Thermostable Protease Research Articles

A novel simple fluorometric protease assay for monitoring hydrolysis of proteins in real time

Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases.

The First Pseudomonas Phage vB_PseuGesM_254 Active against Proteolytic Pseudomonas gessardii Strains

Bacteria of the Pseudomonas genus, including the Pseudomonas gessardii subgroup, play an important role in the environmental microbial communities. Psychrotolerant isolates of P. gessardii can produce thermostable proteases and lipases. When contaminating refrigerated raw milk, these bacteria spoil it by producing enzymes resistant to pasteurization. One possible way to prevent spoilage of raw milk is to use Pseudomonas lytic phages specific to undesirable P. gessardii isolates. The first phage, Pseudomonas vB_PseuGesM_254, was isolated and characterized, which is active against several proteolytic P. gessardii strains. This lytic myophage can infect and lyse its host strain at 24 °C and at low temperature (8 °C); so, it has the potential to prevent contamination of raw milk. The vB_PseuGesM_254 genome, 95,072 bp, shows a low level of intergenomic similarity with the genomes of known phages. Comparative proteomic ViPTree analysis indicated that vB_PseuGesM_254 is associated with a large group of Pseudomonas phages that are members of the Skurskavirinae and Gorskivirinae subfamilies and the Nankokuvirus genus. The alignment constructed using ViPTree shows that the vB_PseuGesM_254 genome has a large inversion between ~53,100 and ~70,700 bp, which is possibly a distinctive feature of a new taxonomic unit within this large group of Pseudomonas phages.

Characterization of a thermostable protease from Bacillus subtilis BSP strain

This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca2+ and Fe2+ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H2O2). Characteristics, such as tolerance to high SDS and H2O2 concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.

Purification and Characterization of a Small Thermostable Protease from Streptomyces sp. CNXK100.

Proteases derived from Streptomyces demonstrate numerous commendable properties, rendering it extensively applicable in biotechnology and various industrial sectors. This study focused on the purification and characterization of the thermostable protease obtained from Streptomyces sp. CNXK100. The purified protease exhibited an estimated molecular weight of 27 kDa, with optimal activity at 75°C and pH 8.0. Notably, the enzyme remained active even without any metal ions and fully active in the presence of Na+, K+, Mg2+, and Cu2+metal ions. The kinetic parameters were determined with a KM value of 3.13 mg/ml and a Vmax value of 3.28 × 106 U/mg. Furthermore, the protease has demonstrated notable stability when subjected to a treatment temperature of up to 65°C for 60 minutes, and across a broad pH range extending from 5.0 to 10.0. This protease also demonstrated resilience against a spectrum of harsh conditions, including exposure to organic solvents, surfactants, bleaching agents, and proteolytic enzymes. Additionally, the enzyme maintained its activity following treatment with commercial detergents, accomplishing complete thrombus lysis at a concentration of 2.50 mg/ml within 4 hours. Remarkably, the protease exhibited stability in terms of activity and protein concentration for 70 days at 4°C. These findings underscore the potential industrial applications of the thermostable protease from Streptomyces sp. CNXK100.

In-silico characterization of a thermophilic serine protease via homology modeling, docking and molecular dynamics simulations

One of the major categories of industrial enzymes, proteases is crucial to the survival of living things. The purpose of this research was to newly thermostable protease from the thermophilum Geobacillus stearothermophilus. With the conserved catalytic tetrad, protease (Protease JJ) is closely related to the serine proteases from the subtilisin S8 peptidase, according to phylogenetic tree analysis. The tertiary structure of Protease JJ was predicted structurally using RoseTTAFold, and it is a sandwich structure overall. Homology modeling validation showed Protease JJ was modeled in X-ray’s protein areas, and it has gained a favored Ramachandran graph regarding Phi/Psi angels. Protease JJ showed structure stability through Molecular dynamics simulation in the presence of Tween20 and Methanol in 1% concentration. Also, Protease JJ exhibited thermal stability at 60 to 90 °C so that amino acid exposure of Protease JJ was low and constant throughout the MD simulation. Docking results of Protease JJ with BSA and βcasein were simulated via MD and it was found that Protease JJ could interact with both BSA and βcasein strongly. MM/PBSA analysis showed Protease JJ may be involved via more amino acids with BSA as well as established more interaction hydrogen bonds. Overall, evidence suggests Protease JJ probably has merit for future experimental investigation as a thermostable protease.Communicated by Ramaswamy H. Sarma

Molecular Cloning, Characterization, and Application of Organic Solvent-Stable and Detergent-Compatible Thermostable Alkaline Protease from Geobacillus thermoglucosidasius SKF4.

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80°C. In addition, the enzyme showed a half-life of 15 h at 80°C, and there was a 60% increase in its activity at 10 mM Ca2+ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.

Increased Expression Levels of Thermophilic Serine Protease TTHA0724 through Signal Peptide Screening in Bacillus subtilis and Applications of the Enzyme.

The thermostable protease TTHA0724 derived from Thermus thermophilus HB8 is an ideal industrial washing enzyme due to its thermophilic characteristics; although it can be expressed in Escherichia coli via pET-22b, high yields are difficult to achieve, leading to frequent autolysis of the host. This paper details the development of a signal peptide library in the expression system of B. subtilis and the optimization of signal peptides for enhanced extracellular expression of TTHA0724. When B. subtilis was used as the host and the optimized signal peptide was used, the expression level of TTHA0724 was 16.7 times higher compared with E. coli. B. subtilis as an expression host does not change the characteristics of TTHA0724. The potential application fields of TTHA0724 are studied. TTHA0724 can be used as a detergent additive at 60 °C, which can sterilize and eliminate mites while thoroughly cleaning protein stains. Soybean meal enzymatic hydrolysis with TTHA0724 at a high temperature produced a higher content of antioxidant peptides. These results indicate that TTHA0724 has great potential for industrial applications.

Understanding the influence of genotype and temperature on proteolytic activity in distinct barley genotypes.

This study aimed at investigating the effects of genotype and temperatures on the proteolytic activity in green malt of 48 barley genotypes, including 19 mutants, 15 hulled, 4 hulless, and 10 wild using enzyme assays based on casein, as substrate. During malting, insoluble barley protein must be hydrolyzed into soluble peptides and free amino acids to supply the brewing yeast with sufficient nutrients to grow rapidly and metabolize glucose and other sugars into alcohol through fermentation. However, the relatively hot temperatures employed during kilning usually denature the proteolytic enzymes due to their thermolabile nature. Even though the hydrolytic activity of most of the proteases is destroyed during the kilning process, the malt includes a small fraction of thermostable proteases that can further degrade protein in the subsequent mashing process. Considering the higher temperature range employed in industrial kilning and mashing, three temperatures (37, 50, and 70°C) were selected to identify the genotypes possessing high activity at the higher range of temperatures as well as thermostable variant of the enzyme. The proteolytic activity in all the genotypes declined after 50°C depicting its optimum temperature. Overall proteolytic activity was observed to be positively correlated with the amino acids and negatively correlated with protein content. Three mutant (BL2086, BL2091, and BL2079) and one wild (WS 237) genotypes possessing proteolytic activity in a higher range at all the studied temperatures have the potential to be exploited in the breeding programs for incorporating trait of thermostable proteolytic activity into low malting efficiency cultivars. PRACTICAL APPLICATION: The optimal hydrolytic activities of carbohydrases and proteases during mashing are essential for producing high-quality wort from malted barley to ensure that hydrolyzed molecules are available to brewers' yeast to support fermentative metabolism. In this study, several barley cultivars were grown under identical environmental conditions but assayed at different temperatures. As result, four genotypes had been obtained that possessed optimal proteolytic activities at a higher temperature range and can be of great interest to breeders and maltsters for altering wort amino acid profiles and better exposure of starch to mashing enzymes, thereby increasing the fermentable sugar yield from the malt.

Purification and characterization of a robust thermostable protease isolated from Bacillus subtilis strain HR02 as an extremozyme.

Since the hot water of Genow, a village in Isin rural district in the central district of Bandar Abbas, Hormozgan Province, Iran, has a rich source of thermophilic bacteria, the current study aimed to find a new thermophilic protease enzyme with suitable properties to be used in different industries. Water and sediment samples were collected from the hot water of Genow, and finally, 20 colonies were isolated. Among these isolated colonies, two bacterial strains grew on the skim milk agar medium, and a clear halo was formed around the colony, which was accurately identified by 16S rRNA gene sequence analysis. The comparison of 16S rRNA gene sequence analyses of isolated strains HR01 and HR02 with registered sequences of 16S rRNA genes in NCBI showed that the two isolates had the most similarity to Bacillus sonorensis and Bacillus subtilis, respectively. Among the two bacterial strains, the highest enzymatic activity was observed in B. subtilis strain HR02, from which the protease purification process was performed. A putative native B. subtilis strain HR02 protease (BSHR02PR) was purified by the UNO Q-6 ionic exchange chromatography method. Biochemical analyses revealed a monomeric enzyme, BsHR02Pro, with a molecular weight of 25 kDa, showing the maximum activity at 70°C and pH8.0. Moreover, the purified enzyme was stable up to 80 °C and in a pH range of 6.0-12.0. The steady-state kinetic analysis for colloidal casein showed that the Km , Vmax and kcat values of the purified enzyme were 25.7μM, 93.2μM min-1 and 2.18 s-1 , respectively. The hot water of Genow is a rich source of protease-producing bacteria. Sediments are a better source for the isolation of these types of bacteria than spring water. Overall, our results demonstrated a potential bacterial enzyme BsHR02Pro as a suitable catalyst to be used in the various industries.

Phlorotannin Extracts from Ascophyllum nodosum Inhibited Proteases Activities and Structural Changes from Apostichopus japonicus

Fresh Apostichopus japonicas (A. japonicas) are easy to autolyze through the actions of endogenous proteases under certain circumstances, further inducing a decline in A. japonicas product quality and business value. To solve this serious problem in the A. japonicas industry, we investigated the inhibition of phlorotannin extracts (PhTE) on the activities and structural changes of crude endogenous proteinase (CEP) from A. japonicas. The results of enzyme activity and SDS-PAGE indicated that PhTE could inhibit enzyme activity in a dose dependent manner. Analysis of multiple spectroscopic methods, scanning electron microscopy, and differential scanning calorimetry indicated that PhTE promoted the cross-linking and aggregation of CEP and increased the particle size of the enzyme, thus changing the structure of the enzyme protein and decreasing the thermostability of protease. In conclusion, PhTE might be an effective inhibitor on endogenous proteinase in A. japonicas, which opened up a new way to prevent the A. japonicas autolysis.

Purification and characterization of thermostable serine alkaline protease from Geobacillus sp. DS3 isolated from Sikidang crater, Dieng plateau, Central Java, Indonesia

Thermostable proteases that optimally withstand the high‐temperature conditions of thermophilic bacteria could be produced and purified, which would be highly beneficial for use in industry. Geobacillus sp. is a thermophilic bacterium that can be found in various environmental conditions. The goal of this study was to isolate and characterize thermostable serine protease that had been produced by thermophilic Geobacillus sp. strain DS3. The proteolytic index was measured in a solid medium. The expression of protease was optimized by Geobacillus sp. DS3 at 50 °C for 18 h. Targeted protease was purified using ammonium sulfate (40‐60%) and DEAE Sephadex A‐25 resin. Using SDS‐PAGE, the molecular weight of the enzyme was predicted to be around 32 kDa. Purified thermostable protease was highly activated at 70 °C, pH 9.6 stable for 1 h, and inhibited by PMSF. Therefore, this enzyme is classified as a thermostable alkaline serine protease. Its kinetic study revealed specific activity of 0.41 U/mg (Vmax) and 0.25 mg/mL (KM). Overall, a thermostable alkaline serine protease from Geobacillus sp. DS3 showed high activity at high temperatures and alkaline pH, which is vital for application in industries such as leather processing and detergent formulation.

Isolation, purification and biochemical characterization of a thermophilic alkaline protease from hot water spring bacteria

Search for a new alkaline and thermostable protease is crucial to explore their industrial applications. Therefore, a thermophilic, alkaliphilic bacteria producing serine alkaline protease was isolated from a hot water spring. Isolated bacteria were identified as B. licheniformis strain 9AkScKu3 as per the results revealed from biochemical studies and 16s rRNA gene sequencing analysis. The purification fold of the enzyme was 23.91 with yield of 18.54%. The purified protease was optimally active at pH 9 and a temperature of 50°C. The enzyme exhibited thermal stability up to 70ºC. Metal ions Ca2+, Mg2+ and Cu2+ enhanced the activity of enzyme. The enzyme exhibited compatibility with 3% surfactants showing more than 100% stability with SDS, tween 20 and triton-x100. Enzyme retained 100% activity with 5% (v/v) H2O2 and organic solvents like toluene and n-hexane at 10% (v/v) concentration. Thus isolated thermophilic alkaline protease is a promising enzyme applicable in detergent formulation.

The effect of extracellular protease secreted by Pseudomonas fluorescens W3 on the quality of UHT milk

The heat-resistant protease produced by psychrotrophic bacteria is harmful to the quality of UHT milk, and the complex structure formed by fat and protein also affects the hydrolysis of protein by protease. The protease secreted by Pseudomonas fluorescens W3 was added to whole milk and skimmed milk before UHT treatment to analyze its effect on the quality of two kinds of UHT milk. During the preservation of UHT milk, increases in proteolysis, viscosity, hardness, sediment content, zeta potential, and particle size were observed. Extracellular protease reduced the whiteness, pH, and cohesiveness of UHT milk during the storage. The protease secreted by P. fluorescens W3 was easier to hydrolyze casein than whey protein, and κ-casein of casein was easily hydrolyzed by the protease. Milk fat accelerated the hydrolysis of casein by protease secreted by P. fluorescens W3, which led to a faster decline in the quality of whole UHT milk. Novelty impact statement Pseudomonas fluorescens W3 can secrete a thermostable protease AprX with a molecular weight of about 45 kDa. The protease AprX has stronger hydrolysis ability on κ-casein than other caseins. Fat can promote the activity of protease AprX, so whole milk is more susceptible to the damage of protease AprX.

Thermostable Protease, Amylase and Lipase Enzymes of Thermophilic Bacteria Isolated from Egyptian Hot Springs

Thermostable Protease, Amylase and Lipase Enzymes of Thermophilic Bacteria Isolated from Egyptian Hot Springs

PROTEASE ACTIVITY OF THERMOPHILIC BACTERIA FROM LEJJA HOT SPRINGS IN SOPPENG SOUTH SULAWESI

This research aimed to observe quantitatively the effect of temperature and pH on protease activities from thermophilic bacteria collected from Lejja—Hot springs. Spectrophotometry on the casein substrate was used to test the proteolytic activity of the crude protease. Mixtures of enzyme and casein were incubated at various temperatures and pH for 20 minutes. The absorbance of tyrosine from protein hydrolysis was determined by spectrophotometry on λ 280 nm. Temperature and pH impacted on protease activity were determined at temperature (60; 65; 70; 75; 80; 85 and 90oC) and pH (6.0; 6.5; 7.0; 7.5; 8, 0; 8,5 and 9,0). Results showed that treatment of temperature variations and pH had a significant effect on protease activity. Crude extract of Bacillus licheniformis protease showed that the highest activity at 80oC and pH 7.5 was 0.1303 unit/ml/minute. Bacillus stearoformis showed the highest enzymatic activity of the protease at 85oC, and pH 7.5 was 0.1226 Unit/ ml/minute. In comparison, Bacillus coagulans reached optimum activity at 75oC, and pH 7.5 was 0.2052 Unit/ml/minute. Isolates of Bacillus licheniformis, B. coagulans and B. stearoformis are bacteria that produce thermostable protease enzymes that can be developed as a source of genes and as a producer of the enzyme itself.

PROTEASE ACTIVITY OF THERMOPHILIC BACTERIA FROM LEJJA HOT SPRINGS IN SOPPENG SOUTH SULAWESI

This research aimed to observe quantitatively the effect of temperature and pH on protease activities from thermophilic bacteria collected from Lejja—Hot springs.  Spectrophotometry on the casein substrate was used to test the proteolytic activity of the crude protease.  Mixtures of enzyme and casein were incubated at various temperatures and pH for 20 minutes. The absorbance of tyrosine from protein hydrolysis was determined by spectrophotometry on λ 280 nm. Temperature and pH impacted on protease activity were determined at temperature (60; 65; 70; 75; 80; 85 and 90oC) and pH (6.0; 6.5; 7.0; 7.5; 8, 0; 8,5 and 9,0). Results showed that treatment of temperature variations and pH had a significant effect on protease activity. Crude extract of Bacillus licheniformis protease showed that the highest activity at 80oC and pH 7.5 was 0.1303 unit/ml/minute. Bacillus stearoformis showed the highest enzymatic activity of the protease at 85oC, and pH 7.5 was 0.1226 Unit/ ml/minute. In comparison, Bacillus coagulans reached optimum activity at 75oC, and pH 7.5 was 0.2052 Unit/ml/minute. Isolates of Bacillus licheniformis, B. coagulans and B. stearoformis are bacteria that produce thermostable protease enzymes that can be developed as a source of genes and as a producer of the enzyme itself.

Understanding the Basis of Occurrence, Biosynthesis, and Implications of Thermostable Alkaline Proteases.

The group of hydrolytic enzymes synonymously known as proteases is predominantly most favored for the class of industrial enzymes. The present work focuses on the thermostable nature of these proteolytic enzymes that occur naturally among mesophilic and thermophilic microbes. The broad thermo-active feature (40-80°C), ease of cultivation, maintenance, and bulk production are the key features associated with these enzymes. Detailing of contemporary production technologies, and controllable operational parameters including the purification strategies, are the key features that justify their industrial dominance as biocatalysts. In addition, the rigorous research inputs by protein engineering and enzyme immobilization studies add up to the thermo-catalytic features and application capabilities of these enzymes. The work summarizes key features of microbial proteases that make them numero-uno for laundry, biomaterials, waste management, food and feed, tannery, and medical as well as pharmaceutical industries. The quest for novel and/or designed and engineered thermostable protease from unexplored sources is highly stimulating and will address the ever-increasing industrial demands.

Isolation of Thermostable Protease Producing Bacteria from the Microbial Mats

As thermostable protease has more commercial value in different industries, the aim of this study was to search for such an enzyme producing bacteria from the microbial mats. Investigation was continued on the isolate for its ability to produce mass amount of enzyme and its activity under suitable optimized conditions. Different parameters including cheap carbon and nitrogen substrates, inoculum size and temperature was selected to optimize the enzyme production conditions. Initially five different isolates from two microbial mats collected from different sources were analyzed for its ability to produce thermostable protease after exposing to higher temperature incubation conditions. Test culture tentatively named as 1F from microbial mat-1 was selected as more enzyme producer among the ten isolates. The organism was selected based on the zone of clearance on skim milk agar by the isolate, that indicating more protease production. Under each optimization parameter, each type of carbon (Lactose), and nitrogen (yeast extract) source showed more enzyme production and activity respectively. About 1% inoculum size and a thermostable temperature of 45°C produced significant amount of enzyme and its activity. The obtained results emphasized the need for thermostable protease for different commercial industries in the existing and near future.

Integration of hydroxyapatite from fish scales and polyethersulfone membrane for protease separation from Bacillus subtilis

ABSTRACT In the present study, an ion exchange membrane for thermostable alkaline protease separation from Bacillus subtilis was prepared via integration of hydroxyapatite from fish scales (FSHAp) and polyether sulfone membrane. The prepared membranes were characterized in terms of morphology, surface chemistry, ion exchange capacity, water uptake and fouling analysis. The performance of ion exchange membranes were evaluated by protease separation at different pH (3, 5, 7, 9, and 11) and ionic strength (0.05, 0.1, 0.15, 0.2, and 0.25 M). Characterization study revealed that PES/FSHAp-1000 membrane promoted the highest performance with a high permeability coefficient of about 64.0 L/m2.h.bar and membrane porosity around 90.1%. It also had a high ion exchange capacity of around 7.09 meq/g and water uptake for about 97.08%. This membrane’s hydrophilicity was also improved by 53%, and its flux recovery ratio (FRR) was about 97.79%. Fourier transform infra-red Spectroscopy (FTIR) and X-ray- diffractometer (XRD) ascertained the successful integration of FSHAp onto the membrane’s surface. Optimum recovery of protease (92.30%) obtained when the elution process performed using a glycine-NaOH buffer at pH 11 and 2.0 M ionic strength.

Optimization of thermostable proteases production under agro-wastes solid-state fermentation by a new thermophilic Mycothermus thermophilus isolated from a hydrothermal spring Hammam Debagh, Algeria

The present work investigates for the first time the presence and isolation of the thermophilic fungi from hydrothermal spring situated at the locality of Guelma, in the Northeast of Algeria. The production of the thermostable proteases and the optimization of culture conditions under agro-wastes solid-state fermentation to achieve optimal production capacity were explored. A statistical experimental approach consisting of two designs was used to determine the optimum culture conditions and to attain the greatest enzyme production. Besides, different agricultural wastes were initially evaluated as a substrate, whereby wheat bran was selected for enzyme production by the isolate under solid-state conditions. The isolate thermophilic fungi were identified as Mycothermus thermophilus by sequencing the ITS region of the rDNA (NCBI Accession No: MK770356.1). Among the various screened variables: the temperature, the inoculum size, and the moisture were proved to have the most significant effects on protease activity. Employing two-level fractional Plackett–Burman and a Box–Behnken designs statistical approach helped in identifying optimum values of screened factors and their interactions. The analysis showed up 6.17-fold improvement in the production of proteases (~1187.03 U/mL) was achieved under the optimal conditions of moisture content 47%, inoculum 5 × 105 spores/g, and temperature at 42 °C. These significant findings highlight the importance of the statistical design in isolation of Mycothermus thermophilus species from a specific location as well as identifying the optimal culture conditions for maximum yield.