乳平板初筛结果确定供试菌株,对选定菌株进行表型及16S rRNA/26S rRNA序列同源性分析,确定产酶菌株的进化地位。用Folin酚显色法复筛,并研究最佳产酶菌株所产蛋白酶的酶学性质。[结果]从6株供试菌株中筛选出一株高产酶菌株P-D-10,初步鉴定隶属于Pseudomonas。酶学性质的初步研究显示,P-D-10产生的蛋白酶最适反应pH在8.5左右;在0～60℃均有催化活性;具有10和40℃2个最适酶活温度;热稳定性较差,在70℃水浴10 min后剩余酶活力仅为20.14%。除了Tween-80对该酶酶活力有促进作用外,其他金属离子、还原剂、表面活性剂和金属离子螯合剂对该酶酶活力均有不同程度的抑制作用。[结论]筛选获得的高产酶菌株P-D-10隶属于Pseudomonas,是一株具有研究与应用潜力的产低温蛋白酶菌株。