Displaying publications 1 - 20 of 35 in total

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  1. Fulltext Recombinant Production and Characterization of an Extracellular Subtilisin-Like Serine Protease from Acinetobacter baumannii of Fermented Food Origin
    Muhammed NS, Hussin N, Lim AS, Jonet MA, Mohamad SE, Jamaluddin H
    Protein J, 2021 06;40(3):419-435.
    PMID: 33870461 DOI: 10.1007/s10930-021-09986-5
    Acinetobacter baumannii is a ubiquitous bacteria that is increasingly becoming a formidable nosocomial pathogen. Due to its clinical relevance, studies on the bacteria's secretory molecules especially extracellular proteases are of interest primarily in relation to the enzyme's role in virulence. Besides, favorable properties that extracellular proteases possess may be exploited for commercial use thus there is a need to investigate extracellular proteases from Acinetobacter baumannii to gain insights into their catalytic properties. In this study, an extracellular subtilisin-like serine protease from Acinetobacter baumannii designated as SPSFQ that was isolated from fermented food was recombinantly expressed and characterized. The mature catalytically active form of SPSFQ shared a high percentage sequence identity of 99% to extracellular proteases from clinical isolates of Acinetobacter baumannii and Klebsiella pneumoniae as well as a moderately high percentage identity to other bacterial proteases with known keratinolytic and collagenolytic activity. The homology model of mature SPSFQ revealed its structure is composed of 10 β-strands, 8 α-helices, and connecting loops resembling a typical architecture of subtilisin-like α/β motif. SPSFQ is catalytically active at an optimum temperature of 40 °C and pH 9. Its activity is stimulated in the presence of Ca2+ and severely inhibited in the presence of PMSF. SPSFQ also displayed the ability to degrade several tissue-associated protein substrates such as keratin, collagen, and fibrin. Accordingly, our study shed light on the catalytic properties of a previously uncharacterized extracellular serine protease from Acinetobacter baumannii that warrants further investigations into its potential role as a virulence factor in pathogenicity and commercial applications.
    Matched MeSH terms: Subtilisins/biosynthesis; Subtilisins/genetics; Subtilisins/isolation & purification; Subtilisins/chemistry
  2. Fruit-specific expression of papaya subtilase gene
    Othman R, Nuraziyan A
    J Plant Physiol, 2010 Jan 15;167(2):131-7.
    PMID: 19729222 DOI: 10.1016/j.jplph.2009.07.015
    Subtilisin-like serine proteases (EC 3.4.21) consist of a widespread family of enzymes that is involved in various processes including in plants. The full-length cDNA (CpSUB1) and the corresponding genomic DNA for papaya subtilase have been obtained using rapid amplification of cDNA ends (RACEs) and PCR primer walking techniques, respectively. The cDNA clone contains an open reading frame of 2316bp encoding 772 amino acids with a calculated molecular mass of 82.6kDa and an isoelectric point (pI) of 8.97. The CpSUB1 gene is composed of nine exons and eight introns. The amino acid sequence encoded by CpSUB1 shared high identity (>60%) with the amino acid sequence of other plant subtilisin-like proteases. Sequence analysis of CpSUB1 revealed the presence of a possible signal peptide (25 amino acid residues) and an NH(2)-terminal prosequence (88 amino acid residues). In addition, papaya subtilase possesses the characteristic subtilisin catalytic triad amino acids namely Asp, His and Ser, together with the substrate-binding site, Asn. DNA hybridization analysis showed that subtilase gene exists as a single copy in the papaya genome. RNA hybridization analyses showed that expression of the subtilase transcripts was only detected in mesocarp but not in non-fruit tissues. Gene expression in fruit tissues reached the highest level during the ripening stage at which the fruits undergo dramatic softening process. Subsequently, pro-subtilase ( approximately 80kDa) was expressed as recombinant pro-enzyme ( approximately 97kDa), which was used to generate antiserum against papaya subtilase, anti-sub. Protein gel blot analysis using anti-sub towards total protein extracted from all ripening stages revealed that a protein with a molecular mass of approximately 70kDa reacted with the antiserum. Hence both RNA hybridization and protein gel blot analyses confirmed the presence of subtilase during papaya fruit ripening, pointing to its possible involvement in this important process.
    Matched MeSH terms: Subtilisins/genetics; Subtilisins/metabolism*
  3. Novel antihypertensive peptides from lupin protein hydrolysate: An in-silico identification and molecular docking studies
    Fadimu GJ, Gan CY, Olalere OA, Farahnaky A, Gill H, Truong T
    Food Chem, 2023 May 01;407:135082.
    PMID: 36493485 DOI: 10.1016/j.foodchem.2022.135082
    Application of non-thermal treatment to proteins prior to enzymatic hydrolysis can facilitate the release of novel bioactive peptides (BPs) with unique biological activities. In this study, lupin protein isolate was pre-treated with ultrasound and hydrolysed using alcalase and flavourzyme to produce alcalase hydrolysate (ACT) and flavourzyme hydrolysate(FCT). These hydrolysates were fractionated into 1, 5, and 10 kDa molecular weight fractions using a membrane ultrafiltration technique. The in vitro angiotensin-converting enzyme (ACE) studies revealed that unfractionated ACT (IC50 = 3.21 mg mL-1) and FCT (IC50 = 3.32 mg mL-1) were more active inhibitors of ACE in comparison to their ultrafiltrated fractions with IC50 values ranging from 6.09 to 7.45 mg mL-1. Molecular docking analysis predicted three unique peptides from ACT (AIPPGIPY, SVPGCT, and QGAGG) and FCT (AIPINNPGKL, SGNQGP, and PPGIP) as potential ACE inhibitors. Thus, unique BPs with ACE inhibitory effects might be generated from ultrasonicated lupin protein.
    Matched MeSH terms: Subtilisins/metabolism
  4. Fulltext Bitterness of green mussel (Perna viridis) hydrolysate as influenced by the degree of hydrolysis
    Normah, I., Siti Hafsah, M.S., Nurul Izzaira, A.
    International Food Research Journal, 2013;20(5):2261-2268.
    MyJurnal
    Green mussel (Perna viridis) was hydrolysed with alcalase under two different conditions consisting of pH7, E/S5% or pH 9, E/S 3% at 60°C for two hours. Hydrolysis at pH 9, E/S3% resulted in a higher degree of hydrolysis (DH) than pH7, E/S5% with degree of hydrolysis of 37.00% and 28.33%, respectively. The green mussel hydrolysates were characterized by molecular weight of
    Matched MeSH terms: Subtilisins
  5. Fulltext Physicochemical properties of mud clam (Polymesoda erosa) hydrolysates obtained using different microbial enzymes
    Normah, I., Noorasma, M.
    International Food Research Journal, 2015;22(3):1103-1111.
    MyJurnal
    Physicochemical properties of mud clam (Polymesoda erosa) hydrolysate produced using two microbial enzymes; alcalase and flavourzyme were determined. Hydrolysis using alcalase at 20.28% degree of hydrolysis (DH) resulted in 25.06 % yield and 45.37% protein while flavourzyme hydrolysis showed 22.93 % DH, 46.67 % protein and 30.68 % yield. Both hydrolysates were yellowish. Better emulsifying properties, foaming properties and water and oil holding capacity were exhibited by flavourzyme hydrolysate compared to the alcalase hydrolysate. However, in terms of amino acid composition, alcalase hydrolysate contained higher amino acid composition (75.06%) than flavourzyme hydrolysate (62.37%). The study suggested that mud clam hydrolysate had the potential to be used in food formulations for human consumption.
    Matched MeSH terms: Subtilisins
  6. Fulltext Optimization of enzymatic hydrolysis of tilapia (Oreochromis niloticus) by- product using response surface methodology
    Roslan, J., Mustapa Kamal, S.M., Md. Yunos, K.F., Abdullah, N.
    International Food Research Journal, 2015;22(3):1117-1123.
    MyJurnal
    Fish protein hydrolysate was recovered from tilapia by-product (TB) through enzymatic hydrolysis using alcalase enzyme. Hydrolysis reaction of TB was monitored according to the degree of hydrolysis (DH) by employing O-phtaldialdehyde (OPA) method. Optimization process for obtaining high yield of TB protein hydrolysate was performed using response surface methodology (RSM) by optimizing a combination of four independent variables namely, pH (6.5-8.5), temperature (55-70oC), substrate concentration (10-17.5% w/v), and enzyme concentration (1.5-3.5% w/w) with (DH) as a response. The optimum enzymatic hydrolysis conditions were obtained at pH 7.5, temperature of 60oC, substrate concentration of 15% (w/v) and 2.5% (w/w) of enzyme concentration and yielded about 20.20% of DH after hydrolyzing for 120 min. RSM generated model predicted that 20.42% of DH could be achieved at these conditions and this model was valid based on the DH value obtained from the experimental study (20.31%) which was quite similar with the predicted value. High yield of DH obtained from the optimization process could produce fish protein hydrolysate with good nutritional and functional properties.
    Matched MeSH terms: Subtilisins
  7. Fulltext Porcine placenta hydrolysate as an alternate functional food ingredient: In vitro antioxidant and antibacterial assessments
    Laosam P, Panpipat W, Yusakul G, Cheong LZ, Chaijan M
    PLoS One, 2021;16(10):e0258445.
    PMID: 34695136 DOI: 10.1371/journal.pone.0258445
    The production of bioactive peptides from animal-based raw materials highly depends on enzymatic hydrolysis. Porcine placenta is an underutilized biomass in Thailand's pig farms, yet it is still a source of proteins and beneficial compounds. Porcine placenta could be used as a protein substrate for the production of enzymatic hydrolysate, which could be employed as a functional food ingredient in the future. The goal of this study was to enzymatically produce porcine placenta hydrolysates (PPH) using three commercial enzymes (Alcalase, Flavouzyme, and papain) and evaluate their in vitro antioxidant and antibacterial activity. The degree of hydrolysis (DH) increased as the enzyme load and hydrolysis time increased, but the DH was governed by the enzyme class. The maximum DH was found after using 10% enzyme for 20 min of hydrolysis (36.60%, 31.40%, and 29.81% for Alcalase, Flavouzyme, and papain). Depending on the enzyme type and DH, peptides of various sizes (0.40-323.56 kDa) were detected in all PPH. PPH created with Alcalase had an excellent reducing capacity and metal chelating ability (p < 0.05), whereas PPH made with Flavourzyme and Papain had higher DPPH• and ABTS•+ inhibitory activities (p < 0.05). Papain-derived PPH also had a strong antibacterial effect against Staphylococcus aureus and Escherichia coli, with clear zone values of 17.20 mm and 14.00 mm, respectively (p < 0.05). When PPH was transported via a gastrointestinal tract model system, its antioxidative characteristics were altered. PPH's properties and bioactivities were thus influenced by the enzyme type, enzyme concentration, and hydrolysis time used. Therefore, PPH produced from porcine placenta can be categorized as an antioxidant and antibacterial alternative.
    Matched MeSH terms: Subtilisins/metabolism; Subtilisins/chemistry
  8. Fulltext Angiotensin-I converting enzyme (ACE) inhibitory peptides from chicken skin gelatin hydrolysate and its antihypertensive effect in spontaneously hypertensive rats
    Sarbon, N.M., Howell, N.K., Wan Ahmad, W.A.N.
    International Food Research Journal, 2019;26(3):903-911.
    MyJurnal
    Chicken skin gelatin hydrolysates and peptides with angiotensin converting enzyme inhibitory (ACEI) activity were produced enzymatically using alcalase, pronase E, and collagenase before fractionation into
    Matched MeSH terms: Subtilisins
  9. Enzymatic hydrolysis: Sialylated mucin (SiaMuc) glycoprotein of edible swiftlet's nest (ESN) and its molecular weight distribution as bioactive ESN SiaMuc-glycopeptide hydrolysate
    Hui Yan T, Lim SJ, Babji AS, Rawi MH, Sarbini SR
    Int J Biol Macromol, 2021 Apr 01;175:422-431.
    PMID: 33561458 DOI: 10.1016/j.ijbiomac.2021.02.007
    Bioactive edible swiftlet's nest (ESN) sialylated-mucin (SiaMuc) hydrolysate is produced by alcalase hydrolysis. Enzymatic hydrolysis of ESN breakdown high-valued ESN SiaMuc-glycoprotein into bioactive SiaMuc-glycopeptide. This is a breakthrough for the issue of insolubility and low extraction rate in ESN, and even increases the bioavailability of ESN nutritional functionality and health benefits. Hydrolysis of ESN SiaMuc-glycoprotein was performed for 1 to 4 h and its effect on physicochemical properties, molecular weight (MW) distribution, SiaMuc-glycoprotein and glycopeptide integrity were determined. Other than improvement in solubility and bioavailability as SiaMuc-glycopeptide, results from SDS-PAGE revealed that MW of SiaMuc-glycoprotein decreased from 42.0-148.8 kDa to 17.7-142.7 kDa with increasing hydrolysis period. Further hydrolysis from maximized DH (90 min) showed an insignificant effect on the MW of ESN SiaMuc-glycopeptide and remained constant at 15.2 kDa. This highlights that enzymatic hydrolysis only influences macro SiaMuc-glycoprotein fractions (142.7, 115.3 and 102.7 kDa), while the majority of SiaMuc-glycopeptide fractions from 36.6-98.6 kDa remained intact. Conclusively, alcalase hydrolysis of ESN showed high recovery in the form of bioactive ESN SiaMuc-glycopeptide. Therefore, enzymatic biotechnology is an economic alternative applicable on ESN that broaden industrial utilization by reducing the MW without destroying the quality of bioactive SiaMuc-glycoprotein.
    Matched MeSH terms: Subtilisins
  10. Fulltext Optimization of enzymatic protein hydrolysis from silver catfish (Pangasius sp.) frame
    Amiza, M.A., Nurul Ashikin, S., Faazaz, A.L.
    International Food Research Journal, 2011;18(2):775-781.
    MyJurnal
    This study aims to determine the combined effects of hydrolysis time, temperature, pH and ratio of enzyme to substrate on the degree of hydrolysis (DH) of silver catfish frame using Response Surface Methodology. The proximate compositions of silver catfish frame and silver catfish hydrolysate powder were determined as well. The effects of independent factors were described using a three-level factors Face Centered Central Composite design. The suggested hydrolysis conditions for obtaining the optimum DH using Alcalase® were – temperature of 55oC, hydrolysis time of 163 min, pH of substrate at 9.45 and an enzyme concentration of 2.0%. The generated model showed a quadratic fit with experimental data. Proximate analyses revealed that silver catfish frame contained 25.02% protein, 68.21% fat and 7.08% ash. While silver catfish frame hydrolysate powder contained 65.05% protein, 32.92% fat and 0.86% ash. The protein recovery in silver catfish frame hydrolysate was as high as 71.6%.
    Matched MeSH terms: Subtilisins
  11. Fulltext Enzymatic preparation of palm kernel expeller protein hydrolysate (PKEPH)
    Ng, K.L., Mohd Khan, A.
    International Food Research Journal, 2012;19(2):721-725.
    MyJurnal
    Utilization of palm kernel expeller (PKE), a palm oil milling by-product, may be diversified through the exploitation of its protein component. The PKE protein could be effectively extracted using an alkaline
    solution and followed by enzymatic hydrolysis to produce PKE protein hydrolysates or crude PKE peptide. The extraction of PKE protein was successfully carried out using an alkaline solution at pH11, at ratio of 1:10 (g/ml), PKE powder to alkaline solution with continuous shaking, 150 rpm, in a water bath operating at 50°C for 30 min. The extracted protein powder (PKEP) had 68.50±3.08% crude protein, 0.54±0.03% fat and 0.73±0.02% ash. The freeze-dried PKEP was re-suspend in particular buffer and hydrolyzed with proteolytic enzymes (Alcalase® 2.4L, Flavourzyme® 500MG, pepsin or trypsin) to obtain PKEP hydrolysate (PKEPH). The effect of enzyme concentration (0, 2, 4, 6, 8 & 10%) and time of hydrolysis (0, 6, 12, 24, 48 h) was studied to determine the most efficient hydrolytic conditions. Results showed that all enzymes tested were capable of hydrolyzing the PKEP and producing hydrolysates with different degree of hydrolysis (DH%). At 8.0% concentration, Alcalase®2.4L hydrolyzed PKEP into the highest DH (75.96%) hydrolysate (PKEPH) after 1h hydrolysis. Although only with 2.0% Alcalase 2.4 L concentration, it was sufficient to produce PKEP hydrolysate of 81.35% DH %, but it required 12 h to hydrolyze the protein. Pepsin was relatively the least efficient protease to hydrolyze the PKEP.
    Matched MeSH terms: Subtilisins
  12. Fulltext Degree of hydrolysis and free tryptophan content of Skipjack Tuna (Katsuwonus pelamis) protein hydrolysates produced with different type of industrial proteases
    Herpandi, Huda, N., Rosma, A., Wan Nadiah W. A.
    International Food Research Journal, 2012;19(3):863-867.
    MyJurnal
    Protein-rich by-products from the canning industry, especially dark flesh of skipjack, have limited uses due to several factors such as darken color, susceptibility to oxidation and off flavour. Protein hydrolysates from skipjack dark flesh was produced with different type of industrial proteases (Alcalase®2.4L FG, Protamex®, Neutrase®1.5MG and Flavourzyme®500MG) for 60, 120, 180 and 240 min with level of proteases used of 0.5, 1, 1.5 and 2% per weight of raw material. The degree of hydrolysis and free tryptophan content of hydrolysate were investigated. The results shows longer time with higher concentration of enzyme has increased the degree of hydrolysis. Alcalase®2.4L FG had the highest degree of hydrolysis among all proteases followed by Protamex®, Flavourzyme®500MG and Neutrase® 1.5MG. All enzymes increase free tryptophan content linearly with the increament of protease enzyme level. The longer the hydrolysis time, the higher the content of free tryptophan produced.
    Matched MeSH terms: Subtilisins
  13. Fulltext Functional and antioxidant properties of angelwing clam (Pholas orientalis) hydrolysate produced using alcalase
    Normah Ismail, Juliana Mahmod, Awatif Khairul Fatihin Mustafa Kamal
    Scientific Research Journal, 2014;11(1):0-0.
    MyJurnal
    In this study, Hydrolysate from angelwing clam (Pholas orientalis) was produced at 0, 1, 2 and 3 hrs and E/S ratio of 0.5 and 3% using alcalase where the pH and temperature were kept constant at pH 8.5 and 60°C, respectively. The hydrolysates were analysed for antioxidant and functional properties such as solubility, emulsifying properties and water and oil holding capacity. Degree of hydrolysis (DH), yield, functional and antioxidant properties were influenced by the hydrolysis time and E/S ratio. Higher enzyme concentration (E/S 3%) and longer hydrolysis time increased the DH. Yield was higher at E/S 3% but reduced with hydrolysis time. Longer hydrolysis time produced more soluble hydrolysate and higher metal chelating activity but lower in emulsifying properties and DPPH activity. Higher enzyme concentration resulted in increase only in solubility and metal chelating activity. This study revealed that enzymatic hydrolysis using alcalase should be performed at shorter hydrolysis time using intermediate concentration of enzyme (E/S between 0.5 to 3%) in order to produce angelwing clam hydrolysate with collectively good functional and antioxidant properties
    Matched MeSH terms: Subtilisins
  14. Fulltext Preparation and physicochemical characterization of fish skin gelatine hydrolysate from shortfin scad (Decapterus macrosoma)
    Rasli, H.I., Sarbon, N.M.
    International Food Research Journal, 2019;26(1):287-294.
    MyJurnal
    Enzymatic hydrolysis of proteins is an important bioprocess method to prepare bioactive peptides with many functionality and health benefits. The aims of the present work were to prepare and determine the physicochemical characteristics of gelatine hydrolysate from skin of shortfin scad (SSGH) via hydrolysis using alcalase. Analyses on chemical composition, molecular weight by SDS PAGE, protein concentration, amino acid composition, Fourier Transform Infrared Spectroscopic features, and solubility of SSGH were thus performed. The yield of SSGH obtained was 51.01% (d.b.). The chemical compositions of SSGH for moisture, protein, fat, and ash were 13.82%, 90.05%, 1.95%, and 12.48%, respectively. SSGH showed low molecular weight (
    Matched MeSH terms: Subtilisins
  15. Fulltext Effects of degree of hydrolysis on physicochemical properties of cobia (Rachycentron canadum) frame hydrolysate
    Amiza, M.A., Kong, Y.L., Faazaz, A.L.
    International Food Research Journal, 2012;19(1):199-206.
    MyJurnal
    The effect of degree of hydrolysis (DH) on the physicochemical properties of cobia frame hydrolysate was determined. Three levels of degree of hydrolysis of cobia frame hydrolysate were studied, which were 53%, 71% and 96%. After enzymatic hydrolysis using Alcalase®, the samples were spray-dried. Cobia hydrolysate powder samples were analyzed for their proximate analysis and physicochemical properties. The proximate analysis showed significant differences in fat and ash content only. DH96 hydrolysate showed desirable essential amino acid profile for human requirement except for methionine and isoleucine. The study found that cobia frame hydrolysate had good colour, emulsifying capacity and excellent foaming properties. However, there were no significant differences in water-holding capacity, oil-holding capacity and peptide solubility among the hydrolysate samples. This study suggested that cobia frame hydrolysate is a potential ingredient and foaming agent for food industry.
    Matched MeSH terms: Subtilisins
  16. Identification and molecular docking study of novel cholesterol esterase inhibitory peptides from camel milk proteins
    Mudgil P, Baby B, Ngoh YY, Vijayan R, Gan CY, Maqsood S
    J Dairy Sci, 2019 Dec;102(12):10748-10759.
    PMID: 31548068 DOI: 10.3168/jds.2019-16520
    Novel bioactive peptides from camel milk protein hydrolysates (CMPH) were identified and tested for inhibition of cholesterol esterase (CEase), and their possible binding mechanisms were elucidated by molecular docking. Papain-generated CMPH showed the highest degree of hydrolysis. All CMPH produced upon enzymatic degradation demonstrated a dramatic enhancement of CEase inhibition compared with intact camel milk proteins, with papain-generated hydrolysate P9 displaying the highest inhibition. Peptide identification and their modeling through PepSite 2 revealed that among 20 potential bioactive peptides in alcalase-generated hydrolysate A9, only 3 peptides, with sequences KFQWGY, SQDWSFY, and YWYPPQ, showed the highest binding toward CEase catalytic sites. Among 43 peptides in 9-h papain-generated hydrolysate P9, 4 peptides were found to be potent CEase inhibitors. Molecular docking revealed that WPMLQPKVM, CLSPLQMR, MYQQWKFL, and CLSPLQFR from P9 hydrolysates were able to bind to the active site of CEase with good docking scores and molecular mechanics-generalized born surface area binding energies. Overall, this is the first study reporting CEase inhibitory potential of peptides generated from milk proteins.
    Matched MeSH terms: Subtilisins/chemistry
  17. Physicochemical and functional properties of Alcalase-extracted protein hydrolysate from oil palm leaves
    Hau EH, Teh SS, Yeo SK, Mah SH
    J Sci Food Agric, 2022 Jan 15;102(1):233-240.
    PMID: 34081335 DOI: 10.1002/jsfa.11350
    BACKGROUND: The oil palm tree produces 90% of wastes and the limited usage of these wastes causes a major disposal problem in the mills. Nevertheless, these by-products have a large amount of nutritional components. Thus, the present study aimed to determine the physicochemical and functional properties of protein hydrolysates (PH) from oil palm leaves (OPL) extracted using different concentrations of Alcalase (0-10%) at 2 h of hydrolysis time.

    RESULTS: Fourier transform infrared spectral analyses showed that the enzymatic hydrolysis altered functional groups of OPL where a secondary amine was present in the PH. Changes were also observed in the thermal stability where the enthalpy heat obtained for PH (933.93-1142.57 J g-1 ) was much lower than OPL (7854.11 J g-1 ). The results showed that the PH extracted by 8% Alcalase exhibited absolute zeta potential, as well as a high emulsifying activity index (70.64 m2  g-1 of protein) and emulsion stability index (60.58 min). Furthermore, this PH showed higher solubility (96.32%) and emulsifying properties compared to other PHs. It is also comparable with commercial plant proteins, indicating that 8% Alcalase is an optimum concentration for hydrolysis.

    CONCLUSION: In summary, the physicochemical and functional properties of PH extracted from OPL showed good functional properties, suggesting that it can be used as an alternative plant protein in food industries. © 2021 Society of Chemical Industry.

    Matched MeSH terms: Subtilisins/chemistry
  18. Preparation, characterization, immobilization, and molecular docking analysis of a novel detergent-stable subtilisin-like serine protease from Streptomyces mutabilis strain TN-X30
    Mechri S, Allala F, Bouacem K, Hasnaoui I, Gwaithan H, Chalbi TB, et al.
    Int J Biol Macromol, 2022 Dec 01;222(Pt A):1326-1342.
    PMID: 36242508 DOI: 10.1016/j.ijbiomac.2022.09.161
    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.
    Matched MeSH terms: Subtilisins/metabolism
  19. Fulltext physicochemical properties of undulated surf clam (Paphia undulata) HYDROLYSATE AS AFFECTED BY DEGREE OF HYDROLYSIS
    YAP JAA YEE, AMIZA MAT AMIN
    UMT Journal of Undergraduate Research, 2020;2(3):15-26.
    MyJurnal
    This study aimed to determine the physicochemical properties of undulated surf clam (Paphia undulata) hydrolysate as affected by the degree of hydrolysis (DH). Three levels of DH of undulated surf clam hydrolysate were prepared which were DH 36.57% (without any enzymatic hydrolysis), DH 58.25% (0.5% Alcalase®; 5 min; pH 7.5; 60ºC) and DH 91.26% (1% Alcalase®; 30 min; pH 7.5; 60ºC). After protein hydrolysis, the undulated surf clam hydrolysates were centrifuged, and their supernatants were freeze-dried. This study found that the protein hydrolysate with lower DH (DH 36.57%) gave lower protein content and higher ash and fat contents compared to other samples (DH 58.25% and DH 91.26%). However, the carbohydrate content is similar in all samples (16.56-20.04%). This study also found that foaming properties (29.43-67.50%), emulsifying capacity (11.94-110.52%) and peptide solubility (57.61-94.08%) were affected by the DH. As DH increased, the emulsifying capacity decreased, while peptide solubility increased. While the foaming capacity increased with increasing DH until it reached a maximum value and level off afterwards. For colour parameters, although there were differences between L*, a* and b* values for all three samples, a fluctuating pattern was noted with DH. DH also did not affect the water-holding and oil-holding capacity of undulated surf clam hydrolysate. This study shows that certain physicochemical properties of undulated surf clam hydrolysate can be tailored by adjusting the degree of hydrolysis.
    Matched MeSH terms: Subtilisins
  20. Fulltext Characteristics of threadfin bream (nemipterus japonicas) hydrolysate produced using bilimbi (averrhoa bilimbi l.) protease and alcalase
    Normah, I., Nur Anati, J.
    International Food Research Journal, 2015;22(6):2259-2266.
    MyJurnal
    Threadfin bream (Nemipterus japonicas) muscle was hydrolysed using protease extracted from
    bilimbi (Averrhoa bilimbi L.) fruit. This study was performed in order to compare the efficiency of bilimbi protease in producing threadfin bream protein hydrolysate with the commercial protease; alcalase 2.4 L. Initially, protease was extracted and then purified using 40% ammonium sulfate precipitation method. The proteolytic activity of the crude extract and purified protease was determined. Precipitation using 40% ammonium sulfate resulted in bilimbi protease specific activity of 2.36 U/mg and 23.13% recovery. Threadfin bream hydrolysate was prepared based on the pH-stat method by hydrolysis for 2 hrs. Hydrolysis using bilimbi protease produced 34.76% degree of hydrolysis (DH) and 3.75% yield while hydrolysis using alcalase resulted in 86.6% DH with 22.78% yield. Alcalase hydrolysate showed higher solubility than bilimbi protease hydrolysate at pH 7 with 70.87 and 32.16% solubility, respectively. Results also showed that protein content of threadfin bream hydrolysate produced using alcalase was higher (86.86%) than those produced using bilimbi protease (22.12%). However, both hydrolysates showed low moisture content between 3.93 to 7.00%. The molecular weight distribution analysis using SDS–PAGE indicated the distribution of smaller peptides especially in alcalase hydrolysate. Overall, the results showed that alcalase is more efficient enzyme choice than bilimbi protease for preparing threadfin bream hydrolysates. However, both hydrolysates could play an important role thus contribute to the food industry.
    Matched MeSH terms: Subtilisins
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