Exploring the molecular mechanism of cold-adaption of an alkaline protease mutant by molecular dynamics simulations and residue interaction network.

Psychrophilic proteases have attracted enormous attention in past decades, due to their high catalytic activity at low temperatures in a wide range of industrial processes, especially in the detergent and leather industries. Among them, H5 is an alkaline protease mutant, which featuring psychrophilic-like behavior, but the reasons that H5 with higher activity at low temperatures are still poorly understood. Herein, the molecular dynamics (MD) simulations combined with residue interaction network (RIN) were utilized to investigate the mechanisms of the cold-adaption of mutant H5. The results demonstrated that two loops involved in the substrate binding G100-S104 and S125-S129 in H5 had higher mobility, and the distance enlargement between the two loops modulated the substrate's accessibility compared with wild type counterpart. Besides, H5 enhanced conformational flexibility by weakening salt bridges and increasing interaction with the solvent. In particular, the absence of Lys251-Asp197-Arg247 salt bridge network may contribute to the structural mobility. Based on the free energy landscape and molecular mechanics Poisson-Boltzmann surface area of the wild type and H5, it was elucidated that H5 possessed a large population of interconvertible conformations, resulting in the weaker substrate binding free energy. The calculated RIN topology parameters such as the average degree, average cluster coefficient, and average path length further verified that the mutant H5 attenuated residue-to-residue interactions. The investigation of the mechanisms by which how the residue mutation affects the stability and activity of enzymes provides a theoretical basis for the development of cold-adapted protease.

Development of a real-time loop-mediated isothermal amplification method for monitoring Pseudomonas lurida in raw milk throughout the year of pasture.

Introduction: The psychrophilic bacterium Pseudomonas lurida (P. lurida) and its thermostable alkaline proteases can seriously damage raw milk quality. Methods: In this study, specific primers were designed for P. lurida's gyrB and aprX genes, and a real-time loop-mediated isothermal amplification (RealAmp) rapid detection method was developed for the early monitoring of P. lurida and its proteases in raw milk. A phylogenetic tree of the gyrB and aprX genes of P. lurida was constructed to analyze the homology of the design sequence of the RealAmp primer. The DNA of 2 strains of P. lurida and 44 strains of non-P. lurida were detected via RealAmp to analyze the specificity of the primer. Results: It was found that aprX-positive proteases were produced by P. lurida-positive strains only when Pseudomonas fluorescens was negative. The dissociation temperatures of gyrB and aprX in the RealAmp-amplified products were approximately 85.0°C and 90.0°C, respectively. Moreover, DNA was detected through a 10-fold dilution of P. lurida in a pure bacterial solution and artificially contaminated skimmed milk. The limit of detection of P. lurida DNA copy number in the pure bacterial solution was 8.6 copies/µL and that in the 10% skimmed milk was 5.5 copies/µL. Further, 144 raw milk samples throughout the year from three farms in Hebei province were analyzed using RealAmp. The highest detection rate of P. lurida was 56% in the first and third quarters, and that of proteases was 36% in the second quarter. The detection rates of P. lurida and its proteases were the highest in samples collected from pasture 2 (52 and 46%, respectively), and the ability of P. lurida to produce proteases reached 88%. Discussion: In conclusion, RealAmp established an early and rapid method for the detection of P. lurida and its proteases in raw milk samples, allowing the identification and control of contamination sources in a timely manner to ensure the quality of milk and dairy products.

Fungal alkaline proteases and their potential applications in different industries.

The consumption of various enzymes in industrial applications around the world has increased immensely. Nowadays, industries are more focused on incorporating microbial enzymes in multiple processes to avoid the hazardous effects of chemicals. Among these commercially exploited enzymes, proteases are the most abundantly used enzymes in different industries. Numerous bacterial alkaline proteases have been studied widely and are commercially available; however, fungi exhibit a broader variety of proteases than bacteria. Additionally, since fungi are often recognized as generally regarded as safe (GRAS), using them as enzyme producers is safer than using bacteria. Fungal alkaline proteases are appealing models for industrial use because of their distinct spectrum of action and enormous diversity in terms of being active under alkaline range of pH. Unlike bacteria, fungi are less studied for alkaline protease production. Moreover, group of fungi growing at alkaline pH has remained unexplored for their capability for the production of commercially valuable products that are stable at alkaline pH. The current review focuses on the detailed classification of proteases, the production of alkaline proteases from different fungi by fermentation (submerged and solid-state), and their potential applications in detergent, leather, food, pharmaceutical industries along with their important role in silk degumming, waste management and silver recovery processes. Furthermore, the promising role of alkali-tolerant and alkaliphilic fungi in enzyme production has been discussed briefly. This will highlight the need for more research on fungi growing at alkaline pH and their biotechnological potential.

A meta-analysis for assessing the contributions of trypsin and chymotrypsin as the two major endoproteases in protein hydrolysis in fish intestine.

For the majority of fish species, regardless of being gastric or agastric, trypsin and chymotrypsin are known as the two main alkaline proteases responsible for the initial stage of protein hydrolysis in the fish intestine. Although the critical role of these proteases for protein hydrolysis in fish intestine is without doubt, the relative input of each enzyme in protein hydrolysis is still unclear. Data used in the present study has been retrieved from a bibliographic search using the Dimensions application (https://app.dimensions.ai/discover/publication tool). Retrieved articles were carefully inspected to identify whether they contained the description of the development of ontogenetic activities for trypsin, chymotrypsin, and total alkaline proteases in fish intestine. From the list of consulted articles, 21 studies were chosen based on correlation coefficients (Pearson correlation test), and four groups of fish were identified with high significant correlation between 1) the activity of chymotrypsin and total alkaline proteases; 2) the activity of trypsin, chymotrypsin, and total alkaline proteases; 3) the activity of trypsin and total alkaline proteases, and 4) mainly negative correlation between trypsin, chymotrypsin, and total alkaline proteases. These results indicated that the relative inputs of trypsin and chymotrypsin in protein hydrolysis may vary significantly among different fish species, which is a crucial point for proper understanding of species-specific digestive traits in both natural and aquaculture scenarios.

Multiple Modular Engineering of Bacillus Amyloliquefaciens Cell Factories for Enhanced Production of Alkaline Proteases From B. Clausii.

Bacillus amyloliquefaciens is a generally recognized as safe (GRAS) microorganism that presents great potential for the production of heterologous proteins. In this study, we performed genomic and comparative transcriptome to investigate the critical modular in B. amyloliquefaciens on the production of heterologous alkaline proteases (AprE). After investigation, it was concluded that the key modules affecting the production of alkaline protease were the sporulation germination module (Module I), extracellular protease synthesis module (Module II), and extracellular polysaccharide synthesis module (Module III) in B. amyloliquefaciens. In Module I, AprE yield for mutant BA ΔsigF was 25.3% greater than that of BA Δupp. Combining Module I synergistically with mutation of extracellular proteases in Module II significantly increased AprE production by 36.1% compared with production by BA Δupp. In Module III, the mutation of genes controlling extracellular polysaccharides reduced the viscosity and the accumulation of sediment, and increased the rate of dissolved oxygen in fermentation. Moreover, AprE production was 39.6% higher than in BA Δupp when Modules I, II and III were engineered in combination. This study provides modular engineering strategies for the modification of B. amyloliquefaciens for the production of alkaline proteases.

Multipotential Alkaline Protease From a Novel Pyxidicoccus sp. 252: Ecofriendly Replacement to Various Chemical Processes.

A newly isolated alkaline protease-producing myxobacterium was isolated from soil. The strain was identified as Pyxidicoccus sp. S252 on the basis of 16S rRNA sequence analysis. The extracellular alkaline proteases produced by isolate S252 (PyCP) was optimally active in the pH range of 11.0-12.0 and temperature range of 40-50°C The zymogram of PyCP showed six caseinolytic protease bands. The proteases were stable in the pH range of 8.0-10.0 and temperature range of 40-50°C. The activity of PyCP was enhanced in the presence of Na+, Mg2+, Cu2+, Tween-20, and hydrogen peroxide (H2O2) (hydrogen peroxide), whereas in Triton X-100, glycerol, ethylenediaminetetraacetic acid (EDTA), and Co2+, it was stable. PyCP showed a potential in various applications. The addition of PyCP in the commercial detergent enhanced the wash performance of the detergent by efficiently removing the stains of tomato ketchup and coffee. PyCP efficiently hydrolyzed the gelatin layer on X-ray film to release the embedded silver. PyCP also showed potent dehairing of goat skin and also efficiently deproteinized sea shell waste indicating its application in chitin extraction. Thus, the results of the present study indicate that Pyxidicoccus sp. S252 proteases have the potential to be used as an ecofriendly replacement of chemicals in several industrial processes.

Marine microbial alkaline protease: An efficient and essential tool for various industrial applications.

With the modern world focusing on environmental friendly products, more and more chemical processes are being replaced by enzymatic methods. Alkaline proteases (APases) place more than 50% of the total world enzyme production. Marine microorganisms are capable of producing an extensive spectrum of APases which have important ecological roles and promising industrial applications. Marine microbial APases can meet the required market demand for various industrial processes due to their strong specificity, mild reaction conditions, environmental friendliness and easy inactivation or control in comparison with chemical catalysts. In this review, a bird's-eye view on recent research works in the field of APase production from marine microorganisms as well as their potential industrial applications. The effect of various physical and chemical parameters on marine microbial APase is discussed. Isolation, purification, optimum pH and temperature of marine microbial APases are also reported. We anticipate that this review will provide an outline of potential industrial application of marine microbial APases and open new avenues to help the academicians, researchers and industrialists.

Alkaline serine protease from the new halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R: purification and properties.

Herein, we report the purification and characterization of an alkaline protease from the alkaliphilic Salipaludibacillus agaradhaerens (formerly Bacillus agaradhaerens) strain AK-R, which was previously isolated from Egyptian soda lakes. The purification procedures resulted in enzyme purification up to 13.3-fold, with a recovery yield of 16.3% and a specific activity of 3488 U/mg protein. AK-R protease was a monomeric protein with an estimated molecular weight of 33.0 kDa. The optimum pH and temperature for AK-R protease were pH 10 and 60 °C, respectively. The enzyme thermostability was significantly enhanced in the presence of CaCl2 by approximately 1.3-fold. Moreover, under optimal conditions, the K m and V max values of the enzyme were 2.63 mg/ml and 4166.7 U/mg, respectively. PMSF caused complete inhibition of the enzyme activity, suggesting that AK-R belongs to the serine protease family. In addition, the enzyme was completely inhibited by EDTA, revealing the requirement of metal ions for AK-R protease activity; hence, it can be classified as a metalloprotease. AK-R protease is a mostly thiol-independent enzyme, since thiol reductants such as ß-mercaptoethanol and dithiothreitol had no effect on the enzyme activity. AK-R protease exhibited high stability in several organic solvents, including butanol, amyl alcohol, dimethyl ether, toluene, diethyl ether and methanol. Moreover, AK-R protease showed significant stability to a variety of surfactants and commercial detergents. The features and properties of AK-R alkaline protease are favourable and suggest its potential applications in various industries, particularly in the laundry detergent industry.

Proteolytic and amylolytic enzymes from a newly isolated Bacillus mojavensis SA: Characterization and applications as laundry detergent additive and in leather processing.

The present work aims to study the simultaneous production of highly alkaline proteases and thermostable α-amylases by a newly isolated bacterium Bacillus mojavensis SA. The optimum pH and temperature of amylase activity were 9.0 and 55°C, respectively, while those of the proteolytic activity were 12.0 and 60°C, respectively. Both α-amylase and protease enzymes showed a high stability towards a wide range of pH and temperature. Furthermore, SA crude enzymes were relatively stable towards non-ionic (Tween 20, Tween 80 and Triton X-100) and anionic (SDS) surfactants, as well as oxidizing agents. Both activities were improved by the presence of polyethylene glycol 4000 and glycerol. Additionally, the crude enzymes showed excellent stability against various solid and liquid detergents. Wash performance analysis revealed that the SA crude enzymes exhibited a remarkable efficiency in the removal of a variety type of stains, such as blood, chocolate, coffee and oil. On the other side, SA proteases revealed a potential dehairing activity of animal hide without chemical assistance or fibrous proteins hydrolysis. Thus, considering their promising properties, B. mojavensis SA crude enzymes could be used in several biotechnological bioprocesses.

Chitin extraction from blue crab (Portunus segnis) and shrimp (Penaeus kerathurus) shells using digestive alkaline proteases from P. segnis viscera.

Since chitin is closely associated with proteins, deproteinization is a crucial step in the process of extracting chitin. Thus, this research aimed to extract chitin from Portunus segnis and Penaeus kerathurus shells by means of crude digestive alkaline proteases from the viscera of P. segnis, regarding deproteinization step, as an alternative to chemical treatment. Casein zymography revealed that five caseinolytic proteases bands exist, suggesting the presence of at least five different major proteases. The optimum pH and temperature for protease activity were pH 8.0 and 60°C, respectively, using casein as a substrate. The crude enzymes extract was highly stable at low temperatures and over a wide range of pH from 6.0 to 12.0. The crude alkaline protease extract was found to be effective in the deproteinization of blue crab and shrimp shells, to produce chitin. The best efficiency in deproteinization (84.69±0.65% for blue crab shells and 91.06±1.40% for shrimp shells) was achieved with an E/S ratio of 5U/mg of proteins after 3h incubation at 50°C. These results suggest that enzymatic deproteinization of crab and shrimp wastes by fish endogenous alkaline proteases could be a potential alternative in the chitin production process.

Alkaline proteases from a newly isolated Micromonospora chaiyaphumensis S103: Characterization and application as a detergent additive and for chitin extraction from shrimp shell waste.

The present study was undertaken to characterize the extracellular thermostable serine alkaline proteases from newly actinomycete strain Micromonospora chaiyaphumensis S103 and to describe their evaluation in commercial detergents and shrimp waste deproteinization. This proteolytic crude extract was active and stable in alkaline solution. It was extremely stable in the pH range of 5.0-12.0. The optimum pH and temperature were 8.0 and 70°C, respectively, using casein as a substrate. The thermoactivity and thermostability of proteases were enhanced by the addition of 5mM Ca2+. Proteases from S103 were also used for shrimp wastes deproteinization in the process of chitin preparation. The percent of protein removal after 3h hydrolysis at 45°C with an enzyme/substrate ratio of 20U/mg had reached 93%. Furthermore, S103 crude enzyme was stable towards several organic solvents and retained 100% of its original activity after 90days of incubation in the presence of methanol, hexane, acetone, and DMSO. These properties make S103 proteases an ideal choice for application in detergent formulations, chitin production, and enzymatic peptide synthesis.

Surfactant- and oxidant-stable alkaline proteases from Bacillus invictae: Characterization and potential applications in chitin extraction and as a detergent additive.

A newly alkaline proteases producing strain was isolated from sea water. The strain was identified as Bacillus invictae on the basis of biochemical characteristics and 16S rRNA sequence analysis. The crude protease activity showed an optimal activity at approximately 60°C and in wide pH interval ranging from 9.0 to 11.0. At least six clear caseinolytic protease bands were observed in a zymogram. Phenylmethylsulfonyl fluoride (PMSF), a serine-protease inhibitor, was found to inhibit completely the protease activity. The crude alkaline proteases showed high stability toward solid and liquid detergents. Furthermore, wash performance analysis revealed that the crude enzyme could effectively remove blood stain when added to commercial detergent. In addition, the crude proteases were found to be effective in the deproteinization of shrimp shell waste. The percent of protein removal after 3h of hydrolysis at 50°C with an E/S ratio of 10U/mg of protein or after fermentation by the strain were about 76% and 82%, respectively. Thus, the results of the present study showed that the crude proteases of B. invectae could be effectively used in several industrial applications, as an eco-friendly agent.

Gelatin hydrolysates from farmed Giant catfish skin using alkaline proteases and its antioxidative function of simulated gastro-intestinal digestion.

This work aims to evaluate the ability of different alkaline proteases to prepare active gelatin hydrolysates. Fish skin gelatin was hydrolysed by visceral alkaline-proteases from Giant catfish, commercial trypsin, and Izyme AL®. All antioxidant activity indices of the hydrolysates increased with increasing degree of hydrolysis (P<0.05). The hydrolysates obtained with Izyme AL® and visceral alkaline-proteases showed the highest and lowest radical scavenging capacity, while prepared with commercial trypsin was the most effective in reducing ferric ions and showed the best metal chelating properties. The hydrolysate obtained with Izyme AL® showed the lowest iron reducing ability, but provided the highest average molecular weight (⩾ 7 kDa), followed by commercial trypsin (2.2 kDa) and visceral alkaline-proteases (1.75 kDa). After in vitro gastrointestinal digestion, the hydrolysates showed significant higher radical scavenging, reducing ferric ions and chelating activities. Gelatin hydrolysates, from fish skin, could serve as a potential source of functional food ingredients for health promotion.

Digestive alkaline proteases from thornback ray (Raja clavata): Characteristics and applications.

This study describes the characterization of a crude protease extract from thornback ray (Raja clavata) and its evaluation in liquid detergent and in deproteinizattion of shrimp waste. At least five clear caseinolytic proteases bands were observed in a zymogram. The crude protease showed optimum activity at pH 8.0 and 50 °C, and it was highly stable over pH range from 8.0 to 11.0. Proteolytic enzymes were very stable in non-ionic surfactants and in the presence of oxidizing agents, maintaining 70% of their activity after incubation for 1 h at 30 °C in the presence of 1% sodium perborate. In addition, they showed high stability and compatibility with various liquid laundry-detergents available in the Tunisian market. The crude extract retained 100% of its activity after preincubation for 60 min at 30 °C in the presence of Nadhif Perfect, Textil and Carrefour laundry detergents. Further, proteases from R. clavata viscera were used for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3 h hydrolysis at 45 °C with an enzyme/substrate ratio of 30 U/mg of proteins was 74%. These results suggest that enzymatic deproteinization of shrimp wastes by fish endogenous alkaline proteases could be applicable to the chitin production process.

The biotechnological potential of subtilisin-like fibrinolytic enzyme from a newly isolated Lactobacillus plantarum KSK-II in blood destaining and antimicrobials.

An antimicrobial oxidative- and SDS-stable fibrinolytic alkaline protease designated as KSK-II was produced by Lactobacillus plantarum KSK-II isolated from kishk, a traditional Egyptian food. Maximum enzyme productivity was obtained in medium containing 1% lactose and 0.5% soybean flour as carbon and nitrogen sources, respectively. Purification of enzyme increased its specific activity to 1,140-fold with a recovery of 33% and molecular weight of 43.6 kDa. Enzyme activity was totally lost in the presence of ethylenediaminetetraacetic acid and was restored after addition of Fe(2+) suggesting that KSK-II is a metalloprotease and Fe(2+) acts as cofactor. Enzyme hydrolyzed not only the natural proteins but also synthetic substrates, particularly Suc-Ala-Ala-Pro-Phe-pNA. KSK-II can hydrolyze the Lys-X easier than Arg-X; thus, it was considered as a subtilisin-family protease. Its apparent Km , Vmax , and Kcat were 0.41 mM, 6.4 µmol mg(-1) min(-1) , and 28.0 s(-1) , respectively. KSK-II is industrially important from the perspectives of its maximal activity at 50°C (stable up to 70°C), ability to function at alkaline pH (10.0), stability at broad pH ranges (7.5-12.0) in addition to its stability toward SDS, H2 O2 , organic solvents, and detergents. We emphasize for the first time the potential of fibrinolytic activity for alkaline proteases used in detergents especially in blood destaining.

Feeding habits and ontogenic changes in digestive enzyme patterns in five freshwater teleosts.

Feeding habits and the activity of digestive enzymes (total alkaline proteases, α-amylase and lipase) from dace Leuciscus leuciscus, roach Rutilus rutilus, Prussian carp Carassius auratus gibelio, perch Perca fluviatilis and pikeperch Sander lucioperca fry were studied in the Malye Chany Lake-Kargat Estuary (western Siberia, Russia). The diet of fry from all studied species was mainly composed of chironomid larvae and zooplanktonic organisms (i.e. cladocera and copepoda), whereas carnivorous species such as P. fluviatilis and S. lucioperca also preyed on fry from other fishes while detritus and microalgae were also important in the diet of ommivorous species. When comparing diet similarity (Sørensen-Dice index, Q(S)) among fry at different stages of development, both omnivorous and carnivorous species showed a high level of similarity (0.67 < Q(S) < 0.89 and 0.73 < Q(S)< 0.89, respectively). Diet similarity values were in agreement with the overall digestive activity profile analysed by cluster analysis. Diet similarity suggested potential trophic competition when zooplanktonic and benthic prey began to decline towards autumn. The analysis of pancreatic digestive enzymes revealed a correlation among their activities and fry feeding habits with α-amylase:total proteases (A:P) values higher than 1 in omnivorous species and lower (A:P ≤ 1) in carnivorous species.

Savinase, the most suitable enzyme for releasing peptides from lentil (Lens culinaris var. Castellana) protein concentrates with multifunctional properties.

The aim of this study was to produce multifunctional hydrolysates from lentil protein concentrates. Four different proteases (Alcalase, Savinase, Protamex, and Corolase 7089) and different hydrolysis times were evaluated for their degree and pattern of proteolysis and their angiotensin I-converting enzyme (ACE) inhibitory and antioxidant activities. Alcalase and Savinase showed the highest proteolytic effectiveness (P ≤ 0.05), which resulted in higher yield of peptides. The hydrolysate produced by Savinase after 2 h of hydrolysis (S2) displayed the highest ACE-inhibitory (IC50 = 0.18 mg/mL) and antioxidant activity (1.22 µmol of Trolox equiv/mg of protein). Subsequent reverse-phase HPLC-tandem mass spectrometric analysis of 3 kDa permeates of S2 showed 32 peptides, mainly derived from convicilin, vicilin, and legumin containing bioactive amino acid sequences, which makes them potential contributors to ACE-inhibitory and antioxidant activities detected. The ACE-inhibitory and antioxidant activities of S2 were significantly improved after in vitro gastrointestinal digestion (P ≤ 0.05). Multifunctional hydrolysates could encourage value-added utilization of lentil proteins for the formulation of functional foods and nutraceuticals.

Zebra blenny (Salaria basilisca) viscera as a source of solvent-stable proteases: characteristics, potential application in the deproteinization of shrimp wastes and evaluation in liquid laundry commercial detergents.

The present study describes the characterization of crude protease extract from zebra blenny (Salaria basilisca) and its evaluation in liquid detergent and shrimp waste deproteinization. At least five caseinolytic proteases clear bands were observed in zymogram. The crude alkaline protease showed optimum activity at pH 8.0 and 60 °C, and it was highly stable over a wide range of pH from 6.0 to 11.0. Proteolytic enzymes showed extreme stability towards non-ionic surfactants (5 % Tween 80 and 5 % Triton X-100) and oxidizing agents (1 % sodium perborate), and relative stability towards anionic surfactant (1 % Sodium dodecyl sulfate (SDS)). They also showed high stability and compatibility with various laundry liquid detergents from Tunisian market. Furthermore, the crude enzyme was stable towards several organic solvents and retained more than 50 % of its original activity after 30 days of incubation at 30 °C in the presence of 50 % (v/v) dimethylsulfoxide (DMSO). Further, proteases from zebra blenny viscera were found to be effective in the deproteinization of shrimp wastes. The protein removal after 3 h at 40 °C with an enzyme/substrate ratio (E/S) of 5 U/mg protein was about 77 %.

Functional analysis and structure determination of alkaline protease from Aspergillus flavus.

Proteases are one of the highest value commercial enzymes as they have broad applications in food, pharmaceutical, detergent, and dairy industries and serve as vital tools in determination of structure of proteins and polypeptides. Multiple application of these enzymes stimulated interest to discover them with novel properties and considerable advancement of basic research into these enzymes. A broad understanding of the active site of the enzyme and of the mechanism of its inactivation is essential for delineating its structure-function relationship. Primary structure analysis of alkaline protease showed 42% of its content to be alpha helix making it stable for three dimensional structure modeling. Homology model of alkaline protease has been constructed using the X-ray structure (3F7O) as a template and swiss model as the workspace. The model was validated by ProSA, SAVES, PROCHECK, PROSAII and RMSD. The results showed the final refined model is reliable. It has 53% amino acid sequence identity with the template, 0.24 Å as RMSD and has -7.53 as Z-score, the Ramachandran plot analysis showed that conformations for 83.4 % of amino acid residues are within the most favored regions and only 0.4% in the disallowed regions.