Plant Proteases Research Articles

Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal

The deleterious effects of intensive use of chemical fertilizers and pesticides in agriculture has led to the substantial research efforts on finding the alternatives to these agrochemicals. This study was aimed to isolate Bacillus species from soil of different regions of Nepal and screen for their ability to promote plant growth directly or indirectly by testing their ability to produce plant growth hormone indole acetic acid, hydrogen cyanide, ammonia and protease as well as phosphate solubilization. Thirty nine Bacillus strains were isolated from 25 soil samples of different regions of Kathmandu and Chitwan districts of Nepal. These isolates were tested for plant growth promoting traits in vitro. Among the total isolates, about 48.7% were indole acetic acid producers, 38.4% of the isolates showed the ability to solubilize the phosphate, 71.8% were able to produce ammonia and all the isolates had the ability to produce hydrogen cyanide and protease. The isolated strains showed positive results to maximum PGPR traits and exhibited a potential to be used as alternatives to chemical fertilizers and pesticides and could be used as low-cost bio-based technology to promote plant growth in the agricultural sector.

The recombinant plant Bauhinia bauhinioides elastase inhibitor reduces rat thrombus without alterations in hemostatic parameters

The anti-inflammatory effects of the plant protease inhibitor BbCI (Bauhinia bauhinioides cruzipain inhibitor), which blocks elastase, cathepsin G, and L, and proteinase 3 has been demonstrated. Here, we investigated the recombinant rBbCI-His(6) (containing a histidine tail) in an experimental venous thrombosis model of vena cava (VC) ligature in rats, comparing to heparin. We evaluate the effects of the inhibitors (native or recombinant) or heparin on the activated partial thromboplastin time (aPTT) and prothrombin time (PT) in human and rat plasmas. The rats undergoing treatment received a saline solution or increasing concentrations of rBbCI-His(6), heparin, or a mixture of both. After 4 h of ligature VC, thrombus, if present was removed and weighed. aPTT, PT, and cytokines were measured in blood collected by cardiac puncture. aPTT, PT, and bleeding time (BT) were also measured at the time of VC (vena cava) ligature. rBbCI-His(6) (0.45 or 1.40 mg/kg) does not alter aPTT, PT or BT. No differences in coagulation parameters were detected in rBbCI-His(6) treated rats at the time of VC ligature or when the thrombus was removed. There was a significant decrease in the weight of thrombus in the animals of the groups treated with the rBbCI-His(6) (1.40 mg/kg), with the rBbCI-His(6) mixture (1.40 mg/kg) + heparin (50 IU/kg) and heparin (100 IU/kg) in relation to control group (saline). The growth-related oncogene/keratinocyte chemoattractant (GRO/KC) serum levels in rats treated with rBbCI-His(6) (1.40 mg/kg) or heparin (200 IU/kg) were reduced. In the experimental model used, rBbCI-His(6) alone had an antithrombotic effect, not altering blood clotting or bleeding time.

Plant protease as regulator and signaling molecule for enhancing environmental stress-tolerance.

Proteases are ubiquitous in prokaryotes and eukaryotes. Plant proteases are key regulators of various physiological processes, including protein homeostasis, organelle development, senescence, seed germination, protein processing, environmental stress response, and programmed cell death. Proteases are involved in the breakdown of peptide bonds resulting in irreversible posttranslational modification of the protein. Proteases act as signaling molecules that specifically regulate cellular function by cleaving and triggering receptor molecules. Peptides derived from proteolysis regulate ROS signaling under oxidative stress in the plant. It degrades misfolded and abnormal proteins into amino acids to repair the cell damage and regulates the biological process in response to environmental stress. Proteases modulate the biogenesis of phytohormones which control plant growth, development, and environmental stresses. Protein homeostasis, the overall balance between protein synthesis and proteolysis, is required for plant growth and development. Abiotic and biotic stresses are major factors that negatively impact cellular survivability, biomass production, and reduced crop yield potentials. Therefore, the identification of various stress-responsive proteases and their molecular functions may elucidate valuable information for the development of stress-resilient crops with higher yield potentials. However, the understanding of molecular mechanisms of plant protease remains unexplored. This review provides an overview of proteases related to development, signaling, and growth regulation to acclimatize environmental stress in plants.

Papain-Like Cysteine Protease Gene Family in Fig (Ficus carica L.): Genome-Wide Analysis and Expression Patterns.

The papain-like cysteine proteases (PLCPs) are the most abundant family of cysteine proteases in plants, with essential roles in biotic/abiotic stress responses, growth and senescence. Papain, bromelain and ficin are widely used in food, medicine and other industries. In this study, 31 PLCP genes (FcPCLPs) were identified in the fig (Ficus carica L.) genome by HMM search and manual screening, and assigned to one of nine subfamilies based on gene structure and conserved motifs. SAG12 and RD21 were the largest subfamilies with 10 and 7 members, respectively. The FcPCLPs ranged from 1,128 to 5,075 bp in length, containing 1–10 introns, and the coding sequence ranged from 624 to 1,518 bp, encoding 207–505 amino acids. Subcellular localization analysis indicated that 24, 2, and 5 PLCP proteins were targeted to the lysosome/vacuole, cytoplasm and extracellular matrix, respectively. Promoter (2,000 bp upstream) analysis of FcPLCPs revealed a high number of plant hormone and low temperature response elements. RNA-seq revealed differential expression of 17 FcPLCPs in the inflorescence and receptacle, and RD21 subfamily members were the major PLCPs expressed in the fruit; 16 and 5 FcPLCPs responded significantly to ethylene and light, respectively. Proteome analyses revealed 18 and 5 PLCPs in the fruit cell soluble proteome and fruit latex, respectively. Ficins were the major PLCP in fig fruit, with decreased abundance in inflorescences, but increased abundance in receptacles of commercial-ripe fruit. FcRD21B/C and FcALP1 were aligned as the genes encoding the main ficin isoforms. Our study provides valuable multi-omics information on the FcPLCP family and lays the foundation for further functional studies.

In vitro gastrointestinal digestion of proteins in the presence of enzyme supplements: Details of antioxidant and antidiabetic properties

Athletes tend to consume more protein than the daily recommended allowance due to their high energy demanding lifestyle. It is worthwhile to supplement their proteins with external enzymes to increase the bioavailability. We have designed in vitro study of two commercial plant proteases (SEBproX and SEBrolase) and one microbial enzyme formulation (pepzymeAG) to determine their role in digestion and absorption of protein. The study was done under INFOGEST simulated GI conditions using pea, whey, and collagen proteins. Increment in degree of hydrolysis was observed in pepzymeAG assisted digestion and SEBproX assisted digestion as opposed to that of SEBrolase assisted digestion of proteins. Further, bioactive peptides with enhanced antioxidant and antidiabetic properties were found to be released in only pepzymeAG assisted digestion. Lower molecular weight peptides (≤6 kDa) were observed to be generated in the gastric digestion stage of pepzymeAG assisted digestion which may increases the rate of gastric emptying and hence helps in better absorption of protein through the intestine. Overall, pepzymeAG could be a great choice for athletes to get benefits of proteins beyond their common nutritional goals.

Toxicity of plant extracts containing trypsin inhibitor to the larvae of Aedes aegypti

Mosquitoes are vectors of many pathogens and can transmit a range of potentially fatal diseases. Some of these diseases spread via mosquitoes belonging to the genus Aedes include Dengue, Chikungunya, Yellow fever, Zika etc. Conventional methods of mosquito control are not always completely successful. Therefore new strategies need to be adopted for mosquito control. Plant protease inhibitors are promising candidates as they prevent larval growth by inhibiting gut proteases of mosquito larvae. In this study we screened 15 plant extracts containing trypsin inhibitors for their toxicity to the larvae of Aedes aegypti. Leaf extracts from Lawsonia inermis showed the highest mortality with 100% mortality after 48 hours in second, third and in fourth instar larvae. The treatment with extract from Annona muricata leaf led to 100, 96 and 66% mortality after 48 hours in second, third and fourth instar larvae respectively. After Proteinase K treatment of Lawsonia inermis leaf extract, a reduction in the in vitro inhibition of larval gut protease activity from 82 to 6% and a decrease in mortality from 80 to 50% were observed compared to untreated. This revealed that the mortality, at least in part, is due to the presence of a protenaceous plant protease inhibitor in the extract. Expressing the gene coding for such protease inhibitors in micro organisms or algae upon which the mosquito larvae feeds will serve as an alternate method for control of mosquito population.

Stacked insecticidal genes in potatoes exhibit enhanced toxicity against Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

The present study was performed to express stacked insecticidal genes in potato cv. Lady Olympia and Agria to encode resistance against Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say). Bacillus thuringiensis (Bt) gene (cry3A), synthetic hybrid (SN-19) and plant proteinase inhibitor Oryza cystatin II (OCII) cloned in pCAMBIA1301 binary vector in two different combinations as of DS-1 (cry3A + SN-19 genes) and DS-2 (OCII + SN-19 genes) constructs and further transformed to two potato cultivars using Agrobacterium-mediated transformation. All molecular analyses confirmed gene integration and expression in a total of 27 primary transformants in both Agria and Lady Olympia. Insecticidal effects of T0 progeny transgenic potato plants were tested against CPB under laboratory conditions. Transgenic plants of Agria and Lady Olympia transformed with DS-1 and DS-2 constructs caused 100% mortality to all larval stages and adults of CPB. However, 100% mortality of tested insects took a longer time in the adult stage (10–14 days) compared to larval stages (2–6 days). Foliage consumption by L2-L4 larval stages and adults of CPB was significantly reduced in Agria and Lady Olympia plants transformed with DS-1 and DS-2 constructs, as compared to their control plants. Lower foliage consumption of transgenic plants by L1 larval stages was also observed, but the reduction was only statistically significant for some of the tested plants. These promising results indicate that the transgenic potato plants exhibit a high potential in controlling CPB population and are a useful tool in the management of imidacloprid-resistant CPB.

Differences in the Inhibitory Specificity Distinguish the Efficacy of Plant Protease Inhibitors on Mouse Fibrosarcoma.

Metastasis, the primary cause of death from malignant tumors, is facilitated by multiple protease-mediated processes. Thus, effort has been invested in the development of protease inhibitors to prevent metastasis. Here, we investigated the effects of protease inhibitors including the recombinant inhibitors rBbKI (serine protease inhibitor) and rBbCI (serine and cysteine inhibitor) derived from native inhibitors identified in Bauhinia bauhinioides seeds, and EcTI (serine and metalloprotease inhibitor) isolated from the seeds of Enterolobium contortisiliquum on the mouse fibrosarcoma model (lineage L929). rBbKI inhibited 80% of cell viability of L929 cells after 48 h, while EcTI showed similar efficacy after 72 h. Both inhibitors acted in a dose and time-dependent manner. Conversely, rBbCI did not significantly affect the viability of L929 cells. Confocal microscopy revealed the binding of rBbKI and EcTI to the L929 cell surface. rBbKI inhibited approximately 63% of L929 adhesion to fibronectin, in contrast with EcTI and rBbCI, which did not significantly interfere with adhesion. None of the inhibitors interfered with the L929 cell cycle phases. The synthetic peptide RPGLPVRFESPL-NH2, based on the BbKI reactive site, inhibited 45% of the cellular viability of L929, becoming a promising protease inhibitor due to its ease of synthesis.

In vivo tumor growth inhibition by Solanum tuberosum aspartic protease 3 (StAP3) treatment

Solanum tuberosum aspartic Proteases (StAPs) show selective plasma membrane permeabilization, inducing cytotoxicity of cancer cells versus normal cells in vitro. Herein, we aimed to evaluate both StAP3 systemic toxicity and antitumoral activity against human melanoma in vivo. The toxicity of a single high dose of StAP3 (10 µg/g body weight, intraperitoneally) was assessed in a Balb/c mice model. Subcutaneous A375 human melanoma xenografts in athymic nude (nu/nu) mice were induced. Once tumors developed (mean larger dimension = 3.8 ± 0.09 mm), mice were StAP3-treated (6 µg/g body weight, subcutaneously under the tumor at a single dose). For both models, controls were treated with physiologic saline solution. StAP3-treated mice showed a significant inhibition of tumor growth (p < 0.05) compared with controls. No signs of toxicity were detected in StAP3-treated mice in both models. These results suggest the potential of these plant proteases as anticancer agents.

Sesame water-soluble proteins fraction contains endopeptidases and exopeptidases with high activity: A natural source for plant proteases

Recently, the interest in the plant proteases has greatly increased. However, only a few of proteases are isolated from the hugely produced oilseeds for the practical utilizations. In this study, the raw sesame milk prepared from peeled sesame seeds was separated into floating, skim, and precipitate fractions by centrifugation. The predominant aspartic endopeptidases and serine carboxypeptidases, which exerted high synergetic activity at pH 4.5–5 and 50–60 °C, were identified in the skim by the liquid chromatography tandem mass spectrometry, Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis, protease inhibitor assay, trichloroacetic acid–nitrogen soluble index (TCA–NSI), and free amino acid analyses. By incubating the mixture (protein content, 2%) of skim and precipitate at pH 4.5 and 50 °C for 6 h, the TCA–NSI and free amino acids achieved to 38.42% and 3148 mg/L, respectively. Moreover, these proteases efficiently degraded the proteins from soybean, peanut, and bovine milk.

Milk protein hydrolysis by actinidin: Influence of protein source and hydrolysis conditions

The plant protease actinidin has been frequently used in the food industry, but its application in dairy systems remains largely unassessed. The aim of this research was to establish the effect of temperature (15–60 °C), time (0–5 h) and enzyme-to-substrate ratio on the actinidin-induced hydrolysis of proteins in whey protein isolate (WPI), whey protein concentrate (WPC) and milk protein concentrate (MPC), as monitored through the degree of hydrolysis (DH) and SDS-PAGE. The DH increased with increasing temperature and incubation time for all three protein sources. A lower E:S ratio resulted in a greater DH for WPC and WPI, but not for MPC. SDS-PAGE analysis revealed that actinidin mainly acted on α-lactalbumin and αS-caseins in WPI and MPC, respectively.

Meat Tenderization through Plant Proteases- A Mini Review | IJB 2021

Meat Tenderization through Plant Proteases- A Mini Review | IJB 2021

Consequences of 'no-choice, fixed time' reciprocal host plant switches on nutrition and gut serine protease gene expression in Pieris brassicae L. (Lepidoptera: Pieridae).

Rapid adaptive responses were evident from reciprocal host-plant switches on performance, digestive physiology and relative gene expression of gut serine proteases in larvae of crucifer pest P. brassicae transferred from cauliflower (CF, Brassica oleracea var. botrytis, family Brassicaceae) to an alternate host, garden nasturtium, (GN, Tropaeolum majus L., family Tropaeolaceae) and vice-versa under laboratory conditions. Estimation of nutritional indices indicated that larvae of all instars tested consumed the least food and gained less weight on CF-GN diet (significant at p≤0.05) as compared to larvae feeding on CF-CF, GN-GN and GN-CF diets suggesting that the switch to GN was nutritionally less favorable for larval growth. Nevertheless, these larvae, especially fourth instars, were adroit in utilizing and digesting GN as a new host plant type. In vitro protease assays conducted to understand associated physiological responses within twelve hours indicated that levels and properties of gut proteases were significantly influenced by type of natal host-plant consumed, change in diet as well as larval age. Activities of gut trypsins and chymotrypsins in larvae feeding on CF-GN and GN-CF diets were distinct, and represented shifts toward profiles observed in larvae feeding continuously on GN-GN and CF-CF diets respectively. Results with diagnostic protease inhibitors like TLCK, STI and SBBI in these assays and gelatinolytic zymograms indicated complex and contrasting trends in gut serine protease activities in different instars from CF-GN diet versus GN-CF diet, likely due to ingestion of plant protease inhibitors present in the new diet. Cloning and sequencing of serine protease gene fragments expressed in gut tissues of fourth instar P. brassicae revealed diverse transcripts encoding putative trypsins and chymotrypsins belonging to at least ten lineages. Sequences of members of each lineage closely resembled lepidopteran serine protease orthologs including uncharacterized transcripts from Pieris rapae. Differential regulation of serine protease genes (Pbr1-Pbr5) was observed in larval guts of P. brassicae from CF-CF and GN-GN diets while expression of transcripts encoding two putative trypsins (Pbr3 and Pbr5) were significantly different in larvae from CF-GN and GN-CF diets. These results suggested that some gut serine proteases that were differentially expressed in larvae feeding on different species of host plants were also involved in rapid adaptations to dietary switches. A gene encoding nitrile-specifier protein (nsp) likely involved in detoxification of toxic products from interactions of ingested host plant glucosinolates with myrosinases was expressed to similar levels in these larvae. Taken together, these snapshots reflected contrasts in physiological and developmental plasticity of P. brassicae larvae to nutritional challenges from wide dietary switches in the short term and the prominent role of gut serine proteases in rapid dietary adaptations. This study may be useful in designing novel management strategies targeting candidate gut serine proteases of P. brassicae using RNA interference, gene editing or crops with transgenes encoding protease inhibitors from taxonomically-distant host plants.

The impact of chymosin and plant‐derived proteases on the acid‐induced gelation of milk

This work aimed to study milk gel formation during a slow fermentation process and the simultaneous action of different proteases like bovine chymosin, recombinant camel chymosin, ficin and bromelain. The acid‐induced gels with bovine chymosin and recombinant camel chymosin were firmer than the control gels without proteases or the gels added with plant proteases. However, the acid‐induced gels with chymosins had a considerable water release and shrunk in volume as the milk fermentation progressed. In contrast, the acid‐induced gels added with bromelain and ficin were softer and showed less syneresis, even at acidic pHs.

Production and Industrial Application of Microbial Aspartic Protease: A Review

Proteases are one of the predominant groups of industrial enzymes and it represents for about 65% of the total global enzyme market. Proteases of microbial origin have great importance over plant sources because they minimize industrial production costs, increase characteristics of the desired products and widely used in biotechnological process. Among the protease enzymes, aspartic proteases are the most important groups of proteolytic enzymes which are mainly produced by plants, animals and many microorganisms to degrade large polypeptides into peptides and amino acids. Microorganisms are also mainly preferred in the production of aspartic protease since they have most of the characteristics desired for biotechnological application rather than plant protease. Aspartic proteases produced from microbial sources are widely used in pharmaceutical, protein hydrolysis, detergent, cheese-making, photographic, baking, meat, leather, food and beverage industries. Although acid protease is vital to enhance the demands of many food and other industries, there are factors affecting the production of aspartic protease. Hence, aspartic protease production using microorganisms is highly affected by various carbon and nitrogen substrates, divalent metal ions, pH, incubation temperature, time, agitation speeds, age of inoculum and density. This review highlights on the production and applications of microbial aspartic proteases.

Влияние совместной обработки эндофитным штаммом бактерий Bacillus thuringiensis B-5351 и салициловой кислотой на устойчивость растений картофеля к Phytophthora infestans (Mont.) de Bary

Влияние совместной обработки эндофитным штаммом бактерий Bacillus thuringiensis B-5351 и салициловой кислотой на устойчивость растений картофеля к Phytophthora infestans (Mont.) de Bary

Efficacy of Partially Purified Soybean Protease Inhibitor on The Development of Cotton Leafworm, Spodoptera littoralis (Bosid.)

Plant protease inhibitors) PIs) are one of the plant strategies to maintain favorable growth and survival. Soybean trypsin inhibitor is the most well-known of the plant serine proteinase. The present study aims to detect the inhibitory activity of two concentrations of partially purified trypsin inhibitors from seeds of soybean(Glycine max). Comparing the results of feeding larvae on partially purified soybean inhibitors with control, revealed that the larval weight reduction caused by soybean PI was noticed after seven days of treatment. The highest larval weight reduction was obtained due to treatment with SPI at levels SPI 30% and SPI 60%, and there was no significant difference between the two levels. Feeding larvae on SPI 60% caused higher larval mortality about 20% than the control followed by SPI 30% which caused 16% larval mortality. The pupation in treated larvae was delayed two days by comparing to control. The highest weight reduction in pupal weight was 3.5± 0.3 gm at SPI 60% compared with control. Besides, the pupation rate decreased from 83% at SPI 60% to 80% at SPI 30%. Also, the reduction in adult emergency % was recorded at SPI level 30% by 41% and at SPI level 60% by 16% compared with the control. Results reveal that soybean protease inhibitors inhibit the growth and development of S. littoralis, and may be used in integrated pest control strategies.

Optimization of Crude Papaya (Carica papaya) Protease in Soft-Unripened Cheese Preparation

The use of plant protease instead of chymosin for producing cheese has become a trend which is aimed at lacto-vegetarian consumers and religion based ecological markets. In this context, the present investigation was carried out in order to utilize milk clotting enzyme from Papaya (Carica papaya). Numerical optimization study revealed that maximum milk clotting activity was achieved at pH 6.5, temperature 70℃ and enzyme concentration 1 g/1000 ml milk using papaya protease as coagulant. Protein, ash and calcium showed no significant (p&gt;0.05) difference among the cheeses made using different coagulants. However, significantly (p&lt;0.05) higher levels of moisture and ash, and lower levels of fat were observed in the cheese produced by papaya protease compared to that made using rennet. Papaya protease significantly enhanced the spreadability of cheese while the other sensory properties were similar to the control except aftertaste. The results revealed that the papaya latex as crude papaya protease may have potential application for the manufacture of soft-unripened cheese and further could be utilized as a milk coagulant in cheese making.

Extraction, Partial Purification, Characteristics, and Antimicrobial Activity of Plant Protease From Moringa Oleifera Leaves

Introduction: Proteases are hydrolyzing enzymes and are considered to be one of the most important groups of enzymes for industry and are used in leather, pharmaceutical, and food industry along with detergents, and bioremediation processes. The main objectives of this study were (i) to extract, partially purify, and characterize the protease enzyme from Moringa (Moringa oleifera) leaves; and (ii) to investigate the effect of such an enzyme against some pathogenic bacteria.Materials and Methods: This enzyme was extracted in a 0.1 M phosphate buffer pH 7. It was left for 24 hours in a refrigerator and was then filtered using filter paper Whatman no. 41. The aqueous filtrate was used to estimate the proteolytic activity.Results: Purification by ammonium sulfate gave the best results at 50%-70% concentration which had the highest specific activity, highest purification fold with the percentage yield of 45.3%. Gel filtration by Sephadex G-100 gave the best specific activity and the best purification fold with the yield from fraction of 34%-43%. The protease enzyme has optimum pH 7 and temperature 50 oC. The enzyme was thermally stable at 40-70 oC for 20-30 minutes. Some metal ions were activator on the enzyme-like Mn2+ (highest), Ba2+, Ca2+, and Na+. The efficacy of protease enzyme was improved when integration with antibiotic against certain bacterial including Bacillus cereus (S3), Staphylococcus aureus, Salmonella typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes. Also, E. coli O157:H7, L. monocytogenes, and Yersinia enterocolitica did not show any growth at pH 10.Conclusions: To conclude, it can be stated that protease enzyme can be considered as a promising agent, cheap, and safe source which is suitable for using in various industries.

Characterisation of hydrolysate for identifying initial peptide cleavage site of κ-casein by milk coagulating Wrightia tinctoria serine proteases

Research on identifying vegetable milk coagulants has gained momentum in the past few decades. Extent of casein hydrolysis and specific initial hydrolytic site on κ-casein are considered crucial in deciding suitability of plant proteases for cheese making. In the present study, three phase partitioned (TPP) proteases (serine) of Wrightia tinctoria stem were assayed to understand their specific cleavage site on κ-casein. Time dependent analysis of κ-casein hydrolysis by W. tinctoria proteases revealed 2 min incubation to be enough for hydrolysis to begin. Molecular mass of the resulting major peptide was confirmed to be 14,542.52 Da and was further characterised using LC/ESI-MSMS. The initial site of hydrolysis by W. tinctoria proteases on κ-casein was Asn123–Thr124, which is different from the expected Phe105–Met106 of rennin. The study concludes that even with its unique initial site of κ-casein hydrolysis, W. tinctoria proteases could influence milk clotting probably with distinct organoleptic properties.