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Alkaline proteases derived from haloalkaliphiles, extremophilic microorganisms thriving in high-pH and high-salt environments, have garnered significant attention in recent years due to their unique biochemical properties and versatile applications. This review highlights the isolation, characterization, and biotechnological potential of alkaline proteases produced by haloalkaliphilic microorganisms. Haloalkaliphiles, found in soda lakes, saline-sodic soils, and other extreme habitats, have evolved to adapt and thrive under conditions characterized by elevated alkalinity and salinity. Alkaline proteases from these microorganisms exhibit high stability and activity under extreme pH and salinity, making them valuable candidates for various industrial processes. The review provides insights into the biochemical properties of alkaline proteases, including optimal pH, temperature, stability, and substrate specificity. Molecular studies have revealed unique structural features that contribute to the stability of these enzymes under harsh conditions, shedding light on their potential applications in challenging industrial processes. The industrial applications of alkaline proteases from haloalkaliphiles are diverse and encompass several sectors. Their role as powerful biocatalysts in detergent formulations is well-established, where they effectively break down proteinaceous stains under high pH conditions. Additionally, these enzymes find applications in leather processing, food industry, waste treatment, and pharmaceuticals, owing to their ability to operate efficiently in alkaline and saline environments. Their stability under extreme conditions, coupled with advances in biotechnological approaches, positions these enzymes as crucial components in the development of sustainable and efficient processes across various industries. Continued research in this field holds promise for discovering novel enzymes and unlocking their full potential for biotechnological innovation. [ABSTRACT FROM AUTHOR]

Considering the current growing interest in new and improved enzymes for use in a variety of applications, the present study aimed to characterize a novel detergent-stable serine alkaline protease from the extremophilic actinobacterium Microbacterium metallidurans TL13 (MmSP) using a combined in silico and experimental approach. The MmSP showed a close phylogenetic relationship with high molecular weight S8 peptidases of Microbacterium species. Moreover, its physical and chemical parameters computed using Expasy's ProtParam tool revealed that MmSP is hydrophilic, halophilic and thermo-alkali stable. 3D structure modelling and functional prediction of TL13 serine protease resulted in the detection of five characteristic domains: [catalytic subtilase domain, fibronectin (Fn) type-III domain, peptidase inhibitor I9, protease-associated (PA) domain and bacterial Ig-like domain (group 3)], as well as the three amino acid residues [aspartate (D182), histidine (H272) and serine (S604)] in the catalytic subtilase domain. The extremophilic strain TL13 was tested for protease production using agricultural wastes/by-products as carbon substrates. Maximum enzyme activity (390 U/gds) was obtained at 8th day fermentation on potato peel medium. Extracellular extract was concentrated and partially purified using ammonium sulfate precipitation methodology (1.58 folds purification fold). The optimal pH, temperature and salinity of MmSP were 9, 60 °C and 1 M NaCl, respectively. The MmSP protease showed broad pH stability, thermal stability, salt tolerance and detergent compatibility. In order to achieve the maximum stain removal efficacy by the TL 13 serine protease, the operation conditions were optimized using a Box–Behnken Design (BBD) with four variables, namely, time (15–75 min), temperature (30–60 °C), MmSP enzyme concentration (5–10 U/mL) and pH (7–11). The maximum stain removal yield (95 ± 4%) obtained under the optimal enzymatic operation conditions (treatment with 7.5 U/mL of MmSP during 30 min at 32 °C and pH9) was in good agreement with the value predicted by the regression model (98 ± %), which prove the validity of the fitted model. In conclusion, MmSP appears to be a good candidate for industrial applications, particularly in laundry detergent formulations, due to its high hydrophilicity, alkali-halo-stability, detergent compatibility and stain removal efficiency. [ABSTRACT FROM AUTHOR]

Protease is used in various industries and the uprising demand urges for its enhanced production. Herein, strain improvement by mutagenic treatment with ethyl methanesulfonate and ultra-violet light was carried out in proteolytic Bacillus subtilis AKAL7. Mutants were screened through proteolytic clear zone ratio on the skim milk agar. The highest protease producer mutant E29 was selected for enhanced protease production in shake flask fermentation using one-factor-at-a-time (OFAT) and statistical methodology. The OFAT approach resulted in maximum level of protease (161.7 U/mL) after 24 h of fermentation at 30 °C, pH 10 in a basal medium. The statistical Plackett–Burman design predicted peptone, yeast extract, MgSO4, pH, and temperature as the significant factors among eight independent variables for the protease production. The response surface methodology (RSM) determined the optimal concentration of the significant factors for maximum protease production (741.284 U/mL). The experimental protease level (722.7 U/mL), which was ~5-fold of that produced with the basal media, validated the predicted values of RSM. The partial purification resulted in a final ~14-fold purified protease with specific activity of ~9508 U/mg protein and molecular weight ~40 kDa. The protease showed maximum activity at 50 °C and stability up to 45 °C for 1 h. However, 10 mM CaCl2 increased its thermostability. Severe inhibition of protease activity by phenylmethylsulfonyl fluoride and HgCl2 revealed the partially purified protease as serine and cysteine type, which was active at pH 7.0–12.0 with the optima at pH 10.0. The protease could substantially dehair the cow skin within 14 h of treatment and retain 98% activity after 30 days of lyophilization. [ABSTRACT FROM AUTHOR]

The effect of four enzyme preparations (EP), Bacillolysin, Agroprot, Protozyme, and Protozyme C (Russia), on the protein and peptide profiles of the protein isolate isolated from peas of the Focor variety, as well as on its smell and taste, was investigated in this work. It was shown that enzyme treatment can improve the odor characteristics of the isolate. Thus, it was possible to reduce significantly the severity of the bean and herbal smell. At the same time, enzyme treatment also improved the taste of the isolate: it was possible to reduce significantly the severity of disturbing flavors such as leguminous, astringent, bitter, and herbal. The results obtained allowed us to select EP (fungal acid aspartic protease) to improve the organoleptic parameters of pea protein isolates intended for the production of meat and dairy product analogs. [ABSTRACT FROM AUTHOR]

Due to the growing need for alkaline proteases in various industrial applications, the preference for microbial sources, such as bacteria and fungi, has surged in comparison to plant and animal-derived alternatives. These microorganisms which can be isolated from natural alkaline habitats have emerged as promising candidates for large-scale industrial production. To meet this demand, greater attention is being given to improving the enzyme yields by optimizing culture conditions employing various approaches, viz., optimization of environmental and nutritional factors, statistical methodologies for screening, strain improvement, etc. Although acidic and neutral proteases are currently in industrial use, this review focuses on alkaline proteases from various sources because of their catalytic abilities at extreme pH values. While acidic proteases do possess distinctive and valuable catalytic capabilities at extreme pH values, they are predominantly utilized in the food industry for specific applications and are primarily produced by fungi, in contrast to alkaline proteases. Considering this, the present review focuses extensively on various environmental and nutritional factors affecting alkaline protease production in submerged fermentation. Additionally, the purification methodologies adopted, and properties of alkaline proteases obtained from different sources are discussed, and the industrial applications of alkaline proteases are also highlighted. [ABSTRACT FROM AUTHOR]

This study evaluated Streptomyces rochei strain NAM-19 solid-state fermentation of agricultural wastes to produce alkaline protease. Alkaline protease production increased with flaxseed, rice bran, and cheese whey fermentation reaching 147 U/mL at 48 h. Statistical optimization of alkaline protease production was performed using the central composite design (CDD). Results of CDD and the optimization plot showed that 4.59 g/L flaxseed, 4.31 g/L rice bran, 4.17 mL cheese whey, and a vegetative inoculum size of 7.0% increased alkaline protease production by 27.2% reaching 186 U/mL. Using the 20–70% ammonium sulfate fractionation method, the optimally produced enzyme was partially purified to fivefold. The partially purified alkaline protease was then covalently immobilized on a biopolymer carrier, glutaraldehyde-polyethylene-imine-κ-carrageenan (GA-PEI-Carr), with 90% immobilization efficiency. Characterizations revealed that immobilization improved thermostability, reusability, optimum temperature, and sensitivity towards metal ions of the free enzyme. The optimal temperature for free and immobilized enzymes was 40 and 50 °C, respectively. Both enzymes had the same optimum pH of 10. Immobilization increased Km from 19.73 to 26.52 mM and Vmax from 56.7 to 62.5 mmol min−1L−1. The immobilized enzyme retained 35% of its initial activity at 70 °C, while the free enzyme retained only 5%. The immobilized enzyme kept 80% of its initial activity at the 20th cycle. After 7 weeks of storage, the free enzyme lost all its initial activity, whereas the immobilized enzyme retained 50%. The free and immobilized enzymes were able to hydrolyze gelatin, and azo-casein demonstrating different relative activity, 85, 80, 90 and 95%, respectively, compared to casein (100%). [ABSTRACT FROM AUTHOR]

The current study aims to exploit the zero-cost inducer wheat bran (WB), an agro-industrial byproduct, for production of alkaline protease (ALK-PR23) by the hyper producer psychrotolerant Lysinibacillus sphaericus Strain AA6 EMCCN3080 for the first time ever. Incubation temperature (25 °C), yeast extract concentration, agitation speed (150 rpm), and aeration ratio [1 volume (liquid):5 volume (Erlenmeyer flask)] provoked ALK-PR23 production; OVAT inferences. The pH, yeast extract, and (NH4)2SO4 levels substantively triggered ALK-PR23 production as deduced from Plackett–Burman design. Incubation time (3 days) and WB [2% (w/v)] were the optimal values inducing positive significant influence on ALK-PR23 as conferred from steepest ascent experiments. Yeast extract (0.446% w/v), (NH4)2SO4 (0.339% w/v), and pH (6.872) prompted ALK-PR23 (592.5 U/mL) with an impressive 98-fold enhancement; Box-Behnken design and ridge steepest ascent path implications. The laboratory validation of the model achieved 100% of the predicted value. Laboratory data would present an eco-friendly, cheap, efficient approach towards concurrent WB recycling and massive production of alkaline protease (ALK-PR23) from L. sphaericus Strain AA6 EMCCN3080. Present data would greatly encourage unveiling biochemical characteristics of ALK-PR23 in prospective studies. [ABSTRACT FROM AUTHOR]

The effect of four enzyme preparations: bacillolysin, agroprot, protozyme and protozyme C (Russia) on solubility, emulsifying activity, emulsion stability, foaming and foam stability of isolates prepared from two varieties of peas was studied. It is shown that treatment with enzymes can increase the solubility of isolates at pH 5 by more than 7 times, the index of emulsifying activity at pH 5 by 1.5 to 2 times, and at pH 6 by almost 1.5 times; the stability index of the emulsion increased by about 20% at pH 5, and by 1.7 times (in one of the varieties) at pH 6; foaming increased by 2.4 to 3 times at pH 5, and at pH 6 by 1.8 to 3.7 times; foam stability increased by 25 to 33% at pH 5 and by more than 1.5 times (in one of the varieties) at pH 6. The results obtained made it possible to select an enzyme preparation (bacterial alkaline serine protease) to improve the parameters of pea protein isolates intended for the manufacture of analogs of fermented milk products. [ABSTRACT FROM AUTHOR]

Aims: Soil metabolites have a great influence on regulating the growth and development of plants and microbes in forest ecosystems. This study aims to reveal the characteristic metabolite profiles and their enriched metabolic pathways in the rhizosphere soil of Masson pine plantations at three different ages. Method: The rhizosphere soil of Masson pine from 10, 31, and 52 years plantation was collected. The metabolites were analyzed using a soil pseudotargeted metabolomics approach and then the relationships between soil metabolites and chemical properties, enzyme activities were established. Results: A total of 172 rhizosphere soil metabolites in 26 classes were identified. There was a decreasing trend in the total concentration of rhizosphere soil metabolites with increasing stand age. Seventy-two characteristic metabolites were screened, mainly saccharides, fatty acids, amino acids, phenolic acids, polyols and terpenoids. Among them, benzoic acid and galactonic acid contributed the most to the young forest, and stearic acid had the greatest influence on the near-mature forest plantation. Erythritol had great effects on the mature forest plantation. Seven metabolic pathways changed with increasing stand age. Correlation analysis showed that available potassium (AK) and soil alkaline protease (S-ALPT) were significantly positively correlated with phenolic acids, polyhydroxy carboxylic acids, etc. Conclusion: The rhizosphere soil of Masson pine had different metabolite profiles at different developmental stages, and the soil nutrient pools were effectively improved. These results provide a reference for plantation management in Masson pine and a deeper understanding of forest metabolic characteristics. [ABSTRACT FROM AUTHOR]

In this study, antioxidant peptides were prepared by enzymatic digestion of wine lees proteins, and their identification and activity assay was carried out. Firstly, the enzymatic conditions were optimized and the best hydrolysis enzyme was alkaline protease. The optimal enzymatic conditions were: pH 10.5, temperature 45 °C, enzyme addition of 5000 U/g, and the material-to-liquid ratio of 20 mg/mL. The enzymatic digestion products were purified with SephadexG-15 and then the peptide sequences were determined by LC–MS/MS. The peptide sequences were screened by BIOPEP, PepDraw and molecular docking to obtain three antioxidant peptides: ALPHAIL, EWAPGAH, and FDDLYFDR. The solid-phase synthesis of the above three peptides was carried out and the antioxidant activities of the synthesized peptides were measured. The results showed that all three peptides had excellent antioxidant activity. To investigate whether these three antioxidant peptides have other potential biological activities, molecular docking of angiotensin-converting enzyme (ACE) was performed on these three peptides. The molecular docking results showed that ALPHAIL docked well with tACE, and it is speculated that it may have ACE inhibitory activity, and the specific activity needs to be further investigated. [ABSTRACT FROM AUTHOR]

Welan gum, a natural polysaccharide produced by Sphingomonas sp. ATCC 31555, has attracted considerable attention in the scientific community due to its desirable properties. However, challenges, such as high viscosity, residual bacterial cells, carotenoids, and protein complexation, hinder the widespread application of welan gum. In this study, we established a method for the extraction and purification of welan gum using a synergistic approach with lysozyme and alkaline protease. Lysozyme hydrolysis conditions were optimized by applying response surface methodology, and the best results for bacterial cell removal were achieved at 11 000 U/g, 44 °C, and pH 9 after 3 h of treatment. Subsequently, we evaluated protein hydrolysis through computer simulation and identified alkaline protease as the most suitable enzyme. Through experimental investigations, we found that the optimal conditions for alkaline protease hydrolysis were 7500 U/g, 50 °C, pH 10, and 600 rpm. These conditions resulted in a sugar recovery rate of 76.1%, carotenoid removal rate of 89.5%, bacterial removal rate of 95.2%, and protein removal rate of 87.3% after 3 h of hydrolysis. The purified welan gum exhibited high transparency and purity. Structural characterization and antioxidant activity evaluation revealed that enzymatically purified welan gum has potential application prospects. Our study provides valuable insights into the optimal method for the enzymatic extraction and purification of welan gum. Such a method is conducive to the development of the multiple potential applications of welan gum. Key points: • A novel process for the synergistic purification of welan gum using lysozyme and alkaline protease was established. • In silico virtual digestion was employed to select the purification enzyme. • Welan gum with high transparency and purity was obtained. [ABSTRACT FROM AUTHOR]

The study aims to produce a detergent-compatible and alkaline thermophilic protease from a Bacillus strain and to investigate its usability as a detergent bio-additive. The protease-producing bacterium was identified as Bacillus pumilus strain TNP93 according to the 16S rRNA sequence. The bacterium optimally synthesized the protease at 40 °C and pH 10 in 40 h. The raw protease displayed its optimum activity at pH 10 and 60 °C and its stability between pH 6–13 and 30–100 °C for 24 h. The molecular mass of the proteolytic band was estimated to be about 85 kDa. The protease was not inhibited by any of the metal ions used (Ba2+, Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Zn2+). 97 and 90% of its original activity with 5 mM PMSF and EDTA remained. The activity was measured as 84, 124, and 95%, respectively, in the presence of 1% concentrations of Tween 20, Tween 80, and Triton X-100. In addition, all of its activity was preserved when the enzyme was exposed to 5% H2O2. The end products of casein were detected as tyrosine, aspartic acid, glycine, and cysteine by thin-layer chromatography. Considering the wash performance analysis, the mix of 1% commercial detergent and enzyme almost removed all of the protein-based stains (blood and egg yolk albumin). These remarkable findings indicate that the alkaline, thermo-, and oxidant-stable TNP93 protease is a valuable candidate for usage as a biological additive in various laundry detergents. [ABSTRACT FROM AUTHOR]

Microorganisms have long captivated researchers for their potential to produce enzymes with diverse industrial applications. Efficient production of proteases from new strains is crucial as these enzymes play a vital role in breaking down protein bonds, enabling their use in industrial applications. Therefore, a novel Exiguobacterium indicum 1.2.3 was isolated (Istanbul, Turkiye) and characterized in this study. This strain produced alkaline serine protease, which works in lower temperatures (20–40 °C) with casein as a specific substrate. The protease was utterly stable for 3 h at 30 °C. The enzyme was also highly stable in the pH range of 8–11. The optimum activity was obtained at pH 10. The crude enzyme activity was enhanced by various metal ions and retained 147%, 125%, 124%, and 117% of its activity within 1 mM Ca2+, Mn2+, Cu2+, and Mg2+, respectively. The crude enzyme was inactive with phenylmethylsulfonyl fluoride, indicating a serine residue on the active side. The enzyme exhibited a significant proteolytic effect in the presence of surfactants and oxidizing agents. The addition of Tween 80, Triton X-100, and sodium perborate improved enzymatic activity up to 135%, 109%, and 105%, respectively. According to the washing results, the crude enzyme effectively removed the blood on different types of standard pre-stained textiles at 30 °C. In conclusion, Exiguobacterium indicum 1.2.3 is a promising candidate for protease production, with its diverse applications spanning various industrial sectors, particularly detergents. [ABSTRACT FROM AUTHOR]

We are first to report the protease-catalyzed polypeptide synthesis in the natural deep eutectic solvent (DES) choline chloride/glycerol (ChCl:Gly) at a 1:2 molar ratio with 30 vol% of water using subtilisin Carlsberg (alkaline protease from Bacillus licheniformis). Poly-L-phenylalanine, poly-a-L-lysine, and the co-polypeptides poly(L-phenylalanine-a-L-lysine) as model polypeptides are produced from amino acid ester substrates in 40–70% yield and molecular weights ranging from 5 to 14 KDa according to dynamic light scattering (DLS). The natural DES is composed of 30 vol% water, whereas no polypeptides are formed at lower water contents. Poly-L-phenylalanine displays supramolecular self-assembly into homogenous nanotubes in water/isopropyl alcohol mixtures. This enzymatic route to polypeptides has advantages over previous reports in water and polar compressed fluids because of the higher molecular weights and greener process, respectively. [ABSTRACT FROM AUTHOR]

The identification and applicability of bacteria are inconclusive until comprehended with genomic repositories. Our isolate, Exiguobacterium sp. TBG-PICH-001 exhibited excellent halo- and organic solvent tolerance with simultaneous production of alkaline protease/s (0.512 IU/mL). The crude protease (1 IU) showed a 43.57% degradation of whey protein. The bulk proteins in the whey were hydrolyzed to smaller peptides which were evident in the SDS-PAGE profile. With such characteristics, the isolate became interesting for its genomic studies. The TBG-PICH-001 genome was found to be 3.14 Mb in size with 17 contigs and 47.33% GC content. The genome showed 3176 coding genes, and 2699 genes were characterized for their functionality. The Next-Generation-Sequencing of the genome identified only the isolate's genus; hence we attempted to delineate its species position. The genomes of the isolate and other representative Exiguobacterium spp. were compared based on orthologous genes (Orthovenn2 server). A pan-genomic analysis revealed the match of TBG-PICH-001 with 15 uncharacterized Exiguobacterium genomes at the species level. All these collectively matched with Exiguobacterium indicum, and the results were reconfirmed through phylogenetic studies. Further, the Exiguobacterium indicum genomes were engaged for homology studies rendering 11 classes of protease genes. Two putative proteases (Zinc metalloprotease and Serine protease) obtained from homology were checked for PCR amplification using genomic DNA of TBG-PICH-001 and other Exiguobacterium genomes. The results showed amplification only in the Exiguobacterium indicum genome. These protease genes, after sequencing, were matched with the TBG-PICH-001 genome. Their presence in its whole genome experimentally validated the study. [ABSTRACT FROM AUTHOR]

Edible bird's nest (EBN) is a traditional food which was nourishing and functional. Particularly, there is the epidermal growth factor (EGF) in EBN, which is thought to play an important role in promoting skin repair. However, the type and content of EGF in EBN were not determined yet. In this study, the type of EGF in EBN was identified as bird EGF by enzyme-linked immunosorbent assay and this method was validated to be accurate and precise. Moreover, it was found that the content of EGF in raw-unclean EBN, raw-clean EBN and stewed EBN was 3000 pg/g–4000 pg/g and there were no significant differences, which suggested that the batches, origins, forms, stewing temperatures and stewing times of EBN had no effect on the content of EGF in EBN. However, it was due to that enzyme destroyed the primary structure of EGF, the EGF content of neutral protease and trypsin hydrolysates of EBN was lower than that of flavor enzymes, alkaline protease and pepsin hydrolysates of EGF. This study was the first to determine the type and content of EGF in EBN, and provided a theoretical basis for the selection and processing of EBN and using EBN as a source of EGF. [ABSTRACT FROM AUTHOR]

Marine by-products contain valuable protein components that can be extracted and analyzed to determine their quality, composition, and performance. In the present study, Bighead carp (Hypophthalmichthys nobilis) heads were hydrolyzed by Alcalase and extracted alkaline protease from rainbow trout (Oncorhynchus mykiss) viscera and fractionated by membrane ultrafiltration into four molecular size fractions (< 3, 3–10, 10–30, 30 < kDa). Amino acid composition, foaming, emulsifying, and solubility properties of the fractions were characterized. Also, the antioxidant and antibacterial activity of the obtained fractions was assessed via ABTS, DPPH, metal chelating, and disc diffusion methods. Fractions of < 3 kDa showed the best activity in the antioxidant and antibacterial tests, so these fractions were used to develop the edible coats. Edible coatings based on these fractions and developed coats incorporating them into the alginate and starch solution coat were used to preserve rainbow trout fillets during cold storage. Based on obtained results, developed starch or alginate coatings enriched with 3 kDa fractions reduced 2-thiobarbituric acid (TBA), peroxide value (PV), total volatile basic nitrogen (TVB-N), and bacterial growth (TVC and PTC) more effectively than control treatments. According to the findings, these peptide fractions could be used in the development of active packaging as antimicrobial and antioxidant components. [ABSTRACT FROM AUTHOR]

Alkaline protease is widely used in the food, detergent, and pharmaceutical industries because of its comparatively great hydrolysis ability and alkali tolerance. To improve the ability of the recombinant Bacillus licheniformis to produce alkaline protease, single-factor experiments and response surface methodology (RSM) were utilized to determine and develop optimal culture conditions. The results showed that three factors (corn starch content, soybean meal content, and initial medium pH) had significant effects on alkaline protease production (P < 0.05), as determined through the Plackett‒Burman design. The maximum enzyme activity was observed with an optimal medium composition by central composite design (CCD): corn starch, 92.3 g/L; soybean meal, 35.8 g/L; and initial medium pH, 9.58. Under these optimum conditions, the alkaline protease activity of strain BL10::aprE was 15,435.1 U/mL, 82% higher than that in the initial fermentation medium. To further investigate the application of the optimum fermentation medium, the overexpressed strain BL10::aprE/pHYaprE was cultured using the optimized medium to achieve an enzyme activity of 39,233.6 U/mL. The present study achieved the highest enzyme activity of alkaline protease by B. licheniformis at the shake-flask fermentation level, which has important application value for large-scale production. [ABSTRACT FROM AUTHOR]

This study was aimed to obtain calcium-binding peptides with the enzymatic hydrolysis of sheep bone. The peptide of molecular weight from 3 to 10 kDa (SBP3) obtained by alkaline protease had the highest calcium-binding capacities which were mainly due to high levels of residues of Asp, Glu, Arg, Lys, Ser, Leu and Phe. The optimal conditions for the preparation of peptide-calcium chelate (SBP3-Ca) were temperature of 50 °C, pH value of 8, mass ratio of peptide/calcium of 3:1 for 55 min by response surface methodology determined, under which calcium chelating rate of 89.56% was obtained. The spectral results showed that peptides are combined with calcium through the interaction between the amino nitrogen atom and carboxyl oxygen atom. The scanning electron microscope and particle size analyses demonstrated that after peptide was combined with calcium ions, the microstructure was changed and the spatial structure was folded, which reduced the particle size with the formation of irregular particles.SBP3-Ca exhibited excellent stability in the presence of oxalic acid, phytic acid and in vitro simulated gastrointestinal environment. The present study provides a basis for the utilization and development of sheep bone peptide calcium chelate as a functional ingredient. [ABSTRACT FROM AUTHOR]

The effect of organic solvents on alkaline proteases was assessed for native, recombinant, and metagenomically derived alkaline proteases. Their stability and the effects of physicochemical parameters were studied in the presence of hexane. The native enzyme was comparatively more resistant against the organic solvents than the recombinant counterparts. On the other hand, the metagenomically derived alkaline protease was minimally resistant against solvents. A similar trend was apparent for the stability of enzyme in organic solvents. The novelty of this study lies in the fact that the majority of the studies on the solvent tolerance have focused on the mesophilic enzymes, while those from the haloalkaliphilic bacteria have received little attention. The comparative tolerance of the native, recombinant, and metagenomic alkaline proteases against the organic solvent has practical importance. The phylogenetic relatedness among the various protease sequences will be described. [ABSTRACT FROM AUTHOR]

Proteases represent 60% of the enzyme market and around 66% of the proteases are derived from the microbial sources. In the present study, protease enzyme was produced from bacterial isolate Ochrobactrum anthropi KTP9 under submerged fermentation having 17.43 IU/mL activity and 3.626 mg/ml of protein content. Ammonium sulphate precipitation increased the specific activity from 4.80 to 22.45 IU/ ml with 4.67 purification fold. Free protease has optimum pH 8.0, temperature 35 °C and found stable upto 28 days on storage at 4 °C. The immobilization yield and immobilization efficiency were found as 40.54% and 89.72%, respectively. Upon immobilization, shift in temperature (35 to 45 °C) and pH optima (8.0 to 10.0) was observed as compared to free protease. The immobilized protease was found to retain 50% residual activity upto 35 days of storage at 4 °C. Immobilization enzyme can be reused upto 6 cycles with 50% residual activity. The immobilized protease showed 76% protein hydrolysis in 120 min as compare to free KTP9 protease. [ABSTRACT FROM AUTHOR]

Biomimetic scaffolds that induce bone growth in defects have always been the main focus of bone regeneration research. This study was to develop biomineralization scaffolds using eggshell membranes as the bio-template. The structural and mineralizing characteristics of the inner and outer eggshell membrane (IM and OM) before and after alkaline protease and sodium sulfite treatments were investigated. Electron microscopy showed that both the IM and OM had a core-mantel structure. IM had a thinner mantel layer and more uniform fiber network structure than OM, exhibiting small average pore size and high specific surface area. The mineralization of IM and OM only occurred on the fibers surface. However, enzymatic treatment caused partial destruction of the mantel layer, which allowed the formation of mineral crystals. Specifically, the IM and enzyme-treated IM (EIM) fibers had better mineralization potentials than OM and the enzyme-treated OM (EOM), respectively. Hydroxyapatite-accumulated EIM (EIMHA) showed stronger hydrophilicity, higher BSA fixation ability, and simulated tissue fluid absorption ability. Moreover, the EIMHA facilitated the cell adhesion of the MC3T3-E1 cells and showed a strong cell proliferation activity. In summary, eggshell membranes treated with alkaline protease and sodium sulfite has a high potential to be used as a biomimetic mineralization scaffold. [ABSTRACT FROM AUTHOR]

The concurrent extraction of lipids, proteins, and carbohydrates can be achieved by aqueous and enzymatic extraction processes, circumventing the low extractability by mechanical pressing and the use of flammable solvents. The use of alkaline protease, preceded or not by carbohydrase pretreatments, was evaluated on the extractability of oil, protein, and carbohydrates from full-fat chickpea flour and protein functionality. Enzymatic extraction increased oil and protein extractability from 49.8 to 72.0–77.1% and 62.8 to 83.5–86.1%, respectively. Although the carbohydrase pretreatments before the addition of protease did not increase oil and protein extractability, the carbohydrate content of the extracts increased from 7.68 to 9.17−9.33 mg/mL, accompanied by the release of new oligosaccharides in the extracts, as revealed by LC–MS/MS characterization. Enzymatic extraction yielded proteins with significantly higher solubility (25.6 vs. 68.2–73.6%) and digestibility (83.8 vs. 90.79–94.67%). Treatment of the extracts with α-galactosidase completely removed the flatulence-causing oligosaccharides (stachyose and raffinose). This study highlights the effectiveness of environmentally friendly bioprocessing strategies to maximize lipid, protein, and oligosaccharide extractability from full-fat chickpea flour with concurrent improvements in protein solubility and in vitro digestibility, reduction of flatulence related oligosaccharides, and generation of a more diverse pool of oligosaccharides for subsequent prebiotic evaluation. [ABSTRACT FROM AUTHOR]

A carboxypeptidase M32 (CPM32) gene (cpm32) from Bacillus megaterium was cloned. CPM32 exhibited the highest sequence similarity of 79.3% with the same from Priestia veravalensis. CPM32 was fused with the signal peptides AmyQ (SPamyQ), AprE (SPaprE), NprB (SPnprB) in Bacillus subtilis WB600, and the highest CPM32 activity of 5320 U/mL was achieved when it was fused with SPamyQ. The purified enzyme (CPM32), 58.6 kDa, was most active at pH 8.0 and 60 °C, respectively, and it was stable over a broad pH range of 5.0–9.0 and an extensive temperature of 30–60 °C. Co2+ and Zn2+ could activate the enzyme activity by 600% and 334%, respectively. CPM32 could cleave most amino acid residues except Pro from chain's C-terminus. N-benzyloxycarbonyl-l-phenylalanyl-l-tyrosine (Z-Phe-Tyr) was the optimal substrate, for which CPM32 exhibited Km 0.6 mmol/L and kcat/Km 82.2 L/mmol s. Debittering soybean isolates using CPM32 (supplemented with 4% alkaline protease, w/w) resulted in a reduction in bitterness value of 47.6% to a commercially acceptable level. Applied to the hydrolysis of soy isolate protein, the bitterness of the hydrolyzates is significantly reduced and the flavor of the soy protein isolate hydrolysates is effectively improved. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the functional properties of soybean protein isolate (SPI) treated with alkaline protease and high-speed shearing homogenization. Alkaline protease-hydrolyzed SPIs that were characterized by varying degrees of hydrolysis between 0 and 6% were treated with high-speed shearing homogenization to obtain different micro-particulate proteins. The results showed that this combined treatment could significantly reduce the particle size of SPI by markedly degrading the structure of both the 7S and 11S subunits, thereby resulting in a significantly reduced content of β-sheet and β-turn structures. The surface hydrophobicity increased considerably for samples with hydrolysis below the threshold of 2% and then declined gradually above this threshold. Furthermore, the combination of hydrolysis and homogenization significantly improved the emulsion stability of SPI hydrolysates. It also significantly improved the foaming properties of SPI. These results demonstrated that alkaline protease hydrolysis combined with high-speed shearing homogenization represents a promising approach for improving the functional and structural properties of SPI. [ABSTRACT FROM AUTHOR]

Fifty protease-producing strains were screened from sediment of deep-sea cold seep, and divided into four different categories: Bacillus, Pseudoalteromonas, Vibrio, and Alteromonas according to the sequences of 16s rRNA. Their abilities to produce protease, amylase, and lipase were determined, and a Bacillus strain gcc-1 displayed very strong alkaline protease activity and stability under different thermal and acidic conditions. The purification of the protease produced by strain gcc-1 was carried out by precipitation with ammonium sulfate, and sequentially chromatographed by anion exchange column and gel filtration. The purified protease showed a single band at the molecular weight of 28 kDa by SDS-PAGE. The characterization results show that the purified protease exhibited a considerable activity and stability in a wide thermal range of 10–80 °C and a wide acidic range of pH 6.5–11.5, and displayed highest activity at 40 °C and pH 8.5. Notably, the protease still maintained high activity even at low to 10 °C. Furthermore, the protease exhibited good stability in presence of different surfactants, organic solvents, oxidizing agent H2O2, and commercial detergents. Therefore, the protease produced by gcc-1 is a cold active and high stable enzyme, and has a promising potential in laundry detergent as an additive. [ABSTRACT FROM AUTHOR]

Alkaline protease aeruginolysin (APR) is an important virulence factor in the evasion of the immune system by Pseudomonas aeruginosa (P. aeruginosa). The P. aeruginosa genome also encodes the highly potent and specific APR peptide inhibitor (APRin). However, the structural reason for the significant inhibition has not been revealed. Using ab initio molecular simulations, we here investigated the specific interactions between APR and APRin to elucidate which amino acid residues of APRin and APR contribute strongest to the inhibition. Since APR has a Zn2+ ion at the ligand-binding site and histidine and glutamic acid residues are coordinated with Zn2+, it is essential to precisely describe these coordination bonds to elucidate the specific interactions between APR and APRin. Therefore, we employed the ab initio fragment molecular orbital method to investigate the specific interactions at an electronic level. The results revealed that Ser1 and Ser2 at the N-terminus of APRin significantly contribute to the binding between APRin and APR. In particular, Ser1 binds strongly to Zn2+ as well as to the sidechains of His176(Hid), His180(Hid), and His186(Hid) in APR. This is the main reason for the strong interaction between APR and APRin. The results also elucidated significant contributions of the positively charged Arg83 and Arg90 residues of APRin to the binding with APR. These findings may provide information useful for the design of novel small agents as potent APR inhibitors. [ABSTRACT FROM AUTHOR]

The present study was undertaken to investigate the role of potassium nitrate in the modulation of phenolic metabolism and glutathione detoxification pathway in contrasting maize genotypes, with differential response towards waterlogging stress. Waterlogging led to a higher increase in the peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, tyrosine ammonia lyase, glutathione-S-transferase, and glutathione peroxidase activities in seedlings of the tolerant genotype, I 172 as compared to the susceptible one, LM 11. Acid and alkaline protease activities were increased in roots and shoots of both the genotypes, though, I 172 seedlings showed lower protease activity than LM 11, under stress conditions. The contents of phenols, flavonoids, ortho-dihydroxyphenols and reduced glutathione were increased in seedlings of both the genotypes. The content of H2O2 and MDA were higher in LM 11 seedlings, under stress conditions. Foliar application of KNO3 further increased the peroxidase, polyphenol oxidase, phenylalanine ammonia lyase and tyrosine ammonia lyase activities in roots and shoots of both the genotypes with a parallel increase in the content of phenolic compounds. It reduced glutathione-S-transferase, glutathione peroxidase and protease activities in roots and shoots of waterlogged seedlings of both the genotypes. With the foliar treatment of potassium nitrate, the contents of GSH, H2O2 and MDA decreased in roots and shoots of I 172 seedlings at both the growth stages. It may be suggested that both K+ and ions have a significant role in improving the phenolic metabolism and glutathione detoxification pathway leading to the enhanced tolerance of maize towards waterlogging stress. [ABSTRACT FROM AUTHOR]

Objectives: Proteases have gained great attention due to their enormous applications in food, tannery, detergent, photography and many other industries. Proteases rank third position in the production of enzymes. This paper targets to isolate a bacterium with high alkaline protease activity and optimization of its production conditions using Response Surface Methodology (RSM). Results: A bacterium isolated from soil contaminated with detergent exhibited clearance zone on skim milk agar medium with a protease activity of 22 U/ml. The bacterial strain was identified as Bacillus cereus KM05 and optimization of its production conditions were performed using statistical methods. Further optimization with Box Behnken design resulted in an increase in protease activity by 1.5-fold (28.6 U/ml). The protease enzyme was thermotolerant up to 70 °C with stability towards alkaline pH (pH 9). The enzyme was not affected by most of the metal ions and solvents. Moreover, the protease was also compatible with six commercial detergents tested. Densitometric analysis of the destained fabric materials following the detergent-enzyme treatment, revealed a stain removal efficiency of 97%. Conclusion: The alkaline protease enzyme obtained was stable at different conditions with stain removal efficacy. Hence, the present alkaline protease could be used for detergent formulations. [ABSTRACT FROM AUTHOR]

Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus obtained from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7–11) and temperature (40–80 °C) ranges, with maximal hydrolytic activities at pH 10 and at 50 °C temperature. The hydrolytic activity is stimulated by Ca2+, Co2+, Mn2+, and DTT. It is inhibited by Fe2+, Cd2+, Cu2+, EDTA, and PMSF. The SLSP-k is stable in anionic, non-anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicates that this enzyme is suitable for the degradation of poultry waste without the loss of nutritionally essential amino acids which otherwise are lost in hydrothermal processing. Therefore, the proteinase is efficient in environmental friendly bioconversion of animal waste into fertilizers or value added products such as secondary animal feedstuffs. [ABSTRACT FROM AUTHOR]

Protein-glutaminase (PG) is a promising protein deaminase. It only hydrolyzes the side chain amido groups of protein-bound to generate ammonia and protein-L-glutamic acid and does not catalyze any other undesirable changes in protein structures. Deamidation of proteins via PG can influence the solubility, emulsification, foaming, and gelation properties of proteins, which are important properties for some food proteins. Therefore, there is great potential for the application of PG in the food industry. PG is derived from Chryseobacterium proteolyticum (C. proteolyticum); however, wild strains are difficult to industrialize because of their low levels of enzyme production. In this article, we studied different strategies for PG expression in B. subtilis. Results showed that PG produced from C. proteolyticum could be successfully secreted in B. subtilis WB800N, and actively secreted in B. subtilis 168(BS168) or DB403 containing a pro-peptide (pro-PG). The secreted PG from B. subtilis WB800N was inactive unless digested by exogenous proteases, such as trypsin, alkaline protease, and neutral protease. However, active PG was secreted by the self-processing of BS168 and DB403. The specific activity of purified PG reached 20.9 U/mg. PG showed maximum activity at pH 5.5, 55 °C and more than 80% of PG activity was retained within a range of pH 3.5–6.5. When Cbz-Gln-Gly was used as the substrate, PG activity was 31.1 ± 0.9 μM min−1 mg−1. Mg2+, Ca2+, and Zn2+ stabilized and even activated PG activity. These strategies concerning PG expression in B. subtilis and the enzymatic properties of PG provide efficient alternatives for PG research and contribute to the industrial-scale production of PG. [ABSTRACT FROM AUTHOR]

Proteolytic enzymes hold a pivotal position in numerous industrial processes where hydrolysis of protein molecules is required under precise conditions. The emerging trend of biotechnological applications in recent years has witnessed a renewed interest in alkaline serine proteases extending their utility in detergent, leather, textile, food and pharmaceutical industries. A variety of microorganisms have been reported to produce alkaline serine proteases on a large scale, however, extensive research to find an alkaline serine protease with desirable characteristics such as significant catalytic efficiency, expanded stability and broad substrate specificity is still ongoing. Although submerged fermentation dominates the commercial enzyme production, recent reports have emphasized on solid state fermentation technology which can reduce major cost associated with the enzyme production. In the present review, recent research on alkaline serine proteases along with their novel properties and production using solid state fermentation have been discussed. [ABSTRACT FROM AUTHOR]

The aim of this research was to evaluate the possible positive effects of feeding Nile tilapia (Oreochromis niloticus) with a diet containing a commercial probiotic AQUA-PHOTO® [Lactobacillus plantarum (1.51 × 106 CFU g−1) and Bacillus subtilis (1.34 × 107 CFU g−1)] on digestive enzymes' activity and gut microbiome profile. The experimental design was completely randomized, with two treatments [control and probiotic diets (0.08 g.100 g−1 of feed)] and six replicate tanks per group (n = 8 animals per experimental unit, mean weight 8.18 g ± 1.85 g). The 96 fish were distributed into 12 aquaria and fed with the probiotic and control diets for 7 days. After this period, intestine samples (anterior, middle, and posterior portions) were collected from two individuals per tank. The samples were analyzed for assessment of DGGE profile and digestive enzymes' activity (amylase, alkaline protease, and alkaline phosphatase). The results were subjected to analysis of variance (ANOVA) and the means compared by t test (P < 0.05). The addition of B. subtilis and L. plantarum in the diets modulated the intestinal microbial biodiversity, more specifically the species richness and habitability index (P < 0.05). Besides, probiotic feeding also increased specific amylase activity in the anterior gut (P < 0.05). The other variables studied did not show any significant differences between treatments. The oral administration of B. subtilis and L. plantarum (AQUA-PHOTO®) modulates intestinal microbiota profile and increases the amylase enzymatic activity. Therefore, this measure may provide better utilization of carbohydrate sources in the diet, supplying energy for Nile tilapia growth. [ABSTRACT FROM AUTHOR]

Proteases are the most important industrial enzymes capable of hydrolyzing proteins into small peptides and amino acids and used commercially in food, textile and pharmaceutical industries. An alkaline serine protease from Pediococcus acidilactici NCDC 252 was purified using a simple two step purification procedure i.e. ammonium sulphate precipitation and gel filtration chromatography with purification fold of 5.9, a yield and specific activity of 11.8% and 23.3 U mg/ml respectively. Purified enzyme worked optimally at pH 8.5 and 37 °C. The enzyme was stable in the pH range of 6.5–9.5 and up to 50 °C. The Km for BAPNA was 38 µM and Vmax was 10.6 U/mg. Purified enzyme exhibited highest hydrolyzing activity for BAPNA. Inhibition of purified enzyme by soybean trypsin inhibitor, benzamide and β-ME suggested the enzyme to be alkaline serine protease with involvement of cysteine residues either in binding or regulation. In-silico characterization of alkaline serine protease revealed the presence of Ser, His and Asp residues at its catalytic site. This shows the resemblance of alkaline protease under study with other studied serine proteases. [ABSTRACT FROM AUTHOR]

The present study aim was to isolate bacterial strains, producing protease, from soil samples of different industrial wastes and dumping areas of slaughter houses. For this, 25 bacterial isolates forming clear zones in skimmed milk agar plates as a result of casein hydrolysis were selected. One bacterial isolate showing the highest protease activity (400 U/ml) was identified as Bacillus subtilis through 16S rRNA gene sequencing. Full-length alkaline protease gene was amplified, sequenced, cloned in expression vector pT7-7, and transformed in E. coli BL21C. Over-expression of protease gene was determined in isopropyl β-d-1-thiogalactopyranoside (0.5 mM) at 37 °C for 3 h. Expressed alkaline protease was purified and 10 folds increased enzyme activity was determined as compared to the wild type B. subtilis DMA-09. The purified protease was detected on SDS-PAGE as a single band of 31 kDa and was stable at 90 °C and 85% enzyme activity was retained at 100 °C. The optimum enzyme activity was obtained at pH 10 and was enhanced in the presence of 5 mM each Mg2+ and Ca2+ while phenylmethylsulfonyl fluoride (PMSF) inhibited its activity at 0.5 mM. The expressed purified alkaline protease may find potential applications in food and biotechnological industries. [ABSTRACT FROM AUTHOR]

Here we report heterologous expression, enzymatic characterization and structure homology modeling of a subtilisin-like alkaline serine protease (ASP) from Bacillus halodurans C-125. Encoding gene was successfully obtained by PCR and cloned into pMA0911 shuttle vector under the control of strong HpaII promoter and expressed extracellularly. ASP enzyme was successfully expressed in B. subtilis WB800 cell line lacking eight extracellular proteases and produced extracellularly in the culture medium. Km, Vmax and specific activity parameters of the recombinantly produced ASP were identified as 0.2899 mg/ml, 76.12 U/ml and 9500 U/mg, respectively. The purified enzyme revealed remarkable proteolytic activity at highly alkaline conditions with a pH optimum 12.0 and notable thermostability with temperature optimum at 60 °C. Furthermore, substrate-free enzyme revealed remarkable pH stability at pH 12.0 and maintained 93% of its initial activity when incubated at 37 °C for 24 h and 60% of its initial activity upon incubation at 60 °C for 1 h. Theoretically calculated molecular mass of ASP protein was confirmed through SDS-PAGE and western blot analysis (Mw: 28.3 kDa). The secondary and tertiary structures of ASP protein were also identified through homology modeling and further examined in detail. ASP harbors a typical S8/S53 peptidase domain comprising 17 β-sheets and 9 α-helixes within its secondary structure. The structure dynamics analysis of modeled 3D structure further revealed that transient inactivating propeptide chain is the most dynamic region of ASP enzyme with 8.52 Å2 β-Factor value. Additional residue-dependent fluctuation plot analysis also confirmed the elevated structure dynamics patterning of ASP N-terminus which could be the potential prerequisite for the autonomous propeptide removal of alkaline serine peptidases. Yet the functional domain of ASP becomes quite stable after autonomous exclusion of its propeptide. Although the sequence homology between ASP and commercial detergent additive B. lentus protease (PDB ID:1GCI) was moderate (65.4% sequence similarity), their overlaid 3D structures revealed much higher similarity (98.14%) within 0.80 Å RMSD. In conclusions, with remarkable pH stability, notable thermostability and particularly high specific activity at extreme alkaline conditions, the unveiled ASP protein stands out as a novel protease candidate for various industrial sectors such as textile, detergent, leather, feed, waste, pharmaceutical and others. [ABSTRACT FROM AUTHOR]

Protease is a kind of enzyme with different applications in medicine and industry. The limitation of the enzymes in industry is lack of tolerance for harsh conditions (high temperature, pH, and low water condition). This study intends to evaluate in silico study and screen the alkaline serine protease producing bacteria from honey and optimize the production of low water active alkaline serine protease. Plackett–Burman method was applied to improve effective factors on protease production, including pH, inoculum size, temperature, time, soybean protein, KH2PO4, MgSO4, CaCl2, NaCl, Glycerin and glucose concentration. Further, the Box–Behnken method was used to optimize the significant level of selected factors. Besides, in silico study was utilized to create the enzyme structure and investigate the stability in harsh conditions. The results showed that the highest protease production belongs to Bacillus sp. Khoz1 closed Bacillus safensis, Also temperature, glucose and soybean protein concentration were three significant factors for protease production and the optimized level for them were 35 °C, 0.5 g/l, and 38.32 g/l, respectively. The microorganism was able to produce protease until 98.36 U/ml and it was 98.68% similar to B. safensis. The stability of microbial alkaline serine protease was also determined in high pH and temperature conditions. The best stable condition for isolated protease was pH 9 and temperature at 50 °C. The in silico data showed that this protease has similar 3D structure to Bacillus subtilis Subtilisin E and highly charge amino acids on its surface were that caused this enzyme still activate and stable in low water condition. [ABSTRACT FROM AUTHOR]

Proteases are one of the most important biocatalysts with an industrial perspective due to their high production capability, cost-effective, and eco-friendly nature. In this study, an alkaline protease produced by Bacillus brevis (228.31 ± 6.2 U/mL) in liquid-state fermentation at pH 7.0 and 47 °C was further enhanced by optimizing various physical parameters. The maximum protease activity (631.09 ± 3.7 U/mL) was recorded at 45 °C, pH 8.0, and using 3 mL of inoculum size after 72 h. The optimally-produced alkaline protease was immobilized on Ca-alginate beads, and the process was optimized using a central composite design-based response surface methodology. The maximum immobilization yield (> 70%) was achieved using a 2–3.0% gelling agent (Na-alginate) and 2.5–3.0% binder (CaCl2), with 400–600 mg/L of protease concentration. Characterization revealed that immobilization improves the pH and thermal stability of protease, as the maximum activity was recorded at pH 10 and 65 °C. The kinetic studies of protease revealed higher Vmax (454.5 U/mL) and lower Km (0.09 μM) values after Ca-alginate immobilization as compared with the free enzyme (Vmax 333.3 U/mL and Km 0.16 μM). Ca-alginate immobilized protease also possessed good recyclability potential in several consecutive substrate hydrolysis experiments. By applying on goatskin and X-ray film, the immobilized biocatalyst showed improved activity than the free enzyme, revealing its potential biotechnological applications. [ABSTRACT FROM AUTHOR]

KP-43, a 43-kDa alkaline serine protease, is resistant to chemical oxidants and surfactants, making it suitable for use in laundry detergents. An amino acid residue at position 195, in a unique flexible loop that binds a Ca2+ ion, dramatically affects the proteolytic activity and thermal stability of KP-43. In the present study, we obtained 20 variants with substitutions at position 195 and investigated how these residues affect hydrolytic activity toward a macromolecular substrate (casein) and a synthetic tetra-peptide (AAPL). At pH 10, the variant with the highest caseinolytic activity, Tyr195Gln, exhibited 4.4-fold higher activity than the variant with the lowest caseinolytic activity, Tyr195Trp. A significant negative correlation was observed between the hydrophobicity of the residue at position 195 and caseinolytic activity at pH 8–10. At pH 7, the correlation became weak; at pH 6, the correlation reversed to positive. Unlike casein, in the case of hydrolysis of AAPL, no correlation was observed at pH 10 or pH 6. Because the amino acid residue at position 195 is located on the protein surface and considered sufficiently far from the active cleft, the variation in caseinolytic activity between the 20 variants was attributed to changes in interaction efficiency with different states of casein at different pH values. To improve the enzymatic activity, we propose substituting amino acid residues on the protein surface to change the efficiency of interaction with the macromolecular substrates. Key points: • A single amino acid residue on the protein surface markedly changed enzyme activity. • The hydrophobicity of the amino acid residue and enzyme activity had a correlation. • The key amino acid residue for substrate recognition exists on the protein surface. [ABSTRACT FROM AUTHOR]

Excess sludge contains a high amount of protein, which can be recovered to prepare protein foaming agents and other products with high added value. Enzymatic hydrolysis (EH) is a promising technology for the recovery of protein from excess sludge, and ultrasound has been identified as a potential method to assist in sludge disintegration. Ultrasonic pretreatment was combined with alkaline protease hydrolysis to extract protein from excess sludge produced by A2/O treatment (S1) and an oxidation ditch treatment (S2), and the extraction effects and changes in sludge dewatering performance were studied. The effects of the six factors ultrasonic power density, ultrasonication time, enzyme dose, pH, hydrolysis temperature and hydrolysis time were analyzed. The results showed that the ultrasound-enhanced enzymatic method could effectively extract sludge protein. Although the extraction efficiencies for the different municipal sludges were different, their extraction conditions were relatively similar. Considering the protein extraction rate and sludge dewatering performance, the selected extraction conditions were as follows: ultrasonic power density, 1 W/mL; ultrasonication time, 20 min; enzyme dose, 3500 U/g; pH 11; hydrolysis temperature, 60 °C; and hydrolysis time, 3 h. Under these conditions, the protein extraction rate (Rp) of S1 and S2 reached 55.9% and 52.3%, respectively. Moreover, the improvement in sludge dewatering performance (Dw) of S1 and S2 was 49.5% and 52.4%, respectively. Comparison of the protein, polypeptide, and amino acid contents obtained from ultrasound-assisted enzymatic hydrolysis (UEH), EH, and ultrasonic hydrolysis (UH) further demonstrated the beneficial effect of ultrasound application on enzymatic hydrolysis. [ABSTRACT FROM AUTHOR]

In order to achieve high yield of fungal protease in a very cost effective way and to meet its increased market demand, current study deals with the screening of various agro-wastes as carbon source for the production of protease from Rhizopus oryzae (SN5)/NCIM-1447 under solid state fermentation. Substrates and culture parameters such as wheat bran, soybean meal, black-gram husk, rice husk, mixture of wheat bran, soybean meal, nitrogen sources, pH, temperature and incubation time were first optimized with one factor at time strategy and then EVOP factorial and yield of alkaline protease was achieved 412.8 U/gds at 28 °C and pH = 6 after 72 h of fermentation taking wheat bran and soybean as a substrate in 4:1 ratio. Further artificial neural networks (ANN), was trained with data of EVOP and yield of protease was enhanced up to 422.6 U/gds with wheat bran: soyabean in ratio of 70:30, pH 6.2 at 30 °C. The evolved process and Rhizopus oryzae (SN5)/NCIM-1447 strain would be promising for protease production at industrial scale at low cost. [ABSTRACT FROM AUTHOR]

Objectives: The utilization of biotechnology in leather sector has more extensive in modern years; more particular to proteolytic enzymes and employed in several steps of the leather making such as soaking, dehairing, bating, solid waste management etc. The current study evaluates the performance of alkaline protease from Bacillus crolab MTCC 5468 in single soaking of goat skins matrix by comparing with the conventional multiple soaking processes. Results: According to the obtained results, the optimum concentration for maximum rehydration of goat skins was accomplished at 1.0% (v/w) of alkaline protease at duration of 3 h over traditional rehydration method (4–6 h). The moisture level, total protein, chloride content and total organic carbon of enzymatic rehydration was superior to that of conventional rehydration and it was also used to measure the effectiveness of rehydration process. Scanning electron microscopic images of enzymatically processed leather exhibits enhanced opening of fiber bundles and smooth grain surface than conventional method. Furthermore, the alkaline protease treated leather exhibited improved moisture uptake, removal of chlorides and suppleness because of hydrolysis of non-collagenous proteins as indicated by well opened up fiber bundles in histological analysis. Conclusions: The application of alkaline protease in rehydration operation of leather production confirmed scope for diminishing water quantity around 66.6%, soaking duration at 50%, minimizing use of harmful dehairing chemicals at 50–60%, thereby, eliminating the bating operation during pre-tanning. These outcomes suggest that alkaline protease have potential application in rehydration of skins for immense environmental concerns of leather tanning sectors. [ABSTRACT FROM AUTHOR]

A thermostable extracellular alkaline protease (called SAPA) was produced (4600 U/mL) by Anoxybacillus kamchatkensis M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer with a molecular mass of 28 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH2-terminal amino-acid residues showed high homology with those of Bacillus proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70 °C. The sapA gene was cloned and expressed in the extracellular fraction of E. coli. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from Bacillus subtilis WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from Bacillus pumilus CBS and subtilisin A from Bacillus licheniformis, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L. Highlights: A new extracellular Anoxybacillus kamchatkensis protease was purified (SAPA) and characterized. SAPA was a serine protease and a monomer with a molecular mass of ∼28 kDa. Optimum pH and temperature values for activity were pH 11 and 70 °C, respectively. The sapA gene encoding SAPA was cloned, sequenced, and expressed in E. coli. rSAPA is a potential candidate for future application as laundry detergent additive. [ABSTRACT FROM AUTHOR]

Granulocyte colony-stimulating factor (G-CSF) is involved in generation of colonies hematopoietic cells by stimulation of bone marrow cells, resulting in the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. To express and secrete G-CSF in Bacillus system, the G-CSF gene was amplified using gene-specific primers and template DNA (pUNO1-hGCSFb). As a result, the PCR product of 530 bp was obtained, sequenced, and analyzed using the BLAST program. The open reading frame encoded a 204-amino acid polypeptide precursor consisting of the 30-residue signal sequence and a 174-residue mature polypeptide. Recombinant secretion vector (pRBAS-CSF3, 6.96 kb) was constructed using pRBAS vector which contains promoter (0.45 kb) and signal sequence (87 bp) of alkaline protease gene (aprE), ribosome binding site (GGAGAGGG) and transferred to Bacillus subtilis LKS. G-CSF protein was secreted and optimized to increase the productivity (19% of total secreted proteins, 0.12 mg/ml). The culture broth was harvested and followed by metal chelating, ethanol precipitation, and gel chromatography. The purified G-CSF was tested for thermogenic activity in cultured brown adipocytes. G-CSF activated HIB1B brown adipocytes by increasing expression levels of brown fat-specific marker proteins (UCP1 and PRDM16) and genes (Lhx8, Ppargc1, Prdm16, Ucp1, and Zic1) in HIB1B brown adipocytes. This result suggests that G-CSF per se or G-CSF-containing foodstuff can be used to treat obesity. [ABSTRACT FROM AUTHOR]

Abstract The detergent-compatible alkaline protease was produced from the bacterial strain Bacillus sp. APP-07 isolated from Laundromat soil of Solapur, Maharashtra, India. The culture was grown in 1000 ml capacity baffled flask with a working volume of 100 ml and incubated at 55 °C for 33 h on a rotary shaker. After incubation, alkaline protease was partially purified by the sequential method of acetone precipitation followed by nominal molecular weight limit (NMWL) cut-off ultrafiltration using 50 K and 10 K filters. Finally, Sephadex G-100 gel filtration chromatographic purification was performed to obtain 3.12 fold purified alkaline protease enzyme with a 66.67% final yield. The purified enzyme showed 31907.269 units (U) of enzyme activity containing 8741.718 U/mg of specific enzyme activity. The molecular weight of the enzyme was confirmed about 33.0 kDa (kDa) by the SDS-PAGE analysis. The purified enzyme was stable at higher pH and temperature range, with an optimum pH 10.5 and temperature 55 °C. The enzyme showed excellent stability and compatibility in various detergents, surfactants, bleach, and oxidizing agents. The enzyme activity enhanced in the presence of Ca2+, Cu2+, and surfactants, whereas; the phenylmethylsulphonyl fluoride (PMSF) and Diisopropyl fluorophosphate (DFP) completely inhibit the enzymatic activity, which pointed out that the enzyme affiliated to serine-centered metalloproteases family. In conclusion, the remarkable tolerance and stability of the enzyme explored the promising candidature for the several potential applications in the laundry detergents. The sustainability of the enzyme might serve several possible applications in the laundry detergents, leather industries, and other harsh industrial processes. [ABSTRACT FROM AUTHOR]

Abstract Proteases are the hydrolytic enzymes which hydrolyzes peptide bond between proteins with paramount applications in pharmaceutical and industrial sector. Therefore production of proteases with efficient characteristics of biotechnological interest from novel strain is significant. Hence, in this study, an alkaline serine protease produced by Bacillus cereus strain S8 (MTCC NO 11901) was purified and characterized. The alkaline protease was purified by ammonium sulfate precipitation (50%), ion exchange (DEAE-Cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. As a result of this purification, a protein with specific activity of 300U/mg protein was obtained with purification fold 17.04 and recovery percentage of 34.6%. The molecular weight of the purified protease was determined using SDS-PAGE under non-reducing (71 kDa) and reducing conditions (35 kDa and 22 kDa). Zymogram analysis revealed that proteolytic activity was only associated with 22 kDa. These results indicate that existence of the enzyme as dimer in its native state. The molecular weight of the protease (22 kDa) was also determined by gel filtration (Sephadex G-200) chromatography and it was calculated as 21.8 kDa. The optimum activity of the protease was observed at pH 10.0 and temperature 70 °C with great stability towards pH and temperature with casein as a specific substrate. The enzyme was completely inhibited by PMSF and TLCK indicating that it is a serine protease of trypsin type. The enzyme exhibits a great stability towards organic solvents, oxidizing and bleaching agents and it is negatively influenced by Li2+ and Co2+ metal ions. The purified protein was further characterized by Matrix Assisted Laser Desorption Ionization/Mass Spectroscopy (MALDI/MS) analysis which reveals that total number of amino acids is 208 with isoelectric point 9.52. [ABSTRACT FROM AUTHOR]

In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis , Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37 °C for 48 h with a constant agitation of 120 rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1 M ice cold CaCl 2 and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60 s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production. [ABSTRACT FROM AUTHOR]

Study was conducted to recover proteins from pangas (Pangasius pangasius) processing waste (fillet frames) using pH shift method and to characterize the recovered isolates. pH 2.0 from acidic range and pH 13.0 from alkaline range were found to have maximum protein recovery (p < 0.05). During the recovery process, acidic pH (pH 2.0) was found to have minimal effect on proteins resulting in more stable isolates and strong protein gels. Alkaline pH (pH 13.0) caused protein denaturation resulting in less stable proteins and poor gel network. Both acidic and alkaline-aided processing caused significant (p < 0.05) reductions in total lipid, myoglobin, and pigment content thus by resulting in whiter protein isolates and gels. The content of total essential amino acids increased during pH shift processing, indicating the enrichment of essential amino acids. No microbial counts were detected in any of the isolates prepared using acid and alkaline extraction methods. pH shift processing was found to be promising in the utilization of fish processing waste for the recovery of functional proteins from pangas processing waste thus by reducing the supply demand gap as well pollution problems. [ABSTRACT FROM AUTHOR]

The aim of the present study is to investigate the effect of three entomopathogenic fungi (EPF) (Beauveria bassiana, Cladosporium cladosporioides, and Verticillium alfalfae) on the aphid species, Metopolophium dirhodum (Walker) (Hemiptera: Aphididae). The selected EPF were isolated from the agricultural soil of the National Institute of Plant Protection (INPV) in Constantine, Algeria, and were tested against the aphid insects that were collected from the same area. The aphid species M. dirhodum were exposed to each fungal spore suspensions 107 conidia/ml for 10 s. Percent mortality was recorded at 1, 3, 5, and 7 days post treatment. Percentage mortalities, 7 days post treatment, were 95.83, 63.98, and 51.83% by B. bassiana, C. cladosporioides, and V. alfalfae, respectively. The higher protease activities were observed for isolate V. alfalfae with 95 U/ml, followed by B. bassiana with 38.26 U/ml, and finally C. cladosporioides with 35, 65 U/ml. The results presented in this study revealed that there was no relation between high alkaline protease activities and high virulence isolates. [ABSTRACT FROM AUTHOR]

Over a hundred of halophilic/halotolerant microorganisms were screened for alkaline protease production. The bacterium showing the highest enzyme production was characterized and identified as Bacillus halodurans US193 on the basis of 16S rRNA gene analysis. It was alkalophilic, thermophilic and halotolerant since it grew optimally at pH 9.7 and 50 °C with tolerance of up to 125 g NaCl l−1. The alkaline protease was purified 4.9 times with about 40186.1 U/mg as specific activity. It exhibited optimal activity at pH 10, 70 °C and 0.25 M NaCl with perfect stability at wide ranges of pH (6–12), temperatures (30–60 °C) and NaCl concentrations (0–2 M). The serine alkaline protease maintained high stability in the presence of Cu2+, Mg2+, Ba2+ and Ca2+ ions, various organic solvents [50% (v/v)] and ionic and non ionic detergent additives. In addition, it was more compatible with various commercialized detergents than other reported detergent proteases, and was very efficient in blood stain removal. These findings let B. halodurans US193 alkaline protease be an ideal candidate for many industrial processes at harsh conditions, especially as a bio-additive in detergent industry. [ABSTRACT FROM AUTHOR]