Enzymatic Techniques Research Articles

Identification and Characterization of Novel Malto-Oligosaccharide-Forming Amylase AmyCf from Cystobacter sp. Strain CF23.

Malto-oligosaccharides (MOSs) from starch conversion is advantageous for food and pharmaceutical applications. In this study, an efficient malto-oligosaccharide-forming α-amylase AmyCf was identified from myxobacter Cystobacter sp. strain CF23. AmyCf is composed of 417 amino acids with N-terminal 41 amino acids as the signal peptide, and conserved glycoside hydrolase family 13 (GH13) catalytic module and predicted C-terminal domain with β-sheet structure are also identified. Phylogenetic and functional analysis demonstrated that AmyCf is a novel member of GH13_6 subfamily. The special activity of AmyCf toward soluble starch and raw wheat starch is 9249 U/mg and 11 U/mg, respectively. AmyCf has broad substrate specificity toward different types of starches without requiring Ca2+. Under ideal circumstances of 60 °C and pH 7.0, AmyCf hydrolyzes gelatinized starch into maltose and maltotriose and maltotetraose as the main hydrolytic products with more than 80% purity, while maltose and maltotriose are mainly produced from the hydrolysis of raw wheat starch with more than 95% purity. The potential applicability of AmyCf in starch processing is highlighted by its capacity to convert gelatinized starch and raw starch granules into MOSs. This enzymatic conversion technique shows promise for the low-temperature enzymatic conversion of raw starch.

An Overview of Structured Lipid in Food Science: Synthesis Methods, Applications, and Future Prospects

Structured lipids have attracted significant interest in industries, such as food, pharmaceuticals, and cosmetics, because they can be alternatives to traditional lipids. Enzymatic synthesis techniques utilizing lipases have gained attention for structured lipids synthesis because of their site and substrate specificity. However, most lipases discovered to date exhibit sn-1,3 regiospecificity or nonspecificity, limiting the selective range of natural oils and fats that can be used as raw materials. In this review, we provide an overview of the current synthesis methods and applications of structured lipids and the limitations of existing lipases in producing structured lipids. In addition, we discuss innovative approaches, including metagenomics, and machine learning, to discover, and classify new lipases and the use of gene editing technologies for lipase engineering. These efforts aim to overcome the limitations of existing lipases and expand the range of natural oils and fats that can be used for the production of structured lipids. Therefore, this study aims to promote a better understanding of structured lipids as a material with high-added value and provides insight into ongoing research efforts in the field.

On-Line Dual-Active Valves Based Centrifugal Microfluidic Chip for Fully Automated Point-of-Care Immunoassay.

There remains an unmet need for a fully integrated microfluidic platform that can automatically perform multistep and multireagent immunoassays. Here, we proposed a novel online dual-active valve-based centrifugal microfluidic chip, termed DAVM, for fully automatic point-of-care immunoassay. Practically, the puncture valve, one of the dual active valves, is capable of achieving precise, on-demand, sequential release of prestored reagents, while the other valve-reversible active valve enables controlled retention and drainage of the reaction solutions. Thereby, our technology mitigates the challenges of hydrophilic/hydrophobic modifications and unstable valve control performance commonly observed in passive valve controls. As a proof of concept, the indirect enzymatic immunoblotting technique was employed on DAVM for fully automated immunological analysis of eight targets, yielding outcomes within an hour. Furthermore, we conducted a comparative analysis of 28 clinical samples with autoimmune diseases. According to 224 clinical data, the sample testing concordance rate between DAVM and the traditional instrument was 82%, with a target compliance rate of 97%. Therefore, our DAVM system has powerful potential for fully automated immunoassays.

Cleaner and sustainable synthesis of high-quality monoglycerides by use of enzyme technologies: techno-economic and environmental study for monolaurin

Currently, monoglycerides (MG) are produced using a complicated energy-intensive technology that contributes negatively toward greenhouse gas mitigation. This work suggests a cleaner and simpler one-step enzymatic production of α-monolaurin in an inert membrane reactor, where the reaction and enzyme separation are conducted simultaneously in one unit. Candida antarctica lipase (Lipozyme 435) was used to catalyze the esterification reaction between lauric acid and glycerin in a solvent-free system under mild temperatures. Response surface methodology was used to optimize the reaction conditions. The optimal conditions were a molecular sieve of 14.85% w/w, a temperature of 56.95 °C, an enzyme amount of 5.38% w/w, and a molar ratio of 4.75% w/w. The gas chromatography (GC) analysis showed that the α-monolaurin percentage was 49.5% when the enzymatic process (ENZ) was used. The conventional chemical (CHEM) and autocatalytic (AUT) esterification methods were also performed to study their proportional MG yields. The GC results showed the MG percentages of 43.9 and 41.7% for CHEM and AUT, respectively. Economic analysis was also conducted for the suggested enzymatic technique, and the findings were compared with those of the CHEM and AUT technologies. Using a plant capacity of 4950 t/year and 11% interest for the proposed ENZ process, the total capital investment of α-monolaurin production was preferably four times less than that of the CHEM process and three times less than that of the AUT method, presenting investment possibilities. However, the ENZ process showed the least profitability (net profit per day) among the three processes. Nevertheless, the return on investment and net present value for the ENZ process were preferably higher than those of CHEM and AUT because of its interestingly lower inside battery limit plant cost and less energy consumption. The AUT/CHEM processes generated a total carbon dioxide (CO2) exhaust of 678.7 t CO2 eq./year. In contrast, the ENZ process exhausted a total CO2 of only 50 t CO2 eq./year. The present integrated techno-economic and environmental study of α-monolaurin production emphasizes the green and cost benefits of the proposed ENZ technology.Graphical

The use of NexoBrid™ enzymatic debridement and coagulation abnormalities

ObjectivesCurrent evidence on how the use of bromelain-based enzymatic debridement techniques (NexoBrid™) affect patient coagulation is limited. A single patient case report [1] suggests that a patient with 15% total body surface area (TBSA) burn developed decreased coagulation activity following debridement with NexoBrid™ enzymatic debridement (ED). Regional Burns Centres in the United Kingdom may be reluctant to use ED, particularly in larger burns, citing concerns regarding coagulation. At our centre we have routinely used ED on deep partial thickness burns since 2017 including on patients with burns over 15% TBSA. This study aims to investigate whether there is a significant disruption in coagulation in patients undergoing ED with burns > 15% TBSA or admitted to intensive care compared to the standard of care (SOC) which is surgical debridement in theatre. MethodsThis single-centre retrospective study includes all patients with a burn treated with ED at Pinderfields General Hospital Regional Burns Centre intensive care unit (ITU) from 2017 to 2020. Patients were matched to those treated with SOC at the same centre by age, % TBSA burn and presence of inhalational injury. These parameters correlate with the Baux score [9]. Percentage of burn debrided was matched as closely as possible, with coagulation profiles and platelet count taken the day before, the day of and three days following surgery. ResultsThirty-one patients were treated with ED in the intensive care unit between 2017 and 2020. Four patients were excluded due to insufficient records and one patient was anti-coagulated. Twenty-six patients were included and matched as described above. Average age of patients receiving ED was 44 years, the same in the matched group. Average TBSA burn is 35.5% (35.8% in matched group). No statistically significant difference in coagulation was seen between patients undergoing ED compared to SOC when considering prothrombin time (PT), activated partial thromboplastin time (aPTT) and platelet count. Both groups slightly breached the upper limit of normal on day 2 post ED and SOC. There was a slight breach of the lower limit of the average platelet count on day 2 post-ED which was neither statistically nor clinically significant. ConclusionsLarge burns are associated with coagulation abnormalities, therefore isolating a single variable in this cohort is challenging. However, this study found no significant change following ED use when compared to SOC and therefore no convincing evidence that ED is associated with coagulation abnormalities. This study represents one of the largest focusing on coagulation abnormalities following the use of ED, as the current literature is limited. Our study suggests that concerns regarding coagulation abnormalities should not prevent patients with large, deep partial thickness burns or full thickness burns being treated with ED.

Estimation and study effect of some trace elements and some electrolytes in hyperlipidemia patients

Abstract Background: Measuring electrolytes in the blood is considered one of the most common diagnostic techniques. Objectives: To assess sodium, potassium, zinc, and copper levels and examine the correlation between parameters and study their probable relation with hyperlipidemia in Babylon province. Materials and Methods: Sodium, potassium, zinc, and copper were estimated in one hundred subjects; 50 patients with hyperlipidemia and 50 healthy subjects participated in this study. The age of the patients and control groups ranged between 19 and 75 years. Sodium and potassium levels were assessed by colorimetric enzymatic technique, whereas the level of zinc and copper was assessed in serum by the flameless atomic absorption spectrophotometer method. Results: Serum levels of sodium and zinc displayed a significant decrease, whereas serum copper significantly elevated (P < 0.05) in hyperlipidemia patients compared with the control group. Serum potassium observed nonsignificant change (P-value > 0.05) in hyperlipidemia patients. On the other hand, copper and zinc correlated significantly with cholesterol, triglyceride, and sodium. Conclusion: Among hyperlipidemia patients in Babylon province, the variability in some trace elements and some electrolytes concentration indicate a significant relationship with hyperlipidemia.

A focused review on recycling and hydrolysis techniques of polyethylene terephthalate

AbstractPolyethylene terephthalate (PET) is used in textile and packaging industries. The main source of PET production is fossil fuels with limited capacity. Also, PET products are single use that transform into high volumes of wastes, causing ecosystem problems. Recycling is proposed to confront this challenge. The four major PET recycling techniques are mechanical, chemical, pyrolysis, and enzymatic. Mechanical, pyrolysis, and enzymatic techniques have constrained capabilities to manage PET waste. Chemical recycling is the potential path to expanding recycling PET waste with possibility of upcycling and addressing dirty waste streams. Several chemical methods are introduced and discussed in literature. The five major chemical recycling techniques are glycolysis, alcoholysis, aminolysis, ammonolysis, and hydrolysis. This review describes PET depolymerization via these techniques and introduces hydrolysis as the one that can depolymerize PET in an organic‐free solvent environment. Hydrolysis tolerates PET mixed wastes streams including copolymers. It helps avoid challenges attributed to using organic solvents in reaction systems. Moreover, hydrolysis produces terephthalic acid, PET monomer, which has recently gained attention as the initiative monomer for PET production. The review focuses on three forms of hydrolysis—alkaline, neutral, and acid, by presenting background studies, issued patents, and recent trends on application of hydrolysis.

Serum Creatinine Determination: Comparison between Colorimetric (Jaffé) and Enzymatic Methods in Renal Transplant Recipients

Background: Serum creatinine is the most requested parameter in current practice in the context of functional exploration of the kidney. Its contribution to medical care is particularly decisive for kidney transplant patients. The aim of the study is to compare the colorimetric and enzymatic techniques available in our laboratory for the determination of creatinine in samples from kidney transplant patients.
 Methods: Prospective study carried out at the Biochemistry laboratory of the University Hospital Center (CHU) Ibn Rochd in Casablanca Morocco, over a period of 8 months, carried out on a series of 45 samples from kidney transplant patients who simultaneously benefited from a creatinine assay by colorimetric and enzymatic techniques. The samples were taken on dry tubes and analyzed on the Alinity ® Abbott analyzer. The statistical study of correlation was made by the SPSS software with calculation of the p-value and the coefficient of Pearson (r).
 Results: During the study period 45 samples were collected. Seventy percent of the prescriptions came from the Nephrology Department. The mean serum creatinine value by enzymatic technique was 39.04 ± 11.7 mg/l, and by colorimetric technique 38.24 ± 12.4 mg/l. The creatinine level by the two techniques was pathological in 69% of cases and normal in 31%. The correlation between the serum creatinine assay by enzymatic Versus colorimetric technique is positive with r=0.994 and P-value<0.001.
 Conclusion: According to literature, colorimetric techniques are sensitive to interference with pseudochromogenic substances, while enzymatic techniques are standardized due to their specificity and performance but remain more expensive. In our study the correlation between the two techniques was positive with a correlation equation: (y = 0.82 + 1*x). According to our results, no significant difference between the two techniques was observed in our laboratory.

Comparative assessment of primary cancer cell culture techniques and cellular composition analysis in non-small cell lung cancer

Preclinical models are required to study individual therapy responses to improve all cancer treatments, particularly non-small cell lung cancer (NSCLC). Patient-derived explants (PDEs) culture model is of great importance in terms of the possibility of tumor cell culture with the microenvironment, and the development of molecular mechanisms and personalized treatment methods. In our study, primary tumor culture with microenvironment was performed using different methods from tumor tissues obtained from 51 patients with NSCLC. To identify the most efficient method, mechanical, enzymatic, and tumor fluid techniques were applied. While the malignant cell rate was > 95% in 3 of these cases, the cancer-associated fibroblasts (CAF) microenvironment was high in 46 (80–94%) and low in 2 (1–79%). Subtyping of cells obtained from culture was performed using the light microscope and, if necessary, additional immunohistochemical markers. Consequently, using different techniques, here we successfully performed primary cell culture from patients with NSCLC with microenvironment. Depending on the cell type and culture conditions, proliferation rate was shown to be altered.

Does insulin make breast cancer cells resistant to doxorubicin toxicity?

The effects of insulin on the doxorubicin (Dox) sensitivity of breast cancer cell line MCF-7 and its Dox-resistant counterpart MCF-7/Dox were studied and glucose metabolism, content of essential minerals, and the expression of several microRNAs in these cells upon exposure to insulin and Dox were compared. Cell viability colorimetric assay, colorimetric enzymatic technique, flow cytometry, immunocytochemical techniques, inductively-coupled plasma atomic emission spectroscopy, and quantitative polymerase chain reaction were used in the study. We found that insulin in high concentration significantly suppressed Dox toxicity, especially in parental MCF-7 cell line. The increase in proliferative activity triggered by insulin in MCF-7 but not MCF-7/Dox cells occurred in the setting of the increased level of specific binding sites for insulin and increased glucose uptake. Insulin treatment of MCF-7 cells in low and high concentrations resulted in the increase of Mg, Ca, and Zn content while in DOX-resistant cells, only Mg content increased upon exposure to insulin. High concentration of insulin increased the expression of kinase Akt1, P-glycoprotein 1 (P-gp1) and DNA excision repair protein ERCC-1 in MCF-7 cells, while in MCF-7/Dox cells, Akt1 expression decreased, and cytoplasmic expression of P-gp1 increased. In addition, insulin treatment affected expression of miR-122-5p, miR-133a-3p, miR-200b-3p, and miR-320a-3p. The decreased manifestation of biological effects of insulin in Dox-resistant cells could be partly explained by the different patterns of energy metabolism in MCF-7 cells and their Dox-resistant counterpart.

Role of Repeated Sperm Extraction by Enzymatic and Mechanical Mincing Techniques in Azoospemic Patients

Azoospermic patients have the option of undergoing in vitro fertilization procedures, specifically intracytoplasmic sperm injection (ICSI), for which sperm is retrieved from the patient's own testicles and then injected directly into the oocyte. The aim of the study: evaluate the role of repeated sperm extraction by enzymatic and mechanical mincing techniques in azoospemic patients. A total of 32 males with non-obstructive azoospermia participated in the research. Testicular sperm extraction (TESE) was performed under spinal or general anesthesia after a comprehensive history, physical examination, and investigations. After that, the biopsies underwent through a series of mincing processes, both mechanical and enzymatic. Patients gave information including age, body mass index and previous biopsy attempts. In both enzymatic and mechanical methods there was no significant higher sperm recovery in patients with history of previous testicular biopsies (66.7% vs. 38.5%; p=0.209) and (66.7% vs. 30.8%; p=0.102) respectively. Patients who had undergone prior testicular biopsies had marginally higher sperm recovery rates using both enzymatic and mechanical techniques

Enzymatic hydrolysis for the removal of 3- monochloropropanediol esters in edible oils using Candida rugosa lipase in the presence of deep eutectic solvents and nanocellulose

3-monochloropropanediol ester (3-MCPDE) is identified as a food-borne contaminant in edible oils and are classified as a possible carcinogen. This study reports an efficient enzymatic technique for the removal of 3-MCPDE from extra virgin olive oil (EVO) using Candida rugosa lipase (CRL) as the biocatalyst in the presence of choline chloride and fructose-based natural deep eutectic solvent (NADES) and nanocellulose (NC) extracted from almond shells. The validity of the method was confirmed by gas chromatography mass spectrometry (GC-MS) showing adequate precision with relative standard deviation values ≤ 2.37%. The quantification and detection limit are within the permissible levels of 3-MCPD in edible oils. Under optimized conditions of 30 min at 90 °C with 60 µL of phenylboronic acid (PBA), the enzymatic hydrolysis resulted in the removal of 79.8% of spiked 3-MCPD in EVO. No adverse effects of the EVO were detected from this technique with respect to the oil quality testing. The application of DESs and NCs as the support material for the CRL biocatalyst for the removal of 3-MCPDE has yet to be explored. This could have a significant impact on the edible oil industry for producing oils of higher quality free from 3-MCPDE.

Enzymatic hydrolysis extraction and quality assessment of fish oil from Patin catfish (Pangasius Hypophthalmus)

AbstractThis study aimed to characterize the extraction of fish oil and investigate the oil quality of Patin catfish (Pangasius hypophthalmus) using the enzymatic hydrolysis technique. The selected parameters were the concentration of enzymes, temperature, and reaction time. Patin catfish available across Southeast Asia is one of the species most accessible to fast‐growing Malaysian population and one of the most convenient food supply alternatives. In this research, alcalase was used as the source of enzyme to obtain the oil yield percentage. The optimization of the recovery process of Patin catfish oil using enzymatic hydrolysis technique was conducted using the multifactor experiments software Design of Experiments (DoE), which obtained an optimum condition for the oil yield percentage at 55.3°C with a 1.2% enzyme concentration for 3 h. The percentage of oil yield obtained was 10.90%. The fish oil was further characterized via the FTIR spectroscopy analysis at 4000–700 cm−1. The investigation of the oil quality was conducted based on analysis test. The acid value test gives 2.24 mg KOH/g, peroxide with 4 meq/kg, and p‐anisidine analysis leads to 0.895.

Prevalence and specificity of red blood cell antibodies in patients transfused in tertiary hospitals in Burkina Faso.

Sub-Saharan African countries face the challenge of immunological transfusion safety that puts many patients at risk of post-transfusion hemolytic reactions. This is because pre-transfusion testing for irregular/unexpected antibodies that helps to prevent these risks are neither universally available nor accessible. The aim of our study was to determine the prevalence of red blood cell alloantibodies and their specificity in patients transfused in Burkina Faso. This was a cross-sectional study including patients who had received at least one blood transfusion. Indirect antiglobulin testing using LISS-enhanced medium gel column agglutination technique was used for antibodies screening and identification. Enzymatic technique with papain-treated red cell reagent was performed in attempt to solve some difficulties if necessary as well as auto-control test and RH-KEL phenotyping when possible to help antibodies identification. A total of 832 patients were included, 51.6% of whom were female, and the median (IQR) age was 34 (20-49) years. Of these, 43.7% had chronic kidney disease and 20.4% were sickle cell patients. The median (IQR) number of immunisation episodes (blood transfusion and pregnancies) was 3 (2-6) with the median (IQR) number of blood units received per patient of 2 (1-5). The proportion of patients with RBCs antibodies was 6.4% (53/832), with mainly anti-Rh antibodies. A combination of 2 antibodies was found in 7 patients and a combination of 3 antibodies in one patient. Antibodies of unknown specificity (AUS) were encountered in 29%. Independent factors associated with antibody positivity were age (OR=1.02; p=0.026), sickle cell disease (OR=3.23; p=0.017) and receiving more than 10 blood units (OR=7.33; p=0.01). In this study, the proportion of patients with RBC antibodies was quite similar to that observed in Sub-Saharan African countries. However, the availability and accessibility of pre-transfusion compatibility tests as well as the quality of methods used should be improved to ensure the safety of blood transfusions.

Bio-based polymer blend with tunable properties developed from paramylon hexanoate and poly(butylene succinate)

Paramylon hexanoate (PaHe) is an ester derivative of paramylon, a storage polysaccharide photosynthesized by Euglena. In this study, PaHe was blended with poly(butylene succinate) (PBS) by melt-extrusion and characterized for its physical properties. Although the blend was immiscible even in low loading of PaHe (2.5–10 wt%), the elongation at break (from 611 to 980%) and modulus of toughness (from 1260 to 2248 MPa) improved compared to neat PBS. This was attributed to the unique micro-bead morphology in blends with low PaHe loading revealed using selective enzymatic etching technique. In addition, the hydrophobicity of PBS was enhanced as a result of inclusion of hydrophobic acyl groups in the blend system. Furthermore, X-ray analyses of uniaxially stretched films showed high degree of orientation (94–96%) with tensile strengths up to 244 MPa. PBS/PaHe blends also had good thermal stability making it viable for sustainable packaging and mulch-film applications.

Competition between enzymatic and non-enzymatic electrochemical determination of cholesterol

Cholesterol is considered an essential component in animal bodies that plays a vital role in hormone production and creating vitamin D. The elevated blood cholesterol level increases the possibility of heart disease. Consequently, the clinical diagnosis of cholesterol concentration is critical for human health care. The electrochemical sensor is one of the leading techniques for detecting cholesterol in biological fluids with high accuracy. Therefore, this review included comparative insights into the enzymatic and non-enzymatic techniques for electrochemical sensing. We discussed the recent progress in the electrochemical determination of cholesterol via various materials, e.g., metal composites, carbonaceous materials, ionic liquid crystals, and polymers. We explained electrochemical sensing techniques like amperometric, potentiometric, conductometric, and impedimetric methods. Furthermore, various enzyme-immobilization approaches were reported, like surface binding and encapsulation. The synergistic effect between the electrode surface and enzymes was demonstrated to clarify the detection mechanism. The review article summarized the outcome detection limits and linear range of detection for various surfaces for enzymatic and non-enzymatic electrochemical techniques within the last twenty years.

Elucidation of the cellulose nanocrystal-sugar beet pectin interactions for emulsification enhancement

Commercial emulsions are usually stabilized by multi-component formulations, such as particle and biopolymer, rather than single emulsifiers. However, their interactions and contributions to emulsification are complicated and lack elucidations. This work investigates the interaction between cellulose nanocrystal (CNC) and sugar beet pectin (SBP) and their performance in emulsion stabilization. The irreversible adsorption of SBP onto the CNC surface was verified by Quartz Crystal Microbalance with Dissipation (QCM-D) and atomic force microscopy. The molecular structure-based adsorption mechanism was elucidated by the enzymatic hydrolysis technique, revealing that the neutral sugar side chains (rich in protein and ferulic acid) were the main adsorbable components of SBP, which was predominately driven by hydrophobic interactions. At the CNC: SBP ratio of 3:1, the mixture of SBP-coated CNC and free SBP showed synergistic emulsification performance. The incorporation of CNC significantly improved the anchoring rate of SBP at the oil-water interface during emulsification to produce smaller emulsion droplets. The hybrid coverage of SBP-coated CNC and free SBP endowed the droplets with more robust repulsion to maintain long-term stability. These findings provide a feasible and convenient way for modulating the emulsifying properties of CNC by changing the compositional formulation. • Cellulose nanocrystal (CNC) adsorbed with sugar beet pectin (SBP) in solution. • The adsorbable fractions of SBP were neutral sugar, protein, and ferulic acid. • The interaction was predominated by hydrophobic interaction. • CNC and SBP showed a synergistic stabilization effect to stabilized emulsions.

Hydrogenase and Nitrogenase: Key Catalysts in Biohydrogen Production.

Hydrogen with high energy content is considered to be a promising alternative clean energy source. Biohydrogen production through microbes provides a renewable and immense hydrogen supply by utilizing raw materials such as inexhaustible natural sunlight, water, and even organic waste, which is supposed to solve the two problems of "energy supply and environment protection" at the same time. Hydrogenases and nitrogenases are two classes of key enzymes involved in biohydrogen production and can be applied under different biological conditions. Both the research on enzymatic catalytic mechanisms and the innovations of enzymatic techniques are important and necessary for the application of biohydrogen production. In this review, we introduce the enzymatic structures related to biohydrogen production, summarize recent enzymatic and genetic engineering works to enhance hydrogen production, and describe the chemical efforts of novel synthetic artificial enzymes inspired by the two biocatalysts. Continual studies on the two types of enzymes in the future will further improve the efficiency of biohydrogen production and contribute to the economic feasibility of biohydrogen as an energy source.

Advances in the One-Step Approach of Polymeric Materials Using Enzymatic Techniques.

The formulation in which biochemical enzymes are administered in polymer science plays a key role in retaining their catalytic activity. The one-step synthesis of polymers with highly sequence-controlled enzymes is a strategy employed to provide enzymes with higher catalytic activity and thermostability in material sustainability. Enzyme-catalyzed chain growth polymerization reactions using activated monomers, protein-polymer complexation techniques, covalent and non-covalent interaction, and electrostatic interactions can provide means to develop formulations that maintain the stability of the enzyme during complex material processes. Multifarious applications of catalytic enzymes are usually attributed to their efficiency, pH, and temperature, thus, progressing with a critical structure-controlled synthesis of polymer materials. Due to the obvious economics of manufacturing and environmental sustainability, the green synthesis of enzyme-catalyzed materials has attracted significant interest. Several enzymes from microorganisms and plants via enzyme-mediated material synthesis have provided a viable alternative for the appropriate synthesis of polymers, effectively utilizing the one-step approach. This review analyzes more and deeper strategies and material technologies widely used in multi-enzyme cascade platforms for engineering polymer materials, as well as their potential industrial applications, to provide an update on current trends and gaps in the one-step synthesis of materials using catalytic enzymes.

Involvement of branched RG-I pectin with hemicellulose in cell–cell adhesion of tomato during fruit softening

Cell adhesion of four cultivars of tomato fruit, “Micro Tom (MT)”, “Heinz 1706 (H1706)”, “Money Maker (MM)”, “Ailsa Craig (AC)” were evaluated and cell walls were analyzed in order to assess the possible contribution of pectic and hemicellulosic polysaccharides to the softening and altered cell adhesion at two different stages of ripeness. Cell wall material (CWM) and solubilised fractions of green and red ripe fruit were analyzed by chemical, enzymatic techniques. In comparison with the four cultivars of tomato fruits, H1706 and MM are harder than MT and AC at both green and red ripe stage. The ripening-associated solubilisation of rhamnogalacturonan-riched pectic polysaccharides was reduced in H1706 and MM, and the content of side -chain sugars from RG-I reduced by more than 50% in MT and AC. In addition to recognized pectic modifying enzymes, RGase had a good effect on cell separation of H1706 and MM fruit at red ripe stage. The higher RG-I content and branching degree have been associated with increased cell adhesion and reduced cell wall porosity, thus maintained fruit firmness.