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Aspergillus species are known to be very important in human and domestic animal health. Aspergillus species commonly cause severe systemic and skin infections, as well as allergic lung diseases. With the development of PCR techniques, these methods are used to identify and diagnose fungi. DNA extraction from Aspergillus species is difficult because the fungal cell wall structure is very durable and complex. Fungal DNA extraction methods containing proteinase K and liquid nitrogen are widely used to break down the cell wall. However, these methods cause DNA loss during the extraction in Aspergillus species. In this study, on the contrary, the commonly used DNA extraction by means of ammonium hydroxide, which is generally used to break down chitin in DNA extraction of ticks and plants, is used. The efficiency of the cell wall lysis method from A. flavus with ammonium hydroxide was compared with methods containing proteinase K and liquid nitrogen. For this purpose, DNA extraction of A. flavus was tried using three different methods. As a result, the cell wall of A. flavus was lysed using ammonium hydroxide in this study. The obtained DNA's quality, concentration, and PCR performance were sufficient. This method has been evaluated as a faster, more straightforward, and more economical alternative. [ABSTRACT FROM AUTHOR]

[Objective] To augment the expression level of protease K and pave the way for the implementation of efficient enzymatic scaling technology, the co-expression of molecular chaperones was employed to enhance the secretion of the heterologous protease K in Pichia pastoris, and the mechanism underpinning its chlorine-free wool scale stripping efficacy has been meticulously investigated.[Metho]We used the Pichia pastoris expression system to have the tprK gene heterologously expressed. Then at the first time we analyzed the effects of over expressions of molecular chaperone Ssa1, Erj5, Sil1, Hac1, Kar2, Lhs1, and Ydj1, which affect protein folding and quality control, on the expressions and enzymatic activities of TPRK. Also we analyzed the effect of TPRK treatment on wool fibers.[Result] TPRK is expressed in P. pastoris GS115, and its optimal reaction conditions are 65℃ and pH 9.0, with good thermal stability and pH stability. The enzyme activities of recombinant strains overexpressing the ssa1, hac1, erj5, and sil1 genes increased by 36.8%, 20.0%, 17.7%, and 14.8%, respectively. High density fermentation was carried out in a 5 L fermentation tank, and after 72 h of induction, the TPRK enzyme activity reached 77 471.99 U/mL. In the application of wool hydrolysis, TPRK hydrolyzed the inner layer of wool scales to gradually peel them off, achieving a peeling effect. The optimal hydrolysis conditions are: TPRK addition of 300 U/mL, reaction temperature of 55℃, pH 9.0, and reaction time of 2 h.[Conclusion] Overexpression of molecular chaperone Ssa1 can effectively increase the expression of TPRK. Using this TPRK to process wool fibers can effectively remove the wool scale layer, while causing less damage to the core cortex layer of wool. This lays the foundation for the promotion and application of protease K in wool scale removal technology. [ABSTRACT FROM AUTHOR]

The data referring to the value of direct immunofluorescence on formalin-fixed, paraffin-embedded tissue (IF-Paraffin) in the diagnosis of renal diseases is controversial. The aim of this study was to investigate whether renal biopsies evaluated by routine immunofluorescence on frozen tissue (IF-Frozen) would yield adequate findings to confirm diagnoses when the IF-Paraffin technique was applied. To show immunoglobulins, complement components, and light chains, 55 native renal biopsies were subjected to IF-Paraffin and IF-Frozen staining techniques. The intensity of the staining was compared, and the sensitivity and specificity were calculated. The IF-Paraffin technique showed a sensitivity of 89%, 81%, 86%, 30%, 71%, 60%, and 77% for IgG, IgM, IgA, C1q, C3, κ, and λ, respectively, whereas specificity was 91%, 100%, 100%, 96%, 94%, 98%, and 100%. It showed diagnostic findings in 87% of cases. Compared to cases that had both IF-Paraffin and IF-Frozen staining techniques, 43 of 55 showed either equal intensity for the diagnostic immunoglobulin/complement or a little difference. Direct immunofluorescence on formalin-fixed, paraffin-embedded sections cannot replace immunofluorescence on frozen sections in the assessment of renal biopsies, but may be a "salvage technique" when frozen tissue is insufficient or unavailable and must be interpreted with great caution. [ABSTRACT FROM AUTHOR]

One of the most important foods in the world is milk, due to its nutrients and composition. Milk also contains lactic acid bacteria (LABs), which can produce antimicrobial compounds and organic acids; an essential factor in developing different methods to control other bacteria. Another critical factor is that several species of LABs are considered potential probiotics, offering several health benefits. This study aimed to isolate, identify LABs, characterize the antimicrobial susceptibility profile, and evaluate the inhibition potential against Staphylococcus aureus (ATCC 25923), Listeria monocytogenes (ATCC 7644), Escherichia coli (ATCC 25922) and Salmonella Typhimurium (ATCC 14028); as well as verifying the acidification capacity, survival of simulated in vitro gastrointestinal conditions and identification of virulence genes and bacteriocin-producing genes. The study was carried out with LABs previously isolated from milk of cows with clinical mastitis, low and high (Somatic Cells Count) in herds from all regions of Brazil (South, Southeast, Central-West, North, and Northeast). One hundred and ten strains were selected for identification using the MALDI-TOF technique. The genera with the most significant spectrum of action were Lactococcus, Lactobacillus, and Pediococcus in the antimicrobial action test against pathogens, concluding that Lactobacillus showed resistance to several antibiotics. The analysis of antimicrobial action made it possible to verify the potential production of antimicrobial compounds by LABs against the primary pathogens of food origin. The most significant reduction in pH could be observed from 24 h to 48 h, ranging from 6.8 (0 h) to 4.74 (48 h) for Lactobacillus and Lactococcus. All isolates survived tolerance tests at pH=3.0 and bile salts (0.5 %), with survival rates ranging from 34.2 to 69.9 % at pH = 3.0 and above 82 % at 0.5 % bile salts. Lacotococcus and Lactobacillus isolates presented the plnEF and plnNC8 genes. This study contributes to the search for alternatives in the treatment of bacterial infections with a reduction in the use of antibiotics, which, due to the increase in bacterial resistance, is causing difficulties in treating diseases caused by these microorganisms. pH is an important parameter when it comes to food safety and conservation. Tolerance to acid stress and bile salts are important properties in evaluating probiotic potential and the ability to maintain high counts under these conditions. The presence of the plnEF and plnNC8 genes suggests bacteriocin production.

The Rnq1 protein is one of the best-studied yeast prions. It has a large potentially prionogenic C-terminal region of about 250 residues. However, a previous study indicated that only 40 C-terminal residues form a prion structure. Here, we mapped the actual and potential prion structures formed by Rnq1 and its variants truncated from the C-terminus in two [RNQ+] strains using partial proteinase K digestion. The location of these structures differed in most cases from previous predictions by several computer algorithms. Some aggregation patterns observed microscopically for the Rnq1 hybrid proteins differed significantly from those previously observed for Sup35 prion aggregates. The transfer of a prion from the full-sized Rnq1 to its truncated versions caused substantial alteration of prion structures. In contrast to the Sup35 and Swi1, the terminal prionogenic region of 72 residues was not able to efficiently co-aggregate with the full-sized Rnq1 prion. GFP fusion to the Rnq1 C-terminus blocked formation of the prion structure at the Rnq1 C-terminus. Thus, the Rnq1-GFP fusion mostly used in previous studies cannot be considered a faithful tool for studying Rnq1 prion properties. [ABSTRACT FROM AUTHOR]

Early and accurate detection of viruses in children might help prevent transmission and severe diseases. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS‐COV‐2) detection in children was evaluated using saliva specimens with a Proteinase K (PTK)‐based RNA preparation, as saliva collection is a simple and noninvasive procedure, even in young children, with fewer concerns about sample contamination. The saliva‐based PTK and the conventional paired nasopharyngeal aspiration (NPA)‐based detection methods were compared between COVID‐19‐positive and ‐negative children. In addition, the detection rate for SARS‐COV‐2 and the difference between admission and discharge by the saliva‐based PTK method was tested in COVID‐19 patients. The diagnostic accuracy of the saliva‐based PTK method was 98.8% compared to NP swab‐based reverse transcriptase polymerase chain reaction. Saliva samples showed high sensitivity (94.1%) and specificity (100%) when using the PTK method. Furthermore, the saliva‐based PTK method significantly reduced the test processing time by 2 h. Notably, Ct values at discharge increased in saliva samples compared with those at admission, which might indicate patients' clinical conditions or virus activity. In conclusion, the saliva‐based PTK implemented in this study streamlines RNA extraction, making the process faster, safer, and more cost‐effective, demonstrating that this method is a rapid and reliable diagnostic tool for SARS‐CoV‐2 detection in children. [ABSTRACT FROM AUTHOR]

Background: Formalin-fixed paraffin-embedded (FFPE) specimen archives are a valuable source of sample material for molecular biological analysis. However, the DNA isolated from FFPE samples is usually low in concentration and fragmented. Thus, it is necessary to optimize the FFPE DNA extraction protocol to obtain the best results. Proteinase K incubation is undoubtedly crucial in DNA extraction procedures, but this step is often not well explained in the manufacturer’s manual. Purpose: This study aimed to find the optimal duration for proteinase K incubation protocols to achieve the highest DNA yields. Methods: Fifteen paraffin blocks of Oral Squamous Cell Carcinoma (OSCC) specimens were obtained, and the cancerous areas were microdissected into smaller cuts for DNA extraction. The samples were randomly divided into three groups (n=5) and subjected to three different proteinase K incubation protocols: one-hour incubation at 56ºC as per the manufacturer’s instructions (Group I), 24-hour incubation at 56ºC (Group II), and 48 hours at room temperature with an additional four hours at 56ºC (Group III). The extracted DNA was then quantified using a Nanodrop spectrophotometer. The recorded data were analyzed using ANOVA-LSD. Results: The highest DNA concentration was found in Group III (107.74 ± 41.92), which was significantly higher compared to Group II (59.46 ± 30.32) and Group I (6.46 ± 1.97) (p

Keratin-containing wastes (feathers and bristles) make up the majority of all animal wastes, a fast-growing agricultural sector that annually increases production volume. The development of methods for environmentally friendly processing of such waste to obtain valuable resources (amino acids and oligopeptides) is an important task in modern science, including biotechnology. A way to dispose of livestock waste according to the current trends of the development of green economy is the use of microorganisms and their enzymes. The possibility of production of keratinolytic enzymes of Aspergillus clavatus VKPM F-1593 has been studied under submerged conditions with different sources of nitrogen and carbon, including the growth of the producer on animal waste. The highest target activity (96.1 E) was achieved using mixed sources of carbon and nitrogen: an inorganic source (sodium nitrate), an easily digestible organic source (fish meal hydrolysate) and a hard-to-get organic source (ground chicken feather). The variation in the content of different substrates in the fermentation media has made it possible not only to regulate the level of proteolytic activity, but also to reach the peak of producer activity on different days of cultivation. The specific activity of the keratinase of A. clavatus VKPM F-1593 (pI 9.3) with respect to different protein substrates has been compared with the activity of the commercial proteinase K preparation. Both enzymes have shown a similar level of activity with respect to most of the used substrates. However, the protease of A. clavatus VKPM F-1593 has had a greater total proteolytic activity, which confirms the prospective use of this culture for the biodegradation of animal waste. [ABSTRACT FROM AUTHOR]

Abstract Background Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. Results To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. Conclusions RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

Ming-Jun Tsai,1,2 Cheng-Yu Lin,3 Jiří Trousil,4 Calvin T Sung,5 Mei-Hua Lee,3 Jia-You Fang,3,6,7 Shih-Chun Yang8 1School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan; 2Jhong Siao Urological Hospital, Kaohsiung, Taiwan; 3Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan; 4Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, Czech Republic; 5Department of Dermatology, University of California, Irvine, CA, USA; 6Research Center for Food and Cosmetic Safety and Research Center for Chinese Herbal Medicine, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan; 7Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 8Department of Microbiology, Soochow University, Taipei, TaiwanCorrespondence: Jia-You Fang, Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan, Taoyuan, 333, Taiwan, Email fajy@mail.cgu.edu.tw Shih-Chun Yang, Department of Microbiology, Soochow University, 70 Linhsi Road, Shihlin, Taipei, 111, Taiwan, Email scyang@scu.edu.twBackground: Simultaneous anti-Cutibacterium acnes and anti-inflammatory actions are highly beneficial in treating acne vulgaris. In this study, we present novel anti-acne nanovesicles based on liposomes loaded with proteinase K (PK), retinoic acid (RA), and soyaethyl morpholinium ethosulfate (SME) to achieve an effective and safe treatment.Materials and Methods: This study examined in vitro planktonic and biofilm C. acnes elimination, as well as the keratinocyte proliferation suppression by liposomes. The multifunctional liposomes for treating C. acnes in mice were also evaluated.Results: We acquired multifunctional liposomes with a size of 71 nm and zeta potential of 31 mV. The antimicrobial activity of SME was enhanced after liposomal encapsulation according to the reduction of minimum bactericidal concentration (MBC) by 6-fold. The multifunctional liposomes exhibited a synergistically inhibitory effect on biofilm C. acnes colonization compared with the liposomes containing PK or those containing SME individually. The adhesive bacterial colony in the microplate was lessened by 62% after multifunctional liposome intervention. All liposomal formulations tested here demonstrated no cytotoxicity against the normal keratinocytes but inhibited C. acnes-stimulated cell hyperproliferation. The in vitro scratch assay indicated that the liposomal RA—but not free RA—restrained keratinocyte migration. The animal study showed that free RA combined with SME and multifunctional nanovesicles had a similar effect on diminishing C. acnes colonies in the skin. On the other hand, liposomes exhibited superior performance in recovering the impaired skin barrier function than the free control. We also found that RA-loaded nanovesicles had greater skin tolerability than free RA.Conclusion: The cationic liposomes containing dual PK and RA represented a potential treatment to arrest bacterial infection and associated inflammation in acne.Keywords: liposome, acne, Cutibacterium acnes, proteinase K, retinoic acid, skin delivery

Background: Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. Results: To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. Conclusions: RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions. [ABSTRACT FROM AUTHOR]

Mitochondria play a principal role in metabolism, and mitochondrial respiration is an important process for producing adenosine triphosphate. Recently, we showed the possibility that the muscle-specific protein myoglobin (Mb) interacts with mitochondrial complex IV to augment the respiration capacity in skeletal muscles. However, the precise mechanism for the Mb-mediated upregulation remains under debate. The aim of this study was to ascertain whether Mb is truly integrated into the mitochondria of skeletal muscle and to investigate the submitochondrial localization. Isolated mitochondria from rat gastrocnemius muscle were subjected to different proteinase K (PK) concentrations to digest proteins interacting with the outer membrane. Western blotting analysis revealed that the PK digested translocase of outer mitochondrial membrane 20 (Tom20), and the immunoreactivity of Tom20 decreased with the amount of PK used. However, the immunoreactivity of Mb with PK treatment was better preserved, indicating that Mb is integrated into the mitochondria of skeletal muscle. The mitochondrial protease protection assay experiments suggested that Mb localizes within the mitochondria in the inner membrane from the intermembrane space side. These results strongly suggest that Mb inside muscle mitochondria could be implicated in the regulation of mitochondrial respiration via complex IV.

The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy to collect electron diffraction data and determine high-resolution structures from very thin micro- and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A2A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.

The Rnq1 protein is one of the best-studied yeast prions. It has a large potentially prionogenic C-terminal region of about 250 residues. However, a previous study indicated that only 40 C-terminal residues form a prion structure. Here, we mapped the actual and potential prion structures formed by Rnq1 and its variants truncated from the C-terminus in two [RNQ+] strains using partial proteinase K digestion. The location of these structures differed in most cases from previous predictions by several computer algorithms. Some aggregation patterns observed microscopically for the Rnq1 hybrid proteins differed significantly from those previously observed for Sup35 prion aggregates. The transfer of a prion from the full-sized Rnq1 to its truncated versions caused substantial alteration of prion structures. In contrast to the Sup35 and Swi1, the terminal prionogenic region of 72 residues was not able to efficiently co-aggregate with the full-sized Rnq1 prion. GFP fusion to the Rnq1 C-terminus blocked formation of the prion structure at the Rnq1 C-terminus. Thus, the Rnq1-GFP fusion mostly used in previous studies cannot be considered a faithful tool for studying Rnq1 prion properties.

ObjectivesLipoarabinomannan (LAM), an abundant cell wall glycolipid of mycobacteria including Mycobacterium tuberculosis (Mtb), is a promising TB diagnostic marker. The current commercially available urine LAM assays are not sufficiently sensitive, and more novel detection strategies are urgently needed to fill the current diagnostic gap.MethodsA proteinase K-pretreated Concanavalin A (ConA)-based ELISA assay was developed. Diagnostic performance was assessed by several bacterial strains and clinical urine samples.ResultsThe limit of detection (LoD) of the assay against ManLAM was 6 ng/ml. The assay reacted strongly to Mtb H37Rv and M. bovis BCG, intermediately to M. smegmatis mc2155, and weakly to four non-mycobacteria pathogens. This method could distinguish TB patients from healthy controls (HCs) and close contacts (CCs) in 71 urine samples treated with proteinase K, which increases urine LAM antibody reactiveness. In TB+HIV+ and TB+HIV− patients, the sensitivity was 43.8 and 37.5%, respectively, while the specificity was 100.0%. The areas under ROC curves (AUCs) were 0.74 and 0.82, respectively.ConclusionThis study implies that ConA can be paired with antibodies to detect LAM. Proteinase K treatment could effectively enhance the sensitivity by restoring the reactiveness of antibodies to LAM.

The regulation and characterization of nanomaterials in foods are of great interest due to the potential risks associated with their exposure and the increasing number of applications where they are used within the food industry. One factor limiting the scientifically rigorous regulation of nanoparticles in foods is the lack of standardized procedures for the extraction of nanoparticles (NPs) from complex matrices without alteration of their physico-chemical properties. To this end, we tested and optimized two sample preparation approaches (enzymatic- and alkaline-based hydrolyses) in order to extract 40 nm of Ag NP, following their equilibration with a fatty ground beef matrix. NPs were characterized using single particle inductively coupled plasma mass spectrometry (SP-ICP-MS). Fast sample processing times (<20 min) were achieved using ultrasonication to accelerate the matrix degradation. NP losses during the sample preparation were minimized by optimizing the choice of enzymes/chemicals, the use of surfactants, and the product concentration and sonication. The alkaline approach using TMAH (tetramethylammonium hydroxide) was found to have the highest recoveries (over 90%); however, processed samples were found to be less stable than the samples processed using an enzymatic digestion based upon pork pancreatin and lipase (≈60 % recovery). Low method detection limits (MDLs) of 4.8 × 106 particles g−1 with a size detection limit (SDL) of 10.9 nm were achieved for the enzymatic extraction whereas an MDL of 5.7 × 107 particles g−1 and an SDL of 10.5 nm were obtained for the alkaline hydrolysis. [ABSTRACT FROM AUTHOR]

We present a spectrophotometric-based assay to identify enzymes that degrade commercially available bioplastics. Bioplastics comprise aliphatic polyesters with hydrolysis-susceptible ester bonds and are proposed as a replacement for petroleum-based plastics that accumulate in the environment. Unfortunately, many bioplastics can also persist in environments including seawater and waste centers. Our assay involves an overnight incubation of candidate enzyme(s) with plastic, followed by A610 spectrophotometry using 96-well plates to quantify both a reduction in residual plastic and the liberation of degradation by-products. We use the assay to show that Proteinase K and PLA depolymerase, two enzymes that were previously shown to degrade pure polylactic acid plastic, promote a 20–30% breakdown of commercial bioplastic during overnight incubation. We validate our assay and confirm the degradation potential of these enzymes with commercial bioplastic using established mass-loss and scanning electron microscopy methods. We show how the assay can be used to optimize parameters (temperature, co-factors, etc.) to enhance the enzyme-mediated degradation of bioplastics. The assay endpoint products can be coupled with nuclear magnetic resonance (NMR) or other analytical methods to infer the mode of enzymatic activity. Overall, the screening capacity of the spectrophotometric-based assay was demonstrated to be an accurate method to identify bioplastic-degrading enzymes. [ABSTRACT FROM AUTHOR]

[Display omitted] Injectable hydrogels are important in situ forming implants for tissue regeneration at damaged sites. Understanding the behavior of these systems in a complex in vivo environment remains a challenge. Ultrathin films as 2D model systems are expected to provide fundamental insights into formation and (bio)degradation at material-liquid interfaces, and are also applicable as bioresponsive coatings. Hydrogel ultrathin films are prepared by covalently cross-linking four-arm PEG macromers with maleimide end-groups (PEG4MAL) at alkaline pH using two different types of dithiol-bearing cross-linkers – thio-depsipeptide (TDP) or 3,6-Dioxa-1,8-octanedithiol (DODT). This thiol-Michael addition "click" reaction is carried out at the air-water interface using the Langmuir technique. Morphological observation in real time is carried out by Brewster angle microscopy (BAM) and in coatings using atomic force microscopy (AFM). Stability against enzymatic and oxidative degradation is evaluated in the same setup. Non-cross-linked PEG or PEG incubated with cross-linkers at slightly acidic pH desorbs from the interface over time. Cross-linking of PEG at alkaline pH renders 2D hydrogel networks (thickness <1 nm) that are stable against desorption. They are easily transferrable onto solid mica surfaces, forming homogenous coatings as revealed by AFM. The type of dithiol cross-linker used to form the branching centers influences the degradability of these 2D hydrogel networks in the presence of lipase, peroxides, or bases. For example, enzymatic degradation of the 2D hydrogel networks can be switched "on" or "off" depending on the cleavable sites in the cross-linkers. [ABSTRACT FROM AUTHOR]

Helicobacter pylori, a WHO class I carcinogen, is one of the most successful human pathogens colonizing the stomach of over 4.4 billion of the world's population. Antibiotic therapy represents the best solution but poor response rates have hampered the elimination of H. pylori. A growing body of evidence suggests that H. pylori forms biofilms, but the role of this growth mode in infection remains elusive. Here, we demonstrate that H. pylori cells within a biofilm are tolerant to multiple antibiotics in a manner that depends partially on extracellular proteins. Biofilm-forming cells were tolerant to multiple antibiotics that target distinct pathways, including amoxicillin, clarithromycin, and tetracycline. Furthermore, this tolerance was significantly dampened following proteinase K treatment. These data suggest that H. pylori adapts its phenotype during biofilm growth resulting in decreased antibiotic susceptibility but this tolerance can be partially ameliorated by extracellular protease treatment.

Biofilms of sporeformers found in the dairy industry are the major contaminants during processing, as they withstand heat and chemical treatment that are used to control microbes. The present work is aimed to remove these resistant forms of bacterial community (biofilm) present in dairy production lines using ecofriendly agents based on proteinase K (Prot-K) coupled with Zinc oxide nanoparticles (ZnO-NPs). Some metal/metal oxide (Ag, CuO and ZnO) NPs were prepared microbially, and ZnO-NPs were characterized as the most effective ones among them. The produced ZnO-NPs were 15–25 nm in size with spherical shape, and FTIR analysis confirmed the presence of proteins and alkanes surrounding particles as capping agents. Application of Prot-K for eradication (removal) of a model biofilm of mixed sporeformers on food-grade stainless steel resulted in an 83% reduction in the absorbance of crystal violet-stained biofilm. When Prot-K was mixed with the biosynthesized NPs ZnO_G240, the reduction increased to 99.19%. This finding could contribute to an efficient cleaning approach combined with CIP to remove the recalcitrant biofilms in dairy production lines.

Application of thermoplastic fiber-reinforced lightweight composite materials provides a wide range of advantages that are of particular importance for the mobility sector. UD tapes composed of unidirectionally (UD) oriented inorganic fibers embedded in a thermoplastic matrix represent light-weight materials with high tensile strength. This publication addresses recycling aspects of novel UD tape made of a combination of basalt fibers and different PLA (polylactic acid) formulations. The kinetics of enzyme-based separation of polymer from the fiber were investigated. Different types of UD tapes with a thickness of 270–290 µm reinforced with basalt fiber weight ratios ranging between 51 and 63% were incubated at 37 °C in buffer solution (pH 7.4) containing proteinase K. The influence of enzyme concentration, tape weight per incubation tube, proteinase K activators, and tape types on the rate of enzymatic decomposition was investigated. Enzyme activity was measured by analyzing lactate concentration with lactate dehydrogenase and by measuring weight loss of the composite material. The rate of lactate release increased in the first 30 min of incubation and remained stable for at least 90 min. Weight loss of 4% within 4 h was achieved for a tape with 56% (w/w) fiber content. For a sample with a surface area of 3 cm2 in a buffer volume of 10 mL, the rate of lactate release as a function of enzyme concentration reached saturation at 300 µg enzyme/mL. With this enzyme concentration, the rate of lactate release increased in a linear manner for tape surface areas between 1 and 5 cm2. Four tapes with different PLA types were treated with the enzyme for 17 h. Weight loss ranged between 7 and 24%. Urea at a concentration of 0.5% (w/v) increased lactate release by a factor of 9. Pretreatment of tapes in alkaline medium before enzymatic degradation increased weight loss to 14% compared to 5% without pretreatment. It is concluded that enzymatic PLA hydrolysis from UD tapes is a promising technology for the release of basalt fibers after alkaline pretreatment or for the final cleaning of basalt fibers.

Myricetin (MYR) and myricitrin (MYT) are well recognized for their nutraceutical value, such as antioxidant, hypoglycemic, and hypotensive effects. In this work, fluorescence spectroscopy and molecular modeling were adopted to investigate the conformational and stability changes of proteinase K (PK) in the presence of MYR and MYT. The experimental results showed that both MYR and MYT could quench fluorescence emission via a static quenching mechanism. Further investigation demonstrated that both hydrogen bonding and van der Waals forces play significant roles in the binding of complexes, which is consistent with the conclusions of molecular modeling. Synchronous fluorescence spectroscopy, Förster resonance energy transfer, and site-tagged competition experiments were performed to prove that the binding of MYR or MYT to PK could alter its micro-environment and conformation. Molecular docking results revealed that either MYR or MYT spontaneously interacted with PK at a single binding site via hydrogen bonding and hydrophobic interactions, which is consistent with the results of spectroscopic measurements. A 30 ns molecular dynamics simulation was conducted for both PK-MYR and PK-MYT complexes. The calculation results showed that no large structural distortions or interaction changes occurred during the entire simulation time span. The average RMSD changes of PK in PK-MYR and PK-MYT were 2.06 and 2.15 Å, respectively, indicating excellent stability of both complexes. The molecular simulation results suggested that both MYR and MYT could interact with PK spontaneously, which is in agreement with spectroscopic results. This agreement between experimental and theoretical results indicates that the method herein could be feasible and worthwhile for protein–ligand complex studies. [ABSTRACT FROM AUTHOR]

Application of thermoplastic fiber-reinforced lightweight composite materials provides a wide range of advantages that are of particular importance for the mobility sector. UD tapes composed of unidirectionally (UD) oriented inorganic fibers embedded in a thermoplastic matrix represent light-weight materials with high tensile strength. This publication addresses recycling aspects of novel UD tape made of a combination of basalt fibers and different PLA (polylactic acid) formulations. The kinetics of enzyme-based separation of polymer from the fiber were investigated. Different types of UD tapes with a thickness of 270-290 μm reinforced with basalt fiber weight ratios ranging between 51 and 63% were incubated at 37 °C in buffer solution (pH 7.4) containing proteinase K. The influence of enzyme concentration, tape weight per incubation tube, proteinase K activators, and tape types on the rate of enzymatic decomposition was investigated. Enzyme activity was measured by analyzing lactate concentration with lactate dehydrogenase and by measuring weight loss of the composite material. The rate of lactate release increased in the first 30 min of incubation and remained stable for at least 90 min. Weight loss of 4% within 4 h was achieved for a tape with 56% (w/w) fiber content. For a sample with a surface area of 3 cm² in a buffer volume of 10 mL, the rate of lactate release as a function of enzyme concentration reached saturation at 300 μg enzyme/mL. With this enzyme concentration, the rate of lactate release increased in a linear manner for tape surface areas between 1 and 5 cm². Four tapes with different PLA types were treated with the enzyme for 17 h. Weight loss ranged between 7 and 24%. Urea at a concentration of 0.5% (w/v) increased lactate release by a factor of 9. Pretreatment of tapes in alkaline medium before enzymatic degradation increased weight loss to 14% compared to 5% without pretreatment. It is concluded that enzymatic PLA hydrolysis from UD tapes is a promising technology for the release of basalt fibers after alkaline pretreatment or for the final cleaning of basalt fibers. [ABSTRACT FROM AUTHOR]

Optimized and reliable DNA/RNA extraction protocols are a vital tool in clinical practice in the context of molecular testing. Here, we present our successful attempt to enhance the quantity of RNA isolated from clinical specimens, which we originally found challenging (breast and testis). We compared several purification methods with special focus on two AllPrep system-based protocols (QIAGEN). Our data suggest that addition of proteinase K may markedly increase RNA and, in some cases, also DNA yield. The extraction kit used, AllPrep DNA/RNA/miRNA universal kit, provides RNA amounts comparable with the phenol-chloroform extraction method; however, part of the final yield consisted of small RNAs, visible as a thick band in the bioanalyzer gel-like image (5S peak). The 5S peak, albeit in some cases dominating the bioanalyzer image, plays only a small role in RT-qPCR analysis, and Qubit or NanoDrop measurements can still be used as a reliable estimate of starting amounts of mRNA for downstream analyses. In conclusion, we showed that implementing a protocol containing a step of proteinase K digestion markedly increases RNA yield. The AllPrep DNA/RNA/miRNA Universal Kit can be successfully used for simultaneous extraction of DNA and total RNA, irrespective of the tissue of origin, and does not present inconveniences related to phenol-chloroform extraction. [ABSTRACT FROM AUTHOR]

Research background. Various protocols for enzymatic hydrolysis of fish by-products are increasingly tested to ensure value-added products with functional and biological properties important for food, cosmetic and medical applications. In addition, they attempt to minimize waste from industrial processing and environmental requirements. This study aims to establish an efficient protocol based on two-step enzymatic hydrolysis of freshwater fish skin and to evaluate the effect of resulting bioactive peptides on free radical scavenging, redox balance and regulation of fibroblast proliferation and migration. Experimental approach. Pepsin-soluble collagen extracted from silver carp (Hypophthalmichthys molitrix) skin was hydrolyzed by proteinase K at specific sites under controlled conditions. The molecular mass of ultrafiltration permeate was determined by gradient electrophoresis and gel filtration chromatography. The biological activity of intermediate and small size bioactive peptides was evaluated in experimental models in vitro mimicking oxidative stress and skin wound conditions. Results and conclusions. Extracted fish collagen was hydrolysed using proteinase K, the most efficient enzyme for the cleavage of the primary structure of the molecule, as previously found in silico. Established optimal conditions increased the enzyme specificity and the process yield. Bioactive peptides exerted significantly higher scavenging activity on free stable radicals and hydroxyl radicals often found in vivo, compared to fish collagen. They stimulated fibroblast metabolism in a dose-dependent manner and up-regulated cell migration in a scratch wound model. Pretreatment of fibroblasts with induced oxidative stress using optimal concentrations of fish peptides prevented the increase of reactive oxygen species production. In conclusion, bioactive peptides from carp skin demonstrated valuable properties of maintaining redox balance and skin wound healing process improvement, which indicated further potential applications in the development of pharmaceutical and nutraceutical formulations. Novelty and scientific contribution. In this study the enzymatic hydrolysis was applied to isolated protein, in contrast to previous studies using waste tissue with variable composition. Recovered bioactive peptides acted not only as antioxidant agents, but also as regulators of oxidative stress and wound healing processes in skin cell models. Their nutritional and cosmetic application is recommended in novel formulations fighting skin ageing phenomena.

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W–S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24. • In the absence of Ca2+, a mutant Proteinase K exhibited an activity approximately 9.2-fold higher than that of wild-type. • The mutant exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. • The introduction of hydrogen bond and π-π stacking at Ca2+ binding sites resulted in an increased in activity and stability. [ABSTRACT FROM AUTHOR]

Jia-You Fang,1– 3 Wei-Ling Chou,4 Chwan-Fwu Lin,2,3,5 Calvin T Sung,6 Ahmed Alalaiwe,7 Shih-Chun Yang1,8 1Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan; 2Research Center for Food and Cosmetic Safety and Research Center for Chinese Herbal Medicine, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan; 3Department of Anesthesiology, Chang Gung Memorial Hospital, Kweishan, Taoyuan, Taiwan; 4Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung, Taiwan; 5Department of Cosmetic Science, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan; 6Department of Dermatology, University of California, Irvine, CA, USA; 7Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al Kharj, Saudi Arabia; 8Department of Cosmetic Science, Providence University, Taichung, TaiwanCorrespondence: Shih-Chun YangPharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, TaiwanEmail phageyang@gmail.comBackground: The biofilm produced by Cutibacterium acnes is a major infection threat for skin and implanted catheters. Nanoparticles provide a new approach to eradicate biofilms. The present study evaluated the capability of cationic liposomes loaded with DNase I (DNS) and proteinase K (PK) to remove preformed C. acnes biofilms.Methods: DNS and PK were able to target and disassemble the biofilm by degrading extracellular polymer substances (EPS). Soyaethyl morpholinium ethosulfate (SME) was used to render a positive charge and enhance the antibacterial activity of the liposomes.Results: The cationic liposomes containing enzymes yielded monodisperse nanovesicles ranging between 95 and 150 nm. The entrapment efficiency of the enzymes in the liposomes achieved a value of 67– 83%. All liposomal formulations suppressed planktonic C. acnes growth at a minimum inhibitory concentration (MIC) equal to the free SME in the solution. The enzyme in the liposomal form inhibited biofilm growth much better than that in the free form, with the dual enzyme-loaded liposomes demonstrating the greatest inhibition of 54% based on a crystal violet assay. The biofilm-related virulence genes PA380 and PA1035 were downregulated by the combined enzymes in the liposomes but not the individual DNS or PK. Scanning electron microscopy (SEM) and confocal microscopy displayed reduced C. acnes aggregates and biofilm thickness by the liposomal system. The liposomes could penetrate through about 85% of the biofilm thickness. The in vitro pig skin permeation also showed a facile delivery of liposomes into the epidermis, deeper skin strata, and hair follicles. The liposomes exhibited potent activity to eliminate C. acnes colonization in mouse skin and catheters in vivo. The colony-forming units (CFUs) in the catheter treated with the liposomes were reduced by 2 logs compared to the untreated control.Conclusion: The data suggested a safe application of the enzyme-loaded cationic liposomes as antibacterial and antibiofilm agents.Keywords: liposomes, cationic surfactant, DNase I, proteinase K, Cutibacterium acnes, biofilm

ABSTRACT: This research was conducted to efficiently extract Fish Protein Hydrolysate (FPH) from Secutor insidiator using papain and proteinase K enzymes. The degree of hydrolysis was found to be 0.8 and 0.9 for proteinase and papain respectively. The proximate analysis of the prepared FPH indicated the levels of Moisture (5.95% and 4.86%), Protein (92.14% and 89.24%), Fat (0.6% and 1.02%) and Ash (6.23 and 4.79) for papain and proteinase respectively. High solubility index (75 – 80%) was observed for the pH levels of 2, 6, 7, 8 & 10 and low solubility index of 31.75% and 32.18% was observed at pH 4 for proteinase and papain respectively. The molecular weight of the hydrolysates was estimated using SDS-PAGE method. Both samples (FPH1 and FPH2) produced bands at a molecular weight of 15 kDa and the second bands lies in between 25 kDa and 75 kDa. The foaming properties were highest at pH 2 (80 – 85%) and lowest at pH 4 (10–20%). The water binding capacity was observed to be at the highest at 0.2% concentration and there was a gradual decrease from 0.4 to 1.0% concentration of FPH. The oil binding capacity was found to be high at concentrations 0.2 and 1.0% and emulsifying properties were observed to be at the maximum in pH 2 and 10. Among the two Fish Protein Hydrolysates (FPHs) prepared, FPH1 (proteinase) has comparatively better improved functional properties than FPH2 (papain).

Research background. Various protocols for enzymatic hydrolysis of fish by-products are increasingly tested to ensure value-added products with functional and biological properties important for food, cosmetic and medical applications. In addition, they attempt to minimize waste from industrial processing and environmental requirements. This study aims to establish an efficient protocol based on two-step enzymatic hydrolysis of freshwater fish skin and to evaluate the effect of resulting bioactive peptides on free radical scavenging, redox balance and regulation of fibroblast proliferation and migration. Experimental approach. Pepsin-soluble collagen extracted from silver carp (Hypophthalmichthys molitrix) skin was hydrolyzed by proteinase K at specific sites under controlled conditions. The molecular mass of ultrafiltration permeate was determined by gradient electrophoresis and gel filtration chromatography. The biological activity of intermediate and small size bioactive peptides was evaluated in experimental models in vitro mimicking oxidative stress and skin wound conditions. Results and conclusions. Extracted fish collagen was hydrolysed using proteinase K, the most efficient enzyme for the cleavage of the primary structure of the molecule, as previously found in silico. Established optimal conditions increased the enzyme specificity and the process yield. Bioactive peptides exerted significantly higher scavenging activity on free stable radicals and hydroxyl radicals often found in vivo, compared to fish collagen. They stimulated fibroblast metabolism in a dose-dependent manner and up-regulated cell migration in a scratch wound model. Pretreatment of fibroblasts with induced oxidative stress using optimal concentrations of fish peptides prevented the increase of reactive oxygen species production. In conclusion, bioactive peptides from carp skin demonstrated valuable properties of maintaining redox balance and skin wound healing process improvement, which indicated further potential applications in the development of pharmaceutical and nutraceutical formulations. Novelty and scientific contribution. In this study the enzymatic hydrolysis was applied to isolated protein, in contrast to previous studies using waste tissue with variable composition. Recovered bioactive peptides acted not only as antioxidant agents, but also as regulators of oxidative stress and wound healing processes in skin cell models. Their nutritional and cosmetic application is recommended in novel formulations fighting skin ageing phenomena. [ABSTRACT FROM AUTHOR]

The Hancornia speciosa latex has shown angiogenic activity. Angiogenesis plays a major role in wound healing, and materials that stimulate this process could be used to develop drugs. This study aimed to explain the role of proteins in the H. speciosa serum fraction latex in angiogenesis. Hence, this material was treated with proteinase K and the proteins were inactivated. After protein inactivation, angiogenic activity was assessed with the chick chorioallantoic membrane assay. The result showed that the proteins in the serum fraction are responsible for angiogenic activity. Then, the total protein content in the serum fraction and its enzymatic activity were investigated. The low protein content observed in the H. speciosa serum fraction latex suggests that this biomaterial could be used to develop new drugs with a hypoallergenic response. Despite the low protein content, there was a significant enzymatic activity of at least three enzymes in the serum fraction latex: β-1,3 glucanase, β-glucosidase, and proteases. These enzymes seem to influence the healing process, assisting debridement, extracellular matrix remodeling, and collagen deposition, and decreasing the chances of contamination by microorganisms. In conclusion, the enzymes in the H. speciosa serum latex are associated with the angiogenic activity of this biomaterial and may be used to assist the wound healing process.

Amyloidoses are caused by the deposition of amyloid fibrils ascribed to protein misfolding. In this study, we examined the antiamyloidogenic and antioxidative activities of quercetin, a plant flavonol from the flavonoid group of polyphenols, on mouse prion protein (moPrP) with biophysical approaches. As the results show, quercetin binds to the C-terminal region of moPrP, and quercetin binding does not affect the structure of moPrP. However, quercetin binding accelerates moPrP fibrillation and changes the structure of moPrP fibrils. Unlike typical prion fibrils, quercetin-bound fibrils are sensitive to proteinase K and are loosely structured. Moreover, due to high antioxidant activity of flavonoid, quercetin-bound fibrils lack imbalance of free radicals and, therefore, they are nontoxic towards neuroblastoma cells. The quercetin shows its uniqueness from typical antiamyloidogenic drugs which either suppress the development of amyloid or eliminate formed amyloids. Quercetin binding converts moPrP into protease-sensitive and non-cytotoxic fibrils. This work provides a powerful resolution in the advancement of antiamyloidogenic treatment.

The regulation and characterization of nanomaterials in foods are of great interest due to the potential risks associated with their exposure and the increasing number of applications where they are used within the food industry. One factor limiting the scientifically rigorous regulation of nanoparticles in foods is the lack of standardized procedures for the extraction of nanoparticles (NPs) from complex matrices without alteration of their physico-chemical properties. To this end, we tested and optimized two sample preparation approaches (enzymatic- and alkaline-based hydrolyses) in order to extract 40 nm of Ag NP, following their equilibration with a fatty ground beef matrix. NPs were characterized using single particle inductively coupled plasma mass spectrometry (SP-ICP-MS). Fast sample processing times (6 particles g−1 with a size detection limit (SDL) of 10.9 nm were achieved for the enzymatic extraction whereas an MDL of 5.7 × 107 particles g−1 and an SDL of 10.5 nm were obtained for the alkaline hydrolysis.

We present a spectrophotometric-based assay to identify enzymes that degrade commercially available bioplastics. Bioplastics comprise aliphatic polyesters with hydrolysis-susceptible ester bonds and are proposed as a replacement for petroleum-based plastics that accumulate in the environment. Unfortunately, many bioplastics can also persist in environments including seawater and waste centers. Our assay involves an overnight incubation of candidate enzyme(s) with plastic, followed by A610 spectrophotometry using 96-well plates to quantify both a reduction in residual plastic and the liberation of degradation by-products. We use the assay to show that Proteinase K and PLA depolymerase, two enzymes that were previously shown to degrade pure polylactic acid plastic, promote a 20–30% breakdown of commercial bioplastic during overnight incubation. We validate our assay and confirm the degradation potential of these enzymes with commercial bioplastic using established mass-loss and scanning electron microscopy methods. We show how the assay can be used to optimize parameters (temperature, co-factors, etc.) to enhance the enzyme-mediated degradation of bioplastics. The assay endpoint products can be coupled with nuclear magnetic resonance (NMR) or other analytical methods to infer the mode of enzymatic activity. Overall, the screening capacity of the spectrophotometric-based assay was demonstrated to be an accurate method to identify bioplastic-degrading enzymes.

The enzymatic degradation of poly(lactic acid) was catalyzed with Proteinase K and the effect of various factors on the rate of degradation was analyzed quantitatively with the help of appropriate kinetic models. The Michaelis-Menten model was modified for the purpose by considering the heterogeneous nature of the reaction and the denaturation of the enzyme. The results proved that Proteinase K degrades the polymer very efficiently. The rate of degradation increases considerably up to 0.1 mg/ml enzyme concentration, but remains constant at larger values. Temperature has an optimum at around 50 °C that is somewhat higher than the 37 °C extensively used in the literature as the most advantageous temperature. If degradation occurs in the same medium throughout the process, the formation of lactic acid results in the rapid decrease of pH and finally in the denaturation of the enzyme. The dropping of pH below 5 slows down and finally stops degradation completely. The daily change of the medium results in degradation with a constant rate and the entire amount of the polymer can be decomposed mainly into monomer or smaller oligomer fragments. Degradation rate decreases slightly with increasing molecular weight and increasing d -lactide content. The use of appropriate kinetic models allows quantitative analysis and the prediction of the rate of enzymatic degradation of PLA. [ABSTRACT FROM AUTHOR]

[Display omitted] This study investigates the interfacial behavior of the proteinase K enzyme at air–water interface. Adsorption of enzyme on the surface was induced using saline subphase. The surface packing and stability of the enzyme was investigated using of surface pressure-area (π-A) and surface potential-area (ΔV-A) isotherms. Proteinase K enzyme forms film at air-aqueous interface and demonstrates good stability as shown through compression-decompression cycle experiments. To characterize the surface assembly morphology of the interfacial enzymes UV–vis and fluorescence spectroscopic techniques were used. The data revealed that the enzyme Langmuir monolayer has good homogeneity with no evidence of aggregates during compression. The secondary structure of the enzyme at interface was determined to be α-helix using p-polarized infrared-reflection absorption spectroscopy. This was confirmed through Circular dichroism spectra of the enzyme Langmuir-Blodgett (LB) film which showed that the major conformation present were α-helices. [ABSTRACT FROM AUTHOR]

The problem in using the existing biodegradable polymers for day‐to‐day commodity and specialty applications (non‐physiological) is the trade‐off between the degradability and physical properties of polymers. Therefore, the authors study the properties of polyester films made by solution blending of amorphous poly(d,l‐lactide) and semi‐crystalline poly(l,l‐lactide) (PLLA) with an aim to achieve a good balance of mechanical properties and degradability under environmental conditions. The degradation test using proteinase K enzyme shows faster degradation of blends in comparison to homopolymers by weight loss. Faster fragmentation of blends and fragments of lower masses in comparison to PLLA is also seen in immature compost with bulk degradation as the main mechanism of degradation. A detailed investigation shows increased crystallinity and the formation of crystalline stereo‐complex in fragmented samples that may limit degradation after a stage causing microplastics persisting for a longer period. Therefore, further degradation studies in compost for at least 8–10 weeks are recommended. Other environmental sinks, such as activated sludge water, fresh, and seawater, provide either extremely slow or no degradation excluding the use of such blends for applications intended for these sinks. In future, smart solutions are required to enhance the degradation of polylactide in different environmental sinks. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) is one of the main pathogens causing urinary catheter-related infections, and its biofilm has strong resistance to immune clearance and antibiotics. Effective measures should be taken to solve catheter biofilm-related infections fundamentally. In this study, the host defense peptide mimicking peptide polymer was synthesized by rapid ring-opening polymerization of α-amino acid N-carboxyanhydride (NCA), which was initiated by lithium bis(trimethylsilyl)amide (LiHMDS). The resulting polymer showed a narrow molecular weight distribution. Antimicrobial experiment showed that this peptide polymer had a high bactericidal activity against the planktonic and persister cells of methicillin-resistant Staphylococcus aureus (MRSA). Bactericidal mechanism showed that this peptide polymer killed MRSA by destroying the integrity of bacterial cell membrane. Protein is one of the basic components of extracellular polymeric matrix (EPS), which plays an important role in bacterial colonization and biofilm development. The dispersing effect of proteinase K on EPS is beneficial to the penetration of antimicrobial agents into biofilms. 1.25 U/mL proteinase K dispersed mature MRSA biofilms in artificial urine, and the remaining cell viability was about 55% of the control group. The combination of proteinase K and polymer further eradicated mature MRSA biofilms in artificial urine, which confirmed the potential of synergism in eradicating the biofilms inside urinary catheter. The optimal combination reduced the cell viability within biofilms to about 10% of the control group. [ABSTRACT FROM AUTHOR]

Commercial hydrolytic enzymes belonging to different subclasses (several lipases, proteinase k, cutinase) were investigated for their ability to degrade different aliphatic polyesters, i.e., poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBSA), two poly(caprolactone), having two different molecular weights, poly(lactic acid) (PLA) and poly(propylene carbonate) (PPC). The enzyme screening was first carried out by investigating the capacity of fully degrading the target polymers in 24 h, then weight loss measurements of selected polyesters and target enzymes were performed. Solid residues after enzyme degradation were characterized by proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and thermogravimetry (TGA). Liquid fractions were studied via GPC, 1H NMR and high-performance liquid chromatography (HPLC). PCL and PBSA were found to be the most biodegradable polyesters, under the conditions used in this study. PBS was fully degraded only by cutinase, whereas none of the tested enzymes were able to completely degrade PLA and PPC, in the conditions assessed here. Cutinase exhibited the highest hydrolytic activity on PBSA, while lipase from Candida sp. (CALB) on low molecular weight PCL. Chemical analyses on residual solids showed that the enzymatic degradation occurred homogeneously from the surface through an erosion mechanism and did not significantly affect the macromolecular structure and thermal stability. Cleaving action mode for each enzyme (endo- and/or exo-type) on the different polyesters were also proposed based on the evaluation of the degradation products in the liquid fraction. [ABSTRACT FROM AUTHOR]

Bacterial biofilms form when bacteria attach to surfaces and generate an extracellular matrix that embeds and stabilizes a growing community. Detailed visualization and quantitative analysis of biofilm architecture by optical microscopy are limited by the law of diffraction. Expansion Microscopy (ExM) is a novel Super-Resolution technique where specimens are physically enlarged by a factor of ∼4, prior to observation by conventional fluorescence microscopy. ExM requires homogenization of rigid constituents of biological components by enzymatic digestion. We developed an ExM approach capable of expanding 48-h old Proteus mirabilis biofilms 4.3-fold (termed PmbExM), close to the theoretic maximum expansion factor without gross shape distortions. Our protocol, based on lytic and glycoside-hydrolase enzymatic treatments, degrades rigid components in bacteria and extracellular matrix. Our results prove PmbExM to be a versatile and easy-to-use Super-Resolution approach for enabling studies of P. mirabilis biofilm architecture, assembly, and even intracellular features, such as DNA organization. • A novel expansion microscopy protocol for Proteus mirabilis biofilms is presented. • New protocol leads to maximum expansion factor of 4.3 for Proteus mirabilis biofilm. • Super-Resolution approach enables studies of biofilm architecture and assembly. • Super-Resolution protocol reveals novel patterns of subcellular DNA organization. • Technique can be adapted to bacterial species and biofilm research. [ABSTRACT FROM AUTHOR]

Myricetin (MYR) and myricitrin (MYT) are well recognized for their nutraceutical value, such as antioxidant, hypoglycemic, and hypotensive effects. In this work, fluorescence spectroscopy and molecular modeling were adopted to investigate the conformational and stability changes of proteinase K (PK) in the presence of MYR and MYT. The experimental results showed that both MYR and MYT could quench fluorescence emission via a static quenching mechanism. Further investigation demonstrated that both hydrogen bonding and van der Waals forces play significant roles in the binding of complexes, which is consistent with the conclusions of molecular modeling. Synchronous fluorescence spectroscopy, Förster resonance energy transfer, and site-tagged competition experiments were performed to prove that the binding of MYR or MYT to PK could alter its micro-environment and conformation. Molecular docking results revealed that either MYR or MYT spontaneously interacted with PK at a single binding site via hydrogen bonding and hydrophobic interactions, which is consistent with the results of spectroscopic measurements. A 30 ns molecular dynamics simulation was conducted for both PK-MYR and PK-MYT complexes. The calculation results showed that no large structural distortions or interaction changes occurred during the entire simulation time span. The average RMSD changes of PK in PK-MYR and PK-MYT were 2.06 and 2.15 Å, respectively, indicating excellent stability of both complexes. The molecular simulation results suggested that both MYR and MYT could interact with PK spontaneously, which is in agreement with spectroscopic results. This agreement between experimental and theoretical results indicates that the method herein could be feasible and worthwhile for protein–ligand complex studies.

This study aimed to evaluate the effect of proteinase K on mature biofilms of dermatophytes, by assays of metabolic activity and biomass. In addition, the proteinase K–terbinafine and proteinase K–griseofulvin interactions against these biofilms were investigated by the checkerboard assay and scanning electron and confocal microscopy. The biofilms exposed to 32 µg ml−1 of proteinase K had lower metabolic activity and biomass, by 39% and 38%, respectively. Drug interactions were synergistic, with proteinase K reducing the minimum inhibitory concentration of antifungals against dermatophyte biofilms at a concentration of 32 µg ml−1 combined with 128–256 µg ml−1 of terbinafine and griseofulvin. Microscopic images showed a reduction in biofilms exposed to proteinase K, proteinase K–terbinafine and proteinase K–griseofulvin combinations. These findings demonstrate that proteinase K has activity against biofilms of dermatophytes, and the interactions of proteinase K with terbinafine and griseofulvin improve the activity of drugs against mature dermatophyte biofilms. [ABSTRACT FROM AUTHOR]

In the biomonitoring using environmental DNA (eDNA) analysis, the suppression of eDNA degradation has been an important issue to estimate accurately the presence of target species and their eDNA concentrations. A recently proposed eDNA preservation technique requires only the addition of benzalkonium chloride (BAC) to water samples and has been used for various studies as an alternative solution to on-site filtration. However, BAC remaining in filters may adsorb to and inhibit the function of the enzyme used to extract eDNA from filters. In this study, we tested whether eDNA yield is affected by BAC addition immediately before filtration and demonstrated that the BAC in water remaining in filters decreased the eDNA yield significantly. We found that simple pre-centrifugation is effective for removing the remaining water from the filters to prevent the eDNA yield reduction due to the use of BAC. This is the most important finding in the present study. Appropriate eDNA extraction techniques are needed when BAC is used to secure eDNA yields and expand the application of eDNA analysis for biomonitoring. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis is the major bioinsecticide worldwide produced due to the Cry protein activity. Several studies have been done to improve the cost–productivity relation. The neutral protease A (NprA) is the major extracellular protein massively produced during the stationary phase by this bacterium, contributing to the Cry proteins' degradation. Also, the deletion of aprA and nprA genes enhanced the yield of Cry protein, stabilizing it. Therefore, to increase Cry production, one possibility is to degrade the NprA protease in the culture media. In the present study, proteinase K was used to hydrolyze the NprA to increase Cry production. Proteinase K was added during the exponential growth of B. thuringiensis culture. The bacilli and endospores were measured along all culture, while the Cry protein was measured at the end of the culture. The addition of PK affects the bacilli and spore kinetics positively but negatively to the Cry protein (there is no Cry protein detection). Therefore, the gene expression of the cry1Ac, nprX, nprA, and spo0A was measured. The expression of each gene was followed along all culture. Results demonstrated that PK alters both the transcriptional levels and the expression order of the genes. [ABSTRACT FROM AUTHOR]