Crude Protease Research Articles

Production and characterization of crude proteases from Halobacillus salinus strain DZ28 newly isolated from salt lake sediments in Algeria and their use as detergent bioadditives

New Algerian Gram-positive, rod-shaped, endospore-forming, salt-philic bacteria (DZ28 strain) that overproduce extracellular alkaline proteases have been isolated from salt lake deposits in Lake Oubeira, El Taref. Strain DZ28 was assigned as Halobacillus salinus DZ28 on the basis of phenotypic properties and 16S rDNA gene sequencing (ripotyping). The maximum protease activity registered after 36 hours of incubation in optimized medium at 30 ° C was 19,000 U / ml in a shaking bottle culture at 160 rpm. The crude extract protease showed optimal activity at 60 ° C temperature and pH 12. It is actively inhibited by PMSF and DIFP, indicating that it belongs to the serine protease family. Interestingly, the crude extract protease was not only very stable to nonionic surfactants and oxidants, but also showed high stability and compatibility with some commercial detergents. It retaining more than 100% of its initial activity after pre-incubation for 1 h at 40°C with ISIS, followed by Pril (98%), Tide (95%) and Dixan (90%). More curiously, the wash overall performance evaluation discovered that it may dispose of blood-stains remove at 40°C for 1 h with low supplementation (500 U/mL). This is the first report of a protease from Halobacillus salinus and has potential as a promising candidate for future applications as a bioadditive for detergent formulations.

Two-Step Purification and Partial Characterization of Keratinolytic Proteases from Feather Meal Bioconversion by Bacillus sp. P45

This study aimed to purify and partially characterize a keratinolytic protease produced by Bacillus sp. P45 through bioconversion of feather meal. Crude protease extract was purified using a sequence of an aqueous two-phase system (ATPS) in large volume systems (10, 50, and 500 g) to increase obtaining purified enzyme, followed by a diafiltration (DF) step. Purified protease was characterized in terms of protein profile analysis by SDS-PAGE, optimum temperature and pH, thermal deactivation kinetics at different temperatures and pH, and performance in the presence of several salts (NaCl, CaCl2, MnCl2, CaO, C8H5KO4, MgSO4, CuSO4, ZnSO4, and FeCl3) and organic solvents (acetone, ethanol, methanol, acetic acid, diethyl ether, and formaldehyde). ATPS with high capacities resulted in purer protease extract without compromising purity and yields, reaching a purification factor up to 2.6-fold and 6.7-fold in first and second ATPS, respectively, and 4.0-fold in the DF process. Recoveries were up to 79% in both ATPS and reached 84.3% after the DF step. The electrophoretic analysis demonstrated a 25–28 kDa band related to keratinolytic protease. The purified protease’s optimum temperature and pH were 55 °C and 7.5, respectively. The deactivation energy (Ed) value was 118.0 kJ/mol, while D (decimal reduction time) and z (temperature interval required to reduce the D value in one log cycle) values ranged from 6.7 to 237.3 min and from 13.6 to 18.8 °C, respectively. Salts such as CaCl2, CaO, C8H5KO4, and MgSO4 increased the protease activity, while all organic solvents caused its decrease. The results are useful for future studies about ATPS scale-up for enzyme purification and protease application in different industrial processes.

Screening, characterization, and kinetic studies of a serine alkaline protease from kitchen wastewater bacteria P2S1An and evaluation of its application in nutraceutical production.

This present investigation aimed at characterizing the biochemical potential and kinetic study of the protease isolated from kitchen wastewater bacteria, P2S1An. The enzymatic activity was optimum when incubated for 96 h, at 30°C and pH9.0. The enzymatic activity of the purified protease (PrA) was 10.47-folds that of crude protease (S1). PrA was about 35 kDa in molecular weight. The broad pH and thermal stability, chelators, surfactants and solvent tolerance, and favorable thermodynamics suggested the potentiality of the extracted protease PrA. Thermal activity and stability were enhanced in presence of 1-mM Ca2+ ion at high temperatures. The protease was a serine one as its activity was completely diminished in presence of 1-mM PMSF. The Vmax , Km , and Kcat /Km suggested stability and catalytic efficiency of the protease. PrA hydrolyzes fish protein with 26.61 ±0.16% of peptide bond cleavage after 240 min, comparable to Alcalase 2.4L (27.13 ±0.31%). PRACTITIONER POINTS: A serine alkaline protease PrA was extracted from kitchen wastewater bacteria Bacillus tropicus Y14. Protease PrA showed significant activity and stability in a wide temperature and pH range. Protease showed great stability towards additives like metal ions, solvents, surfactants, polyols, and inhibitors. Kinetic study showed that the protease PrA had a prominent affinity and catalytic efficiency for the substrates. PrA hydrolysed fish proteins into short bioactive peptides which signify its potential in the formation of functional food ingredients.

Characteristics and Substrate Specificity of Semi-Purified Bacterial Protease ofBacillusthuringiensisHSFI-12 with Potential as Antithrombotic Agent

Commercial proteases, such as Nattokinase (NK), Staphylokinase (SAK), and Streptokinase (SK) play an important role in the destruction of thrombus, the main cause of death in cardiovascular disease. The latest technology combining enzymes with certain drugs is the target of new research in the thrombolytic area. The first step is to develop protease from Bacillus thuringiensis HSFI-12 bacteria as an antithrombotic agent, characterization of the bacterial enzyme is necessary. This study aims to determine the specificity of protease from Bacillus thuringiensis HSFI-12 to explore its potential as an antithrombotic agent in terms of anticoagulant and fibrinolytic activities. The molecular weight and specificity of bacterial protease were determined with a zymographic method with casein as substrate. Bacillus thuringiensis HSFI-12 was first cultured on Nutrient Agar (NA) media and then on Skim Milk Agar (SMA) media. The obtained crude protease from Skim Milk Broth (SMB) was then concentrated as dialysate. Both crude and dialysate proteases were tested for their specific activity, as well as anticoagulant and fibrinolytic activities. Next, the dialysate’s molecular weight and specificity on the casein substrate were investigated using the zymographic method. As result, protease activity in crude form is lower than that in dialysate, which was 0.5570 ± 0,004 U/mL and 2.1767 ± 0,005 U/mL, respectively. The molecular weight of the obtained bacterial protease was between 117 – 133 kDa and the enzyme is capable of degrading casein as shown on the zymogram. Overall, both crude and dialysate proteases of Bacillus thuringiensis HSFI-12 show potential as an antithrombotic agent for exhibiting anticoagulant and antiplatelet activities. Yet, it could not exhibit direct fibrinolytic activity implying the possibility that the enzyme plays a role as a plasminogen activator, which can dissolve fibrin by activating plasmin.

Effects of cold atmospheric plasma on endogenous enzyme activity and muscle protein oxidation in Trachinotus ovatus

In this study, we investigated the effects of cold atmospheric plasma (CAP) technology on endogenous enzyme characteristics and muscle protein properties of the golden pomfret (Trachinotus ovatus) under different treatment power and time conditions. Results showed that the enzymatic activity of cathepsin B, L, and calpain in crude protease extracts (CPE) decreased significantly as the treatment power and treatment time of CAP increased (p < 0.05). Oxidative degradation of the CPE after exposure to CAP resulted in significant changes in the structure, total sulfhydryl, and carbonyl content of the CPE (p < 0.05). CAP of an appropriate intensity resulted in significant improvements in the color parameters, hydration properties, and textural property parameters of muscle proteins (p < 0.05). These results suggest that CAP, as a non-thermophysical modification technique, can inhibit the activity of endogenous enzymes as well as alter the protein function in food.

Commercial Extraction of Applicable Alkaline Protease and Chitinase by Bacillus thuringiensis dendrolimus IP 4A/4B

Objective: The study aims to produce alkaline protease and chitinase in the same fermentation process in the most economical way possible so as to reduce production costs and to investigate the application of alkaline protease in detergents compatibility. Methods: Inoculums were prepared in nutrient yeast-extract standard medium, grown, and extracted from the solid medium, while the enzyme activities were measured using Lowry protein assay and high-performance liquid chromatography. Results: The culture of Bacillus thuringiensis subspecies dendrolimus IP 4A/4B was performed under solid-state fermentation conditions. The efficiency and performance of extraction using tap water outperformed other solutions. The crude protease enzyme was compatible with some detergents available in the local market, such as Persil, Tide, and Ariel, where the crude enzyme retained its activity at rates that varied between 113%, 110% and 83%. No microbial growth and therefore no chitinase enzymatic activity was observed on the first and second days, while microbial growth and chitinase activity commenced on the third day and reached the peak of the curve on the fourth day and stabilized on the sixth day. The productivity of chitinase in solid-state was superior to submerged fermentation conditions, which were 2.050 and 0.693U (μmole/mL/min). Conclusion: Production of alkaline protease by Bacillus thuringiensis dendrolimus IP/4B is efficient using tap water for extraction. The alkaline protease is compatible with (Arial, Tide and Persil) detergents, and the same bacterial strain also produced chitinase.

Optimization of Crude Protease Production from Bacillus thuringiensis HSFI-12 and Thrombolytic Activity Its Enzyme Dialysate

Cardiovascular disease (CVD) is among the leading causes of death in the world caused by thrombosis. Thrombosis is the formation of excessive blood clots on the walls of blood vessels called thrombus. This could lead to fatal blockage of the heart muscle or brain. Thrombosis can be treated using enzyme-type drugs such as thrombolytic proteases. There have been many studies of bacteria producing thrombolytic enzymes isolated from fermented foodstuffs. However, commercial production of bacterial enzymes to fulfill the need of thrombolytic agents is still limited, causing high price of CVD therapy. Bacillus sp. HSFI-12 (Holothuria scabra Fermented Intestine-12) isolated from the fermented intestine of sea cucumber Holothuria scabra had been reported to have a competitive thrombolytic activity to the commercial Nattokinase. This study aimed to determine bacterial optimum incubation time based on enzyme activity after bacterium molecular identification was done. It also aimed to obtain the dialysate of bacterial crude protease and then determine the thrombolytic activity of the obtained dialysate. Thrombolytic activity was tested on 4 different types of blood (A, B, AB and O). Results of molecular identification showed that strain HSFI-12 shared similarity level of 99.80 %, with Bacillus thuringiensis. Activity of crude protease from the incubated cultures peaked at 48 h of incubation with activity of 191.5 U/mL The activity of concentrated protease after precipitation and dialysis process (dialysate) was 2-times higher by 355,7 U/mL. The percentage of lysis of blood clots produced by crude blood groups A, B, AB and O showed a range of values ​​of 66.423 - 67.656 % while those that produce dialysate are 77.564 - 78.861 %. As conclusion, the best (optimized) incubation time to produce crude enzyme from B. thuringiensis HSFI-12 with the highest activity was 48 h. The process of concentrating the crude thrombolytic protease of B. thuringiensis HSFI-12 increases both activity and thrombolytic ability of the bacterial enzyme. HIGHLIGHTS The most optimum incubation time for protease production from Bacillus thuringiensis is 48 h Partial purification on bacterial crude protease through resulted in 86 % increased activity Partial purification on bacterial crude protease resulted in 17 % increased blood clot lysis ability Clot lysis ability of both crude and dialysate proteases are higher than that of Nattokinase (NK) GRAPHICAL ABSTRACT

Potential Application of HSFI-8 Crude Protease as Meat Tenderizer and Anticoagulant Agent

Potential Application of HSFI-8 Crude Protease as Meat Tenderizer and Anticoagulant Agent

Potential Application of HSFI-8 Crude Protease as Meat Tenderizer and Anticoagulant Agent

Protease enzymes are enzymes that are able to hydrolyze peptide bonds in proteins into simple compounds. The protease enzyme used was derived from fermented sea cucumber digestive organs (HSFI-8). HSFI-8 uses meat tenderizer as well as an anticoagulant agent. The samples used were beef, chicken and tuna whose protein profile was analyzed using SDS-PAGE. Three types of meat were soaked in HSFI-8 at a concentration of 30% v/v for 3 h. In addition, protease enzymes as therapy for the treatment of CVD. The second sample, human blood, was tested for anticoagulant by Lee-White in vitro. Blood samples were treated with the addition of HSFI-8 as an anticoagulant which measured blood clotting time. The results showed that soaking beef using HSFI-8 was able to break the peptide bonds in proteins which were marked by the addition of minor protein bands. There was no change in the amount of protein bands in chicken and cob meat. The results of the anticoagulant test showed that HSFI-8 was able to prolong blood clotting time in samples given the addition of HSFI-8. Although HSFI-8 did not inhibit the clotting of the 10% EDTA anticoagulant, HSFI-8 was good as an anticoagulant.

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.

Preparation, characterization, immobilization, and molecular docking analysis of a novel detergent-stable subtilisin-like serine protease from Streptomyces mutabilis strain TN-X30

We recently described the production of a detergent-biocompatible crude protease from Streptomyces mutabilis strain TN-X30. Here, we describe the purification, characterization, and immobilization of the serine alkaline protease (named SPSM), as well as the cloning, sequencing, and over-expression of its corresponding gene (spSM). Pure enzyme was obtained after ammonium sulphate precipitation followed by heat-treatment and Sephacryl® S-200 column purification. The sequence of the first 26 NH2-terminal residues of SPSM showed a high sequence identity to subtilisin-like serine proteases produced by actinobacteria. The spSM gene was heterologously expressed in Escherichia coli BL21(DE3)pLysS and E. coli BL21-AI™ strains using pTrc99A (rSPSM) and Gateway™ pDEST™ 17 [(His)6-tagged SPSM] vectors, respectively. Results obtained indicated that the (His)6-tagged SPSM showed the highest stability. The SPSM was immobilized using encapsulation and adsorption-encapsulation approaches and three different carriers. Features of SPSM in soluble and immobilized forms were analyzed by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode, X-ray diffraction (XRD), zeta potential measurements, and field emission scanning electron microscopy (FE-SEM). The white clay and kaolin used in this study are eco-friendly binders to alginate-SPSM and show great potential for application of the immobilized SPSM in various industries. Molecular modeling and docking of N-succinyl-l-Phe-l-Ala-l-Ala-l-Phe-p-nitroanilide in the active site of SPSM revealed the involvement of 21 amino acids in substrate binding.

Characterization of Pleurotus citrinopileatus hydrolysates obtained from Actinomucor elegans proteases compared with that by commercial proteases.

Pleurotus citrinopileatus, a nutritious and palatable edible mushroom, can be used as an appropriate material to prepare high-grade flavoring agents. Based on this, the current study aimed to investigate the feasibility of a productive protease system from Actinomucor elegans to prepare P. citrinopileatus hydrolysate (PCH). The Actinomucor elegans crude protease (AECP) was prepared from the solid-state fermentation product of P. citrinopileatus by A. elegans. AECP and four commercial proteases (alcalase, neutrase, papain, and protamex) were applied to acquire five kinds of PCHs. The physical-chemical properties of PCHs as well as its concentrationandcomposition of nonvolatile compounds were comparatively analyzed. Sensory evaluation and electronic tongue analysis were utilized to evaluate sensory characteristics. AECP was found to be the most effective protease, with the highest hydrolysis degree (35.91%) and protein recovery (81.46%). The result of molecular weight distribution indicated that peptides below 500Da were the main fraction of AECP hydrolysates, while AECP hydrolysates showed the highest content of monosodium glutamate-like (20.23 ± 0.16mg/g) and flavor 5'-nucleotide (4.30 ± 0.07mg/g) peptides. In summary, the AECP hydrolysate had superior sensory profiles compared with other hydrolysates. In addition, AECP hydrolysates exhibited favorable kokumi taste in which peptides below 500Da showed the highest correlation with kokumi by the results of partial least-squares regression. These results indicated the feasibility of applying PCHs as flavor additives or seasoning in the food industry. AECP might be used as an alternative enzyme choice because of its low cost and high hydrolysis efficiency. PRACTICAL APPLICATION: Pleurotus citrinopileatus served as a potential raw material for natural seasonings because of its high protein content and appropriate ratio of umami amino acids to total amino acids. Enzymatic hydrolysis was an efficient approach to improve the flavor of P. citrinopileatus, where the choice of enzyme was one of the most critical factors. The research indicated that P. citrinopileatus hydrolysate prepared by A. elegans crude protease (AECP) exhibited an acceptable flavor, which provided theoretical support for the high-value utilization of P. citrinopileatus as food seasoning. AECP might be applied as an alternative enzyme resource because of its low cost and high hydrolysis efficiency.

Enzymatic bioprospecting of endophytic Aspergillus niger isolated from Albizia lebbeck (L.) Benth

Endophytic fungi reside inside plants in a mutualistic relationship with the host and produce numerous bioactive compounds, including extracellular enzymes. A rich diversity of endophytic fungi associated with the medicinal plant Albizia lebbeck (L.) Benth was bio-prospected in the present study. Thirty-three isolates obtained from the plant were assessed for secretion of six different extracellular hydrolytic enzymes. The results showed that 38%, 9%, 69%, 16%, 38%, and 13% of the endophytic isolates could produce amylase, protease, lipase, laccase, cellulase, and gelatinase enzymes, respectively. Overall, 78% of the isolates showed positive results for one or the other enzyme. Aspergillus niger (Isolate 8) isolated from the bark proved to be most efficient in producing all the enzymes and was further exploited for various applications. The crude fungal amylase of the isolate helped in 39.08% reduction in biochemical oxidation demand (BOD) of municipal wastewater. Effective blood stain removal from the white cotton cloth was achieved when crude protease was used in combination with detergent. 13.29% biodegradation of vegetable oil was observed using fungal lipase. Dye decolorization of methylene blue (71.65%) and methyl orange (56.53%) was observed by fungal laccase. Saccharification of bagasse (83.94%) and paper (64.93%) was achieved by using fungal cellulase. Complete extraction of silver from waste X-ray films was achieved by crude gelatinase. The results obtained in the present study can further be strengthened using purified enzymes.

Characterization and application of a crude bacterial protease to produce antioxidant hydrolysates from whey protein

Bacillus sp. CL14 crude protease was partially characterized and applied to obtain antioxidant whey protein isolate (WPI) hydrolysates. Optimal activity occurred at pH 9.0 and 60 °C. Ca2+, Mg2+, and Mn2+ (5 mM) enhanced activity (12–26%), whereas Co2+, Cu2+, Fe2+, and Zn2+ inhibited it (50–94%). At 1% (v/v), Tween 20 and Triton X-100 enhanced activities (21–27%), β-mercaptoethanol decreased it (15%), and dimethyl sulfoxide (DMSO) had no effect. Sodium dodecyl sulfate (SDS; 0.1%, w/v) increased activity by 36%. Complete inhibition by phenylmethylsulfonyl fluoride (PMSF), and 85% inhibition by ethylenediaminotetraacetic acid, indicates its serine protease character and the importance of cations for activity/stability. With 5 mM Ca2+, protease was optimally active at 65 °C and completely stable after 20 min at 40–55 °C. Crude protease preferentially hydrolyzed WPI and soy protein, followed by casein. WPI hydrolysis was then performed (55 °C, pH 9.0, 5 mM Ca2+) for 0–180 min. Contents of trichloroacetic acid (TCA)-soluble proteins in WPI hydrolysates (HWPI) increased from 29% (0 min) to 50–52% (60–180 min), accompanied by enhanced radical scavenging activity (14%, 0 min; ∼34%, 60–180 min) and Fe2+-chelating ability (56%, 0 min; ∼74%, 45–180 min). CL14 protease might represent an alternative biocatalyst to obtain antioxidant hydrolysates from WPI and, potentially, from other food proteins.

Isolation and identification of protease‐producing bacteria from sludge and sediment soil around Adama, Ethiopia

Proteases are enzymes used in industries such the production and processing of detergents, food, leather, and silk. The aim of this study was to isolate and identify protease‐producing bacteria from a sludge disposal site and from sediments. Soil samples were collected separately from the selected area. Samples weighing 1 g were serially diluted and spread onto skim milk agar. A total of 16 bacteria species were isolated from the study samples. Four bacterial isolates showed high proteolytic activity and were selected for enzymatic study based on their zone of proteolysis. The isolates were identified based on biochemical tests. The results indicated that the isolated bacteria were E. coli (99.69%), Pseudomonas putrefaciens (Shewanella putrefaciens) (91.61%), Bacillus carboniphilus (92.78%), and Lysinibacillus sphaericus (98.4%). The crude protease enzymes produced by these bacterial isolates showed promising results for application in dehairing and destaining as detergent additives. Bacillus carboniphilus showed the best level of activity and was selected as the most potent protease‐producing bacteria for both dehairing and destaining ability. Soils from sludge disposal sites and sediments from around tannery wastes could be good sources from which to isolate alkaline protease‐producing bacteria.

Characterization of the Proteolytic Activity of a Halophilic Aspergillus reticulatus Strain SK1-1 Isolated from a Solar Saltern.

Salterns are hypersaline environments that are inhabited by diverse halophilic microorganisms, including fungi. In this study, we isolated a fungal strain SK1-1 from a saltern in the Republic of Korea, which was identified as Asperillus reticulatus. This is the first reported saline-environment-derived A. reticulatus that belongs to the Aspergillus penicillioides clade and encompasses xerophilic fungi. SK1-1 was halophilic, obligately requiring NaCl for growth, with a maximum radial growth of 6%–9% (w/v) NaCl. To facilitate the biotechnological application of halophilic fungi, we screened the SK1-1 strain for proteolytic activity. Proteases have widespread applications in food processing, detergents, textiles, and waste treatment, and halophilic proteases can enable protein degradation in high salt environments. We assessed the proteolytic activity of the extracellular crude enzyme of SK1-1 using azocasein as a substrate. The crude protease exhibited maximum activity at 40–50 °C, pH 9.5–10.5, and in the absence of NaCl. It was also able to retain up to 69% of its maximum activity until 7% NaCl. Protease inhibitor assays showed complete inhibition of the proteolytic activity of crude enzymes by Pefabloc® SC. Our data suggest that the halophilic A. reticulatus strain SK1-1 produces an extracellular alkaline serine protease.

Optimization and Functional Properties of a Protease from a Fish Gut Isolate Pseudomonas sp. PD14 Grown on Fish Processing Wastes Substrate

Extracellular protease production from Pseudomonas sp. PD14 isolated from the gut of rabbitfish, Siganus guttatus, was investigated using fish processing wastes as nitrogen source. Three types of fish processing by-products – namely, heads, bones, and viscera – from yellowstripe scad (Selaroides sp.) and Japanese halfbeak (Hyporhampus sajori), as well as trimmings and viscera from squid (Loligo sp.) were used. Among the fish waste preparations tested, Japanese halfbeak viscera (HV) flour (1.0%) supported the maximum protease production of 365.0 U/mg protein. Effects of media components on protease production in the optimized concentration of HV flour revealed that the protease enzyme can be optimally produced using maltose (0.50%) as a carbon source, at pH 8.0, and incubated for 48 h at 40 °C. Partial characterization of the crude enzyme supernatant showed that the optimum pH and temperature were 8.0 and 50 °C, respectively. Furthermore, the crude protease exhibited thermostability retaining 50% activity at 60 °C and fairly stable up to pH 11.0. Considering the high costs of industrial enzyme production, the use of fish processing wastes for protease production offers a promising future for enzyme biotechnology.

ISOLATION AND SCREENING OF PROTEOLYTIC BACTERIA FROM SIDOARJO SHRIMP PASTE AS PROTEASE SOURCE TO EXTRACT THE COLLAGEN FROM MILKFISH SCALES (CHANOS CHANOS)

This research aimed to isolate protease-producing bacteria from Sidoarjo shrimp paste for extracting collagen from milkfish scales. This study began with isolation, followed by screening and purification of protease-producing bacterial isolates. Further confirmation of the isolates’ proteolytic indices and the crude protease production, the enzymes’ efficacy in extracting collagen from milkfish scales were tested, followed by pathogenicity and identification using 16S rRNA molecular technique. The study has successfully isolated 15 proteolytic bacterial isolates using skimmed milk agar, but only isolates of TR-10, TR-4.1.1, and TR-15.1 exhibited prospective proteolytic activity based on their corresponding proteolytic indices of 2.96 ± 0.06, 3.10 ± 0.10, and 3.71 ± 0.48. Although the proteolytic activity of isolates TR-10 (0.22 ± 0.05 U/mL) and TR-15.1 (1.07 ± 0.14 U/mL) was high in a salt medium using peptone as the nitrogen source, only the former showed satisfactory activity to extract soluble collagen from milkfish scales. Based on the 16SrRNA, the TR-10 isolate was identified as Bacillus megaterium. The non-pathogenicity of the TR-10 bacterium signified its promising role as a protease source for the halal collagen extraction from milkfish scales.

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.

Hydrolysis of peanut (Arachis hypogea L) protein concentrate by fungal crude protease extract: effect on structural, functional and in-vitro protein digestibility.

Peanut protein concentrates (PPCs) were subjected to hydrolysis by crude protease extract (CPE) obtained from three fungi viz; Rhizopus oligosporus, Trichoderma reesei, and Aspergillus oryzae and the effect on structural, functional and in-vitro protein digestibility (IVPD) properties were studied. Particle size was found significantly (p ≤ 0.05) lower in hydrolyzed samples than un-treated samples. Fourier transform infrared spectroscopy (FTIR) spectrum of hydrolyzed samples displayed intense absorbance peaks in the wavelength ranging from 1500 to 2600cm-1. Peanut protein concentrates hydrolyzed by CPE from R. oligosporus showed higher surface hydrophobicity (564.18). Total sulfhydryl content was found lower in all the hydrolyzed samples whereas, reverse trend was observed for exposed sulfhydryl content. The structural changes simultaneously affected the functional and IVPD attributes of hydrolyzed PPCs. In comparison to the PPCs hydrolysed using crude extracts from T. reesei and R. oligosporus, PPCs hydrolysed by A, oryzae showed higher solubility, water and oil binding capacity, foaming capacity and foam stability. Higher IVPD values of 86.70% was also found in PPCs hydrolyzed with CPE of A. oryzae. The study established that CPE hydrolysis of PPCs has potential for scale-up studies and may serve as a cost effective alternative to protein hydrolysis with pure enzymes.