Protein and Peptide Letters - Volume 28, Issue 10, 2021

Background: Phytic acid acts as anti-nutritional factor in food and feed ingredients for monogastric animals as they lack phytases. Objective: Phytase production by Bacillus subtilis subsp. subtilis JJBS250 was studied in solid-state fermentation and its applicability in dephytinization of food. Methods: Bacterial culture was grown in solid state fermentation using wheat bran and various culture conditions were optimized using ‘One variable at a time’ (OVAT) approach. Effects of different substrates (wheat bran, wheat straw, sugarcane bagasse), incubation time (24, 48, 72 and 96 h), incubation temperatures (25, 30, 35 and 40°C), pH (4.0, 5.0, 6.0, 7.0 and 8.0) and moisture content (1:1.5, 1:2.0, 1:2.5 and 1:3) were studied on phytase production. Bacterial phytase was used in dephytinization of food samples. Results: Optimization of phytase production was studied in solid state fermentation (SSF) using ‘One variable at a time’ (OVAT) approach. Bacillus subtilis subsp. subtilis JJBS250 grew well in various agroresidues in SSF and secreted high enzyme titres using wheat bran at 30°C and pH 5.0 after incubation time of 48 h with substrate to moisture ratio of 1:3. Glucose and ammonium sulphate supplementation to wheat bran further enhanced phytase production in SSF. Optimization of phytase production resulted in 2.4-fold improvement in phytase production in solid state fermentation. The enzyme resulted in dephytinization of wheat and rice flours with concomitant release of inorganic phosphate, reducing sugar and soluble protein. Conclusion: Optimization resulted in 2.34-fold enhancement in phytase production by bacterial culture that showed dephytinization of food ingredients with concomitant release of nutritional components. Therefore, phytase of B. subtilis subsp. subtilis JJBS250 could find application in improving nutritional quality of food and feed of monogastric animals.

Background: As a heat-resistant polymerase, Thermus thermophilus (Tth) DNA polymerase can be widely used in Polymerase Chain Reaction (PCR). However, its non-specific amplification phenomenon is serious, which greatly limits development. Objective: In this study, we prepared Tth monoclonal antibodies against Tth DNA polymerase and researched their application in hot-start PCR. Methods: Tth was recombinantly expressed and purified, and used as an antigen to immunize BALB/ c mice to obtain monoclonal antibodies. The qualified monoclonal antibody and Tth were incubated for a period of time at a certain temperature to obtain the hot-start Tth. We tested the polymerase activity and exonuclease activity blocking the performance of hot-start Tth. Finally, the hot-start Tth was applied to one-step RT-PCR. Results: Tth with a purity of >95% was obtained, and ten monoclonal antibodies were obtained by immunization. After incubation, three monoclonal antibodies were identified that could inhibit the polymerase activity of Tth at low temperature. Furthermore, these three antibodies successfully eliminated non-specific amplification in practical applications. Conclusion: Three monoclonal antibodies were successfully validated. Among them, monoclonal antibody 9 had the best overall effect. They possess the function of inhibiting at low temperature and releasing at high temperature, which can be used as Tth polymerase inhibitors in the field of molecular diagnostics.

Background: The incidence of allergy has been increasing at an alarming rate over the last few decades. Objective: Our present study aims to find out the structurally homologous motifs present in different proteinaceous allergens. Methods: Significant number of protein sequences and their corresponding structures of various pollen, fungal, bacterial, and food allergens were retrieved and the sequence and structural identity were analyzed. Results: Intra- and inter-sequence along with their structural analysis of the proteinaceous allergens revealed that no significant relationships exist among them. A few, but not the negligible number of high structural similarities, were observed within different groups of allergens from fungus, angiosperms, and animals (Aves and Mammalia). Conclusion: Our in silico study on thirty-six different allergens showed a significant level of structural similarities among themselves, regardless of their sequences.

Background: Altered expression of N-glycans such as polylactosamine is observed in colon cancer. AHL, a polylactosamine specific lectin from Adenia hondala from a medicinal plant from the Passifloraceae family has been reported earlier. Objective: The aim of the present study is to study the interaction of AHL with human colon cancer epithelial HT-29 cells and colon cancer tissues. Methods: Cell viability was determined by MTT [3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide] assay, while cell surface binding, apoptosis by Annexin-V-PI assay and ROS production using DCFDA [2’,7’ - dichlorofluorescindiacetate] kit method were analysed by flowcytometry, immunohistochemistry was performed using biotinylated AHL, protein purification by affinity chromatography using asialofetuin-coupled Sepharose -4B column. Results: AHL strongly binds to HT-29 cells with a Mean Fluorescence Intensity of 12.4, which could be blocked by competing glycoprotein asialofetuin. AHL inhibits HT-29 cell growth in a dose and time-dependent manner with IC50 of 2.5 μg/mL and differentially binds to human normal and cancerous tissues. AHL induces apoptosis and slight necrosis in HT-29 cells with an increase in the early apoptotic population of 25.1 and 36% for 24 h and 48 h respectively and necrotic population of 1.5 and 4.6% at 24 h and 48 h respectively as revealed by Annexin-V-PI assay. AHL induces the release of Reactive Oxygen Species in HT-29 cells in a dose-dependent manner. Conclusion: To the best of knowledge, this is the first report on lectin from Adenia hondala which is not a RIP with apoptotic and necrotic effects. These findings support the promising potential of AHL in cancer research.

Aims: To encapsulate a purified bacteriocin into a nanovesicles and check its antibacterial effect. Background: Although the use of nano-encapsulated bacteriocins in food matrices is poorly reported, encapsulated nisin can reduce L. monocytogenes counts in whole and skimmed milk and in soft cheese. Objective: The present study deals with the extraction and purification of a bacteriocin from an isolated strain Pediococcus pentosaceus KC692718. A comparative study of the effect of free pediocin and liposome encapsulated pediocin against Listeria sp. was performed. Methods: The purification of the extracted cell free supernatant was subjected to ammonium sulphate precipitation, cation exchange chromatography followed by gel permeation chromatography. The bacteriocin activity and protein concentration were determined using Lowry’s method. The characterization of the pure pediocin was done. Liposome like nanovesicle was constructed and the stability of the liposome encapsulated pediocin was checked. Finally, the antibacterial effect was comparatively studied of the free pediocin, liposome, and liposome encapsulated pediocin simultaneously. Results: The pediocin of 3.6kDa was purified with a specific activity of 898.8. AU/mg. It remained stable from pH 2.0-8.0 was found to be moderately stable above 80°C and remain stable for one month when stored at -20°C. The encapsulated pediocin showed stability since it retained 50% of its initial activity. The encapsulated pediocin showed 89% of encapsulation efficiency. Conclusion: The encapsulated pediocin not only improved pediocin stability but also enhanced the controlled release of the antimicrobial substances, enough for inhibiting the foodborne pathogen L. monocytogenes.

Background: Obesity is a serious health problem that dysregulate Renin-Angiotensin System (RAS) and intestinal microbiota. Objective: The present study aimed to evaluate the Angiotensin-(1-7) [ANG-(1-7)] oral formulation effects on obese mice intestinal microbiota. Methods: Mice were divided into four groups: obese and non-obese treated with ANG-(1-7) and obese and non-obese without ANG-(1-7) during four weeks. Results: We observed a significant decrease in the fasting plasma glucose, total cholesterol, triglycerides, and Low-density lipoprotein levels and increased High-density lipoprotein in animals treated with ANG-(1-7). The histological analysis showed intestinal villi height reduction in mice treated with ANG-(1-7). Additionally, increased Bacteroidetes and decreased Firmicutes (increased Bacteroidetes/ Firmicutes ratio) and Enterobacter cloacae populations were observed in the High-Fat Diet + ANG-(1-7) group. Receptor toll-like 4 (TLR4) intestinal mRNA expression was reduced in the HFD+ANG-(1-7) group. Finally, the intestinal expression of the neutral amino acid transporter (B0AT1) was increased in animals treated with ANG-(1-7), indicating a possible mechanism associated with tryptophan uptake. Conclusion: The results of the present study suggest for the first time an interaction between oral ANG-(1-7) and intestinal microbiota modulation.

Background: Brucellosis is a zoonotic disease that causes serious economic losses due to factors, such as miscarriages and decreased milk yield in animals. Existing live vaccines have some disadvantages, so effective vaccines need to be developed with new technological approaches. Objective: The primary objectives of this study were the expression and purification of recombinant Omp25 fusion protein from B. abortus, and the evaluation of the effect of the Omp25 protein on cell viability and inflammatory response. Methods: The omp25 gene region was amplified by a polymerase chain reaction and cloned into a Pet102/D-TOPO expression vector. The protein expression was carried out using the prokaryotic expression system. The recombinant Omp25 protein was purified with affinity chromatography followed by GPC (Gel Permeation Chromatography). The MTS assay and cytokine-release measurements were carried out to evaluate cell viability and inflammatory response, respectively. Results: It was determined that doses of the recombinant Omp25 protein greater than 0.1 μg/mL are toxic to RAW cells. Doses of 1 μg/mL and lower significantly increased inflammation due to Nitric Oxide (NO) levels. ELISA results showed that IFN-γ was produced in stimulated RAW 264.7 cells at a dose that did not affect the viability (0.05 μg/mL). However, IL-12, which is known to have a dual role in the activation of macrophages, did not show a statistically significant difference at the same dose. Conclusion: Studies on cell viability and Th1-related cytokine release suggest Omp25 protein to be a promising candidate molecule for vaccine development.

Aim: Being the common cause and major burden of deaths globally, timely cancer management is crucial. Background: Thymic immunosuppressive pentapeptide (TIPP) is a novel pentapeptide originally obtained from calf thymic immunosuppressive extract. Previously, TIPP has been proved to suppress the allergic and inflammatory responses in allergic mice via blocking MAP kinases/NF-ΚB signaling pathways. Objective: In this study, in vitro anticancer activity of TIPP was tested on two different types of cancers using MCF-7 and K562 cell lines. Methods: Tumor xenograft models for breast cancer and chronic myeloid leukemia were designed. In vivo anticancer activity of TIPP was investigated on both cancer types. The liver and tumor tissues of the mice were preserved for immunohistochemistry analysis. Results: In vitro anticancer activity of TIPP showed significant inhibition on cell viability of both breast cancer and chronic myeloid leukemia. In vivo anticancer effect of TIPP in both types of cancer models further proved the potent anticancer nature of TIPP. Immunohistochemistry analysis assured that TIPP is a safe drug for normal organs such as the liver. Conclusion: Our present study revealed that TIPP is a potent anticancer drug and an important treatment option for various diseases. Further work is needed to test the flexible and proficient activity of the novel peptide.

Aim: This study aims to perform differential protein expression analysis of serum samples from Oral Squamous Cell Carcinoma (OSCC) patients and healthy controls in search of potential diagnostic and/or prognostic biomarker(s). Objective: OSCC is usually diagnosed late, which results in poor survival and high mortality. Identification of non-invasive prognostic biomarkers is of utmost importance for early diagnosis and proper management of the disease; hence we used a proteomic approach to identify potential biomarkers from serum. Methods: Serum samples (OSCC n=45 and control n=30) were depleted, and proteins were separated using 2-D gel electrophoresis followed by identification by mass spectrometric analysis. Gene expression analysis of identified proteins in malignant and normal tissue was also performed to complement proteomics studies. Results: Among differentially expressed proteins, up-regulation of heat shock protein alpha (HSP90α) from the serum of oral cancer patients was observed. We also observed elevated levels of Haptoglobin (HP) along with downregulation of Type II keratin cytoskeletal 1(KRT1) and serum albumin (ALB) in oral cancer patients. Gene expression studies on identified proteins in malignant and normal tissue revealed a similar pattern with the exception of KRT1. We believe that elevated levels of serum HSP90 alpha might be used as a potential biomarker. Conclusion: Our findings suggest a contribution of HSP90 alpha and other identified proteins in oral pathology as pro/anti-apoptotic modulators, thus considering their potential as predictive biomarkers.

Background: It is well known that alcohol can trigger inflammatory effects in the gastrointestinal tract (GIT), interfering with mucosal homeostasis. Objectives: This study evaluated the effectiveness of Lactococcus lactis treatment in controlling the increase in molecular biomarkers related to allergic inflammation and the effect on the diversity and abundance of the Enterobacteriaceae family in the GIT after high-dose acute administration of ethanol. Methods: Mice received ethanol or saline solution by gavage for four consecutive days, and 24 h after the last administration, the animals were given L. lactis or M17 broth orally ad libitum for two consecutive days. The animals were subsequently sacrificed and dissected. Results: L. lactis treatment was able to restore basal levels of secretory immunoglobulin A in the gastric mucosa, serum total immunoglobulin E, interleukin (IL)-4 production in gastric and intestinal tissues, and IL-10 levels in gastric tissue. L. lactis treatment encouraged the diversification of the Enterobacteriaceae population, particularly the commensal species, in the GIT. Conclusion: This research opens a field of studies regarding the modulatory effect of L. lactis on immunological and microbial changes induced after alcohol intake.

Background: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. Objectives: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. Methods: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression). Results: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. Conclusion: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Background: The need for agonists and antagonists of β2 adrenoceptor (β2AR) is warranted in various human disease conditions, including cancer, cardiovascular and other metabolic disorders. However, the sources of agonists of β2AR are diverse in nature. Interestingly, there is a complete gap in the exploration of agonists of β2AR from serum that is a well-known component of culture media that supports growth and proliferation of normal and cancer cells in vitro. Methods: In this paper, we employed a novel vertical tube gel electrophoresis (VTGE)-assisted purification of intracellular metabolites of MCF-7 cells grown in vitro in complete media with fetal bovine serum (FBS). Intracellular metabolites of MCF-7 cells were then analyzed by LC-HRMS. Identified intracellular tripeptides of FBS origin were evaluated for their molecular interactions with various extracellular and intracellular receptors, including β2AR (PDB ID: 2RH1) by employing molecular docking and molecular dynamics simulations (MDS). A known agonist of β2AR, isoproterenol was used as a positive control in molecular docking and MDS analyses. Results: We report here the identification of a few novel intracellular tripeptides, namely Arg-His- Trp, (PubChem CID-145453842), Pro-Ile-Glu, (PubChem CID-145457492), Cys-Gln-Gln, (PubChem CID-71471965), Glu-Glu-Lys, (PubChem CID-11441068) and Gly-Cys-Leu (PubChem CID-145455600) of FBS origin in MCF-7 cells. Molecular docking and MDS analyses revealed that among these molecules, the tripeptide Arg-His-Trp shows a favorable binding affinity with β2AR (-9.8 Kcal/mol). The agonistic effect of Arg-His-Trp is significant and comparable with that of a known agonist of β2AR, isoproterenol. Conclusion: In conclusion, we identified a unique Arg-His-Trp tripeptide of FBS origin in MCF-7 cells by employing a novel approach. This unique tripeptide Arg-His-Trp is suggested to be a potential agonist of β2AR and it may have applications in the context of various human diseases like bronchial asthma and chronic obstructive pulmonary disease (COPD).