Plant Proteases Research Articles

RD21-like proteases: key effector hubs in plant-pathogen interactions.

Over the past decades, numerous studies have demonstrated that proteases serve as a crucial regulatory mechanism in controlling plant immunity. In this review, we specifically focus on the role of one subfamily of RD21-like papain-like cysteine proteases that carry a C-terminal granulin domain. These proteases share high homology but have been described under very different names in different plant species. We provide a comprehensive overview of the background, endogenous regulation, and subcellular localization of RD21-like proteases in plants. Notably, RD21-like proteases act in immunity against various pathogens and they are targeted by many unrelated pathogen-secreted effectors that inactivate, mislocalise or degrade RD21-like proteases. We highlight open questions and strategies to use this knowledge to develop innovative strategies for crop protection.

Degradation of gluten using plant proteases

The paper describes the fact of the presence of a proteolytic enzyme in the leaves of the natural hybrid variety of grape - Isabella (Vitis Labrusca L.) and the perspective of its practical application for breaking down wheat gluten. As our research has shown, this enzyme effectively degradates the constituent components of the protein fraction of wheat flour-the gluten fraction. It is known that the wheat gluten is a strong allergen for many people, which can initiate a disease such as celiac disease. Protease from Isabella leaves carries out deep hydrolysis of gluten to low molecular weight soluble peptides and amino acids. This effect can be successfully used in the production of gluten-free bread products.

Effects of tannin-tolerant lactic acid bacteria in combination with tannic acid on the fermentation quality, protease activity and bacterial community of stylo silage.

Proteolysis during ensiling primarily occurs due to undesirable microbial and plant protease activities, which reduce the protein supply to ruminant livestock and cause a series of environmental problems. The objective of this study was to investigate the effects of the tannin-tolerant lactic acid bacterium strain Lactiplantibacillus plantarum 4 (LABLP4) in combination with tannic acid (TA) on protein preservation in stylo (Stylosanthes guianensis) silage. The stylos were either ensiled without additives (control) or treated with LABLP4 (106 colony-forming units per gram of fresh matter), 1% (fresh matter basis) TA, 2% TA, LABLP4 + 1% TA and LABLP4 + 2% TA. Fermentation quality, protein composition, protease activity and bacterial diversity were determined at 3, 7, 14 and 31 days of ensiling. The combination of LABLP4 and TA decreased the pH, coliform bacteria count, non-protein nitrogen, ammonia-nitrogen (NH3-N) content and protease activities (P < 0.05) and increased the true protein content (P < 0.05) compared to the control. LABLP4 + TA led to a lower pH and NH3-N content than LABLP4 or TA alone (P < 0.05). On the last day (31 days) of ensiling, LABLP4 + TA increased the relative abundances of Firmicutes and Lactiplantibacillus (P < 0.05), except for the LABLP4 treatment, and decreased the relative abundance of Actinobacteria (P < 0.05). The combination of tannin-tolerant LABLP4 and TA effectively improved the fermentation quality of stylo silage and reduced protein degradation by altering the bacterial community structure. © 2024 Society of Chemical Industry.

Extracellular plant subtilases dampen cold-shock peptide elicitor levels

Recognizing pathogen-associated molecular patterns on the cell surface is crucial for plant immunity. The proteinaceous nature of many of these patterns suggests that secreted proteases play important roles in their formation and stability. Here we demonstrate that the apoplastic subtilase SBT5.2a inactivates the immunogenicity of cold-shock proteins (CSPs) of the bacterial plant pathogen Pseudomonas syringae by cleaving within the immunogenic csp22 epitope. Consequently, mutant plants lacking SBT5.2a activity retain higher levels of csp22, leading to enhanced immune responses and reduced pathogen growth. SBT5.2 sensitivity is influenced by sequence variation surrounding the cleavage site and probably extends to CSPs from other bacterial species. These findings suggest that variations in csp22 stability among bacterial pathogens are a crucial factor in plant–bacteria interactions and that pathogens exploit plant proteases to avoid pattern recognition.

Engineering inhibitory repeat domains of Pin-II type proteinase inhibitors indicate their high structural-functional tolerance to mutagenesis

Plant proteinase inhibitors (PIs) are critical in defending against biotic stress. Most PIs contain an inhibitory repeat domain (IRD), which serves as the functional component, displaying a high degree of sequence and structural conservation. In this study, we examined the structural and functional resilience of IRDs using a combination of computational modeling and experimental validation. We have taken an evolution-based approach to enhance the PIs effectiveness of two previously identified Capsicum annuum IRDs, IRD4 and IRD10. Through in silico site-saturation mutagenesis of IRD4 and IRD10, we identified key sites associated with enhanced PI activity for targeted mutagenesis. Binding energy predictions for a mutant IRD library, tested against target proteases, suggested that positions R11 and N32 in IRD4 and N32 and H33 in IRD10 were promising candidates for further modification to improve inhibitory potential. Subsequent experimental validation revealed that the mutant proteins IRD4_R11K and IRD4_N32S exhibited stronger chymotrypsin inhibition than the wild-type (WT) IRD4. Similarly, the mutants IRD10_N32S and IRD10_H33 N demonstrated improved trypsin inhibition relative to the WT IRD10. These findings indicate that engineered IRD variants can tolerate structural changes while maintaining or enhancing their inhibitory activity against target proteases. Overall, this study demonstrates the potential of engineering PIs to increase their structural and functional resilience, offering new opportunities for biotechnological applications.

Characterization and kinetics of a cathepsin B-inhibiting protein from Musa acuminata Colla peel

Hyperexpression of cathepsin B caused by an imbalance of endogenous inhibitors is involved in multiple pathologies, hence making it a key therapeutic target. Protease inhibitors are effective biomolecules that regulate protease activities and are considered potential therapeutic agents in various diseases. Plant protease inhibitors have been reported as an effective complementary alternative drug. A proteinaceous cathepsin B inhibitor (CBI-BP) has been isolated from Musa acuminata Colla (banana) peel with a molecular weight of 27.9 kDa on SDS-PAGE. The purity of the CBI-BP was confirmed on the native- PAGE. The isolated CBI-BP showed an IC50 value of 8.14 μg and a Ki value of 10.59 μg (0.19 μM). Cathepsin B inhibition kinetics indicated that CBI-BP follows a mixed-type of cathepsin B inhibition. Its inhibition activity was also confirmed by reverse zymography. The inhibitor was stable from pH 2.6–10.0 with maximum activity at pH 7.2, temperature 25–100 °C and exhibited thermostability for 60 min at 70 °C. MALDI/TOF/MS analysis of CBI-BP showed 40 % similarity to the GH18 domain-containing protein (A0A4S8JRM9) from Musa balbisiana. Although in-silico docking studies showed binding of A0A4S8JRM9 to cathepsin B affects the binding energy of the substrate to cathepsin B but is not reported for any anti-cathepsin B activity. This suggests that isolated CBI-BP might be a novel protein with anti-cathepsin B activity. Thus the isolated CBI-BP may be further explored as possible anti-cathepsin B drug.

Green Nanotechnology Through Papain Nanoparticles: Preclinical in vitro and in vivo Evaluation of Imaging Triple-Negative Breast Tumors.

Recent advancements in nanomedicine and nanotechnology have expanded the scope of multifunctional nanostructures, offering innovative solutions for targeted drug delivery and diagnostic agents in oncology and nuclear medicine. Nanoparticles, particularly those derived from natural sources, hold immense potential in overcoming biological barriers to enhance therapeutic efficacy and diagnostic accuracy. Papain, a natural plant protease derived from Carica papaya, emerges as a promising candidate for green nanotechnology-based applications due to its diverse medicinal properties, including anticancer properties. This study presents a novel approach in nanomedicine and oncology, exploring the potential of green nanotechnology by developing and evaluating technetium-99m radiolabeled papain nanoparticles (99mTc-P-NPs) for imaging breast tumors. The study aimed to investigate the efficacy and specificity of these nanoparticles in breast cancer models through preclinical in vitro and in vivo assessments. Papain nanoparticles (P-NPs) were synthesized using a radiation-driven method and underwent thorough characterization, including size, surface morphology, surface charge, and cytotoxicity assessment. Subsequently, P-NPs were radiolabeled with technetium-99m (99mTc), and in vitro and in vivo studies were conducted to evaluate cellular uptake at tumor sites, along with biodistribution, SPECT/CT imaging, autoradiography, and immunohistochemistry assays, using breast cancer models. The synthesized P-NPs exhibited a size mean diameter of 9.3 ± 1.9 nm and a spherical shape. The in vitro cytotoxic activity of native papain and P-NPs showed low cytotoxicity in HUVEC, MDA-MB231, and 4T1 cells. The achieved radiochemical yield was 94.2 ± 3.1% that were sufficiently stable (≥90%) for 6 h. The tumor uptake achieved in the 4T1 model was 2.49 ± 0.32% IA/g at 2h and 1.51 ± 0.20% IA/g at 6h. In the spontaneous breast cancer model, 1.19 ± 0.20% IA/g at 2h and 0.86 ± 0.31% IA/g at 6h. SPECT/CT imaging has shown substantial tumor uptake of the new nanoradiopharmaceutical and clear tumor visualization. 99mTc-P-NPs exhibited a high affinity to tumoral cells confirmed by ex vivo autoradiography and immunohistochemistry assays. The findings underscore the potential of green nanotechnology-driven papain nanoparticles as promising agents for molecular imaging of breast and other tumors through SPECT/CT imaging. The results represent a substantial step forward in the application of papain nanoparticles as carriers of diagnostic and therapeutic radionuclides to deliver diagnostic/therapeutic payloads site-specifically to tumor sites for the development of a new generation of nanoradiopharmaceuticals.

Assessment of Cytotoxic Effects of Plant Protease Inhibitors on Cancerous Cell Lines

Assessment of Cytotoxic Effects of Plant Protease Inhibitors on Cancerous Cell Lines

Intra-chloroplast proteases: A holistic network view of chloroplast proteolysis.

Different proteases and peptidases are present within chloroplasts and nonphotosynthetic plastids to process precursor proteins and to degrade cleaved chloroplast transit peptides and damaged, misfolded, or otherwise unwanted proteins. Collectively, these proteases and peptidases form a proteolysis network, with complementary activities and hierarchies, and build-in redundancies. Furthermore, this network is distributed across the different intra-chloroplast compartments (lumen, thylakoid, stroma, envelope). The challenge is to determine the contributions of each peptidase (system) to this network in chloroplasts and nonphotosynthetic plastids. This will require an understanding of substrate recognition mechanisms, degrons, substrate, and product size limitations, as well as the capacity and degradation kinetics of each protease. Multiple extra-plastidial degradation pathways complement these intra-chloroplast proteases. This review summarizes our current understanding of these intra-chloroplast proteases in Arabidopsis and crop plants with an emphasis on considerations for building a qualitative and quantitative network view.

Anti-SARS-CoV-2 Viral Activity of Sweet Potato Trypsin Inhibitor via Downregulation of TMPRSS2 Activity and ACE2 Expression In Vitro and In Vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever recorded in human history, causing significant losses to both life and economies globally. However, the available evidence is currently insufficient to establish the effectiveness and safety of antiviral drugs or vaccines. The entry of the virus into host cells involves binding to angiotensin-converting enzyme 2 (ACE2), a cell surface receptor, via its spike protein. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a host surface protease, cleaves and activates the virus's S protein, thus promoting viral infection. Plant protease inhibitors play a crucial role in protecting plants against insects and/or microorganisms. The major storage proteins in sweet potato roots include sweet potato trypsin inhibitor (SWTI), which accounts for approximately 60% of the total water-soluble protein and has been found to possess a variety of health-promoting properties, including antioxidant, anti-inflammatory, ACE-inhibitory, and anticancer functions. Our study found that SWTI caused a significant reduction in the expression of the ACE2 and TMPRSS2 proteins, without any adverse effects on cells. Therefore, our findings suggest that the ACE2 and TMPRSS2 axis can be targeted via SWTI to potentially inhibit SARS-CoV-2 infection.

Serine proteinase inhibitors from Nicotiana benthamiana, a nonpreferred host plant, inhibit the growth of Myzus persicae (green peach aphid).

The green peach aphid (Myzus persicae) is a severe agricultural crop pest that has developed resistance to most current control methods, requiring the urgent development of novel strategies. Plant proteinase inhibitors (PINs) are small proteins that protect plants against pathogens and/or herbivores, likely by preventing efficient protein digestion. We identified 67 protease genes in the transcriptomes of three M. persicae lineages (USDA-Red, G002 and G006). Comparison of gene expression levels in aphid guts and whole aphids showed that several proteases, including a highly expressed serine protease, are significantly overexpressed in the guts. Furthermore, we identified three genes encoding serine protease inhibitors (SerPIN-II1, 2 and 3) in Nicotiana benthamiana, which is a nonpreferred host for M. persicae. Using virus-induced gene silencing (VIGS) with a tobacco rattle virus (TRV) vector and overexpression with a turnip mosaic virus (TuMV) vector, we demonstrated that N. benthamiana SerPIN-II1 and SerPIN-II2 cause reduced survival and growth, but do not affect aphid protein content. Likewise, SerPIN-II3 overexpression reduced survival and growth, and serpin-II3 knockout mutations, which we generated using CRISPR/Cas9, increased survival and growth. Protein content was significantly increased in aphids fed on SerPIN-II3 overexpressing plants, yet it was decreased in aphids fed on serpin-II3 mutants. Our results show that three PIN-IIs from N. benthamiana, a nonpreferred host plant, effectively inhibit M. persicae survival and growth, thereby representing a new resource for the development of aphid-resistant crop plants. © 2024 Society of Chemical Industry.

Effects of plant protease inhibitors (Pep-3-EcTI, Pep-BbKI, and Pep-BrTI) versus corticosteroids on inflammation, remodeling, and oxidative stress in an asthma-COPD (ACO) model.

The peptide derived from E. contortisiliquum trypsin inhibitor (Pep-3-EcTI), peptide derived from kallikrein inhibitor isolated from B. bauhinioides (Pep-BbKI), and B. rufa peptide modified from B. bauhinioides (Pep-BrTI) peptides exhibit anti-inflammatory and antioxidant activities, suggesting their potential for treating asthma-chronic obstructive pulmonary disease (COPD) overlap (ACO). We compared the effects of these peptides with dexamethasone (DX) treatment in an ACO model. In this study, 11 groups of male BALB/c mice were pre-treated under different conditions, including sensitization with intraperitoneal injection and inhalation of ovalbumin (OVA), intratracheal instillation of porcine pancreatic elastase (ELA), sensitization with intraperitoneal injection, and various combinations of peptide treatments with Pep-3-EcTI, Pep-BbKI, Pep-BrTI, dexamethasone, and non-treated controls (SAL-saline). Respiratory system resistance, airway resistance, lung tissue resistance, exhaled nitric oxide, linear mean intercept, immune cell counts in the bronchoalveolar lavage fluid, cytokine expression, extracellular matrix remodeling, and oxidative stress in the airways and alveolar septa were evaluated on day 28. Results showed increased respiratory parameters, inflammatory markers, and tissue remodeling in the ACO group compared to controls. Treatment with the peptides or DX attenuated or reversed these responses, with the peptides showing effectiveness in controlling hyperresponsiveness, inflammation, remodeling, and oxidative stress markers. These peptides demonstrated an efficacy comparable to that of corticosteroids in the ACO model. However, this study highlights the need for further research to assess their safety, mechanisms of action, and potential translation to clinical studies before considering these peptides for human use.

Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

Plant Protease Inhibitors as Emerging Antimicrobial Peptide Agents: A Comprehensive Review.

Antimicrobial peptides (AMPs) are important mediator molecules of the innate defense mechanisms in a wide range of living organisms, including bacteria, mammals, and plants. Among them, peptide protease inhibitors (PPIs) from plants play a central role in their defense mechanisms by directly attacking pathogens or by modulating the plant's defense response. The growing prevalence of microbial resistance to currently available antibiotics has intensified the interest concerning these molecules as novel antimicrobial agents. In this scenario, PPIs isolated from a variety of plants have shown potential in inhibiting the growth of pathogenic bacteria, protozoans, and fungal strains, either by interfering with essential biochemical or physiological processes or by altering the permeability of biological membranes of invading organisms. Moreover, these molecules are active inhibitors of a range of proteases, including aspartic, serine, and cysteine types, with some showing particular efficacy as trypsin and chymotrypsin inhibitors. In this review, we provide a comprehensive analysis of the potential of plant-derived PPIs as novel antimicrobial molecules, highlighting their broad-spectrum antimicrobial efficacy, specificity, and minimal toxicity. These natural compounds exhibit diverse mechanisms of action and often multifunctionality, positioning them as promising molecular scaffolds for developing new therapeutic antibacterial agents.

Plant and Arthropod IgE-Binding Papain-like Cysteine Proteases: Multiple Contributions to Allergenicity.

Papain-like cysteine proteases are widespread and can be detected in all domains of life. They share structural and enzymatic properties with the group's namesake member, papain. They show a broad range of protein substrates and are involved in several biological processes. These proteases are widely exploited for food, pharmaceutical, chemical and cosmetic biotechnological applications. However, some of them are known to cause allergic reactions. In this context, the objective of this review is to report an overview of some general properties of papain-like cysteine proteases and to highlight their contributions to allergy reactions observed in humans. For instance, the literature shows that their proteolytic activity can cause an increase in tissue permeability, which favours the crossing of allergens through the skin, intestinal and respiratory barriers. The observation that allergy to PLCPs is mostly detected for inhaled proteins is in line with the reports describing mite homologs, such as Der p 1 and Der f 1, as major allergens showing a frequent correlation between sensitisation and clinical allergic reactions. In contrast, the plant food homologs are often digested in the gastrointestinal tract. Therefore, they only rarely can cause allergic reactions in humans. Accordingly, they are reported mainly as a cause of occupational diseases.

Plant cathepsin B, a versatile protease.

Plant proteases are essential enzymes that play key roles during crucial phases of plant life. Some proteases are mainly involved in general protein turnover and recycle amino acids for protein synthesis. Other proteases are involved in cell signalling, cleave specific substrates and are key players during important genetically controlled molecular processes. Cathepsin B is a cysteine protease that can do both because of its exopeptidase and endopeptidase activities. Animal cathepsin B has been investigated for many years, and much is known about its mode of action and substrate preferences, but much remains to be discovered about this potent protease in plants. Cathepsin B is involved in plant development, germination, senescence, microspore embryogenesis, pathogen defence and responses to abiotic stress, including programmed cell death. This review discusses the structural features, the activity of the enzyme and the differences between the plant and animal forms. We discuss its maturation and subcellular localisation and provide a detailed overview of the involvement of cathepsin B in important plant life processes. A greater understanding of the cell signalling processes involving cathepsin B is needed for applied discoveries in plant biotechnology.

The potential of plant protease enzymes as rennet alternatives for developing halal cheese product: A review

Cheese, a derivative of dairy products made using the enzyme rennet, has received full attention because of the critical point for halalness from the milk coagulation process, which uses the rennet enzyme. Rennet enzymes can be obtained from the stomachs of animals such as cows, pigs, and goats, and they can also be produced from microbes. This very high risk of haram sources or unclean contamination has led to the development of cheese products using plant protease enzymes as a substitute for rennet enzymes. This study aims to highlight plant protease enzymes, characterize the enzymes produced, characterize cheese produced, and the potential of plant protease enzymes in replacing Rennet. Plants that have protease enzymes, such as noni, papaya, pineapple, red ginger, strawberries, pears, biduri, moringa, kiwi, tamarillo, and many other plants, have the potential to replace the rennet enzyme in making cheese. Thus, the doubts (mashbooh) arising from making cheese can be avoided by developing products from raw materials with guaranteed halal quality. Plant ingredients that can replace the rennet enzyme in making cheese are many and varied, for example, noni, papaya, moringa, bidi, pineapple, red ginger, kiwi, tamarillo, pears, Balanites aegyptiaca, strawberries, and many more. Doubtless, the problem of making cheese with Rennet can be avoided by developing cheese products from raw materials guaranteed to be halal. Limitations to plant protease enzymes on cheese production only apply to soft cheese, while it is difficult to produce hard cheese from plant enzymes.

Plant Protease Inhibitors: A Defense Mechanism Against Phytophagous Insects

In the realm of agriculture, the constant threat of pests and pathogens poses a significant challenge to crop yields. Traditional chemical pesticides, while effective, come with drawbacks such as lack of specificity and the development of resistance. This has spurred a growing interest in exploring alternative methods, with a focus on biodegradable biological control agents and natural products. One promising avenue is the use of Plant Protease Inhibitors (PPIs), which act as a defense mechanism against phytophagous insects. PPIs hinder the activity of insect gut digestive enzymes, leading to reduced protein digestion and impeding the growth and survival of insects. The article delves into the various types of PPIs, their mechanisms of action, and their effectiveness in plant defense. Specifically, it explores the Cystatin Superfamily, with a focus on Family–4 Cystatins known as Phytocystatins. These inhibitors, found in a variety of plants, exhibit potential as biopesticides due to their impact on insect proteolysis. The study also discusses the role of phytocystatins in controlling phytophagous arthropods by targeting their essential digestive proteases. In conclusion, the article emphasizes the significant value of phytocystatins in plant defense and suggests their potential integration as a novel tool in Pest Control Management, highlighting the need for improved policies to enhance their adoption in sustainable agriculture.

Proteolytic Activity of Silkworm Thorn (Cudrania tricuspidata) Fruit for Enzymatic Hydrolysis of Food Proteins.

This study aimed to isolate the proteolytic fraction from the silkworm thorn fruit (Cudrania tricuspidata) through ethanol precipitation at different ratios, and to determine its proteolytic activity and optimal activity conditions. Furthermore, the hydrolysis characteristics and antioxidant activity of soy protein isolate (SPI) and whey protein concentrate (WPC) hydrolyzates obtained through the enzymatic hydrolysis of freeze-dried silkworm thorn fruit powder (SF) were evaluated. For isolation and partial purification of proteolytic fraction, the water-solubilized fraction of the silkworm thorn fruit was purified through ethanol precipitation at four different ratios of 1:1, 1:2, 1:4, and 1:6 (v/v). The protein recovery rate, caseinolytic activity, protein pattern, and optimal activity (pH, temperature, and inhibitors) of fractional ethanol precipitate obtained from the silkworm thorn fruit (ESF) were evaluated. The proteolytic fraction obtained from silkworm thorn fruit exhibited a major protein band around 65-70 kDa and showed the highest proteolytic activity at a 1:4 ratio of ethanol precipitation (p < 0.05). The optimal activity of the measured enzyme fraction was determined to be at pH 9.0 and 50 °C, and the proteolytic activity of ESF was almost inhibited by phenyl methyl sulphonyl fluoride (PMSF, 2 mM), a serine protease inhibitor. Compared to Alcalase and papain, extensively used as commercial enzymes, the silkworm thorn fruit powder was less effective in hydrolyzing SPI and WPC. Nevertheless, SPI and WPC hydrolyzates mediated with silkworm thorn fruit powder showed even better antioxidant activities than those mediated with Alcalase and papain. Thus, our results show the potential application of silkworm thorn fruit as a novel source of plant protease for producing human-grade protein hydrolyzates.

Plant proteases and anti-bacterial substances in Allium sativum L. varieties

Allium sativum L. protease still remains largely understudied although new varieties of garlic appear quite often, e.g., lanang garlic. This study tested the antibacterial effect of garlic and the effectiveness of various A. sativum proteases as meat tenderizers. &#x0D; The research involved powder extracts of four varieties of A. sativum: kating, lanang, black garlic, and sin-chung. The degradation kinetics was defined based on the Lineweaver-Burk equation. The degradation zones were measured using sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE). Scan electron microscopy served to test the changes in meat connective tissue.&#x0D; Lanang demonstrated the largest inhibition zones against Escherichia coli (9.75 ± 0.15 mm) and Staphylococcus aureus (1.04 mm). Sin-chung protease degraded beef protein with the highest Vmax of 0.1818 μg/μL/min at 10–22 KDa (small peptide, troponin C, and troponin I), 25–40 KDa (myosin light chain, troponin T, α and β tropomyosin, actin), and 100–140 KDa (protein C). The same garlic variety degraded mutton meat protein at 10–17 KDa (small peptide) and 25–40 KDa (myosin light chain, troponin T, α and β tropomyosin, actin) with Vmax of 0.1135 μg/μL/min.&#x0D; All four A. sativum proteases proved to be quite effective meat tenderizers.