Tailoring surface properties of functionalized graphene papers aiming to enzyme immobilization

The use of enzymes as catalysts requires recovery and reuse to make the process viable. Enzymatic immobilization changes enzyme stability, activity, and specificity. It is very important to explore new substrates for immobilization with appropriate composition and structure to improve the efficiency of the immobilized enzymes. This work explores the use of two different graphene oxide papers, one produced by oxidation route (GO) and the other by electrochemical synthesis (EG), aiming for β-galactosidase immobilization. The chemical and structural properties of these two papers were characterized by Raman spectroscopy, X-ray photoelectron spectroscopy and X-ray diffraction. Atomic force microscopy images showed that EG paper ensured more efficient immobilization of the enzymes on the surface of the paper. Cyclic voltammetry was used to monitor the reaction of conversion of lactose to glucose in the free enzyme solution and graphene paper immobilized enzyme solutions. The cyclic voltammetry analysis showed that immobilized enzymes on GO paper showed an improvement in the activity of β-galactose when compared to free enzyme solution, as well as enzyme immobilized on a glassy carbon electrode.

Fingerprint construction through genotyping by sequencing for applied breeding in Brassica rapa

This study evaluated genotyping by sequencing (GBS) protocol for fingerprinting Brassica rapa and the data derived were more reliable than the re-sequencing data of B. rapa. Of the 10 enzyme solutions used to analyze the numbers of genotypes and single nucleotide polymorphisms (SNPs) in B. rapa, five solutions showed better results, namely: A (HaeIII, 450–500 bp), E (RsaI+HaeIII, 500–550 bp), F (RsaI+HaeIII, 500–600 bp), G (RsaI+HaeIII, ‘All’ fragment), and J (RsaI+EcoRV-HF®, ‘All’ fragment). The five enzyme solutions showed less than 40% similarity in different individuals from various samples, and 90% similarity in between two individuals from one sample. The E enzyme solution was most suitable for fingerprinting B. rapa revealing well-distributed SNPs in the whole genome. Of the 82 highly inbred lines and 18 F1 lines of B. rapa sequenced by GBS in E enzyme solution, known parents of 10 F1 lines were verified and male parents were discovered for 8 F1 lines that had only known female parents. This study provided a valuable method for screening parents for F1 lines in B. rapa for applied breeding through efficient evaluation of GBS with varied library construction strategies.

Safety Evaluation and Whole Genome Sequencing of Aspergillus japonicas PJ01 Reveal Its Potential to Degrade Citrus Segments in Juice Processing

Aspergillus japonicas PJ01 (A. japonicas PJ01) is a strain isolated from the rotten branches. In previ-ous studies, it was shown that it can produce complex enzymes to degrade polysaccharide com-ponents. In this study, we evaluated the safety of its crude enzyme solution. Acute oral toxicity, subchronic toxicity, micronucleus and sperm malformation tests all validated the high biologi-cal safety for the crude enzymes. Secondly, we carried out the citrus segment degradation ex-periment of crude enzyme solution. Compared with the control group, the crude enzyme solu-tion of A. japonicas PJ01 can completely degrade the segments in 50 min, which provides the basis for enzymatic peeling during juice processing. The whole genome sequencing showed that the genome of A. japonicus PJ01 has a GC content of 51.37% with a size of 36204647 bp, and encoded 10070 genes. GO, COG, KEGG and CAZy databases were used in gene annotation analyses. Pathway enrichment showed many genes related to carbohydrate metabolism, rich in genes re-lated to pectinase, xylanase and carboxylcellulase. Therefore, the complex enzyme produced by A. japonicus PJ01 can be used in gizzard juice processing to achieve efficient enzymatic decapsu-lation.

Effect of Enzyme Treatments on Protoplast Isolation from Leaves of Vetiver (Vetiveria spp.)

Protoplast isolation is a first and important step for establishing a new plant with desired traits through protoplast fusion technology. This experiments were conducted to evaluate various concentration of enzymes and incubation time on protoplast yield and viability in two vetiver ecotypes, Kamphaeng Phet 2 (Vetiveria zizanioides Nash) and Prachuap Khiri Khan (V. nemoralis A.Camus). The results revealed that protoplast yields were significantly affected by different enzyme treatments. The highest protoplast yield (6.12x105 protoplasts/ml) and high viability (98.61%) in Kamphaeng Phet 2 was obtained through the process of cell wall digestion when treated with enzyme solution containing 0.5% (w/v) cellulase onozuka R-10 and 0.5% (w/v) macerozyme R-10 in combination. While, the optimal enzyme solution for protoplast isolation from leaves of Prachuap Khiri Khan was the combination of 1.0% (w/v) cellulase onozuka R-10 and 0.4% (w/v) macerozyme R-10, resulting in the highest yield (6.80x105 protoplasts/ml) and viability (96.56%) of protoplasts. Meanwhile, incubation time of 24 h with the optimal enzyme solution resulted in the highest protoplast yields of both ecotypes. Our findings have the potential to generate an efficient protocol to isolate the protoplast from leaves of vetiver which can be used for further research studies in protoplast culture and fusion for vetiver improvement. Keywords: Cellulase onozuka R-10, Macerozyme R-10, Protoplast isolation, Vetiver

Streptomyces sp. K47 alkaline proteases: partial purification and analysis by zymography

Microbial enzymes are used as organic catalysts in different industrial processes. In this study, we aimed to produce and investigate alkaline proteases from a novel actinobacterium strain isolated from a Black Sea marine sediment. The optimal production conditions for Streptomyces sp. K47 alkaline proteases was 4-days incubation at 28ºC in a salt-free medium buffered with 50 mM Tris-HCl buffer (pH 9.0) and containing glucose (1.0%, w v-1) and yeast extract (0.5%, w v-1). The enzyme solution was partially purified using (NH4)2SO4 precipitation (40-70%). After desalting, it was purified 1-84 fold with a recovery of 19.42%. Zymogram analyses revealed the presence of more than one protease enzyme. The enzyme solution exhibited maximum activity at pH 9.0 and 37ºC, remaining stable after a 2-hour incubation at all tested conditions. Streptomyces sp. K47 has the potential to be used in industrial processes because of its ability to produce multiple protease enzymes displaying stability in a broad pH and temperature range.

Effect of eco-enzymes prepared from selected organic waste on domestic waste water treatment

Water Pollution has become a major problem with increasing urbanisation and rapid industrialisation. Despite the abundance of water, pollution causes the water to be less useful and more harmful to health, environment, and life on our planet. In past few years, many researchers have focussed on use of biological & physical treatment methods that are cost-effective and cause no harm to the environment instead of chemical methods. The aim of the present work is to study the effect of organic solid wastes in the form of orange peels, marigold flowers, and neem leaves on domestic wastewater treatment. Eco-enzyme solutions were prepared using Dr. Rosukon’s method from the wastes mentioned which involves mixing jaggery along with the wastes and water in the ratio of 1:3:10. The eco-enzyme solution was then allowed to be prepared through 90 days of fermentation process. The three eco-enzyme solutions – after 10 days of filtration – were then mixed with domestic wastewater samples individually keeping 90 astewater and 10% eco-enzyme solution. The results after 50 days of digestion period suggests that orange eco-enzyme was the most effective in reducing Total Dissolved Solids (TDS) while Marigold eco-enzyme was most effective in reducing Chemical Oxygen Demand (COD).

Enzyme-Assisted Method for Phycobiliproteins Extraction from Porphyra and Evaluation of Their Bioactivity

Due to the poor protein availability of algae in their unprocessed form, development of extraction methods for phycobiliproteins is of great significance. This study aimed to extract phycoerythrin (PE) and phycocyanin (PC) from Porphyra via bacterial enzymatic hydrolysis and to evaluate their bioactivity. To induce enzyme production, Porphyra powder was added into the culture medium of two marine bacterial strains. The pH and enzyme activity of the cultured supernatant, namely crude enzyme solution, were significantly raised. For PE and PC extraction, Porphyra were incubated within crude enzyme solution with homogenization and ultrasonication followed by ultrafiltration process. After distinguishing by fast performance liquid chromatography (FPLC), three major fractions were observed and identified as R-PE, R-PC and small molecular PE by high-performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis (PAGE) analysis. With respect to bioactivity, these three fractions exhibited free radical scavenging and antioxidant activities in a various degree. In addition, the angiotensin-converting-enzyme (ACE) inhibitory activity of both R-PE and R-PC fractions was observed in a concentration-dependent manner. Taken together, the employed process of bacterial enzymatic hydrolysis is suggested to be a feasible method to obtain PE and PC from Porphyra without limiting their bioactivity.

Improvement of Displacement Efficiency in Heavy Oil Reservoirs with Enzyme

In this study, a systematical technique has been developed to experimentally and numerically evaluate the displacement efficiency in heavy oil reservoirs with enzyme under different conditions. Firstly, dynamic interfacial tensions (IFTs) between enzyme solution and heavy oil are measured with a pendant-drop tensiometer, while effects of pressure, temperature, enzyme concentration, and contact time of enzyme and heavy oil on equilibrium IFT were systematically examined and analyzed. After waterflooding, enzyme flooding was carried out in sandpacks to evaluate its potential to enhance heavy oil recovery at high water-cut stage. Numerical simulation was then performed to identify the underlying mechanisms accounting for the enzyme flooding performance. Subsequently, a total of 18 scenarios were designed to simulate and examine effects of the injection modes and temperature on oil recovery. Except for pressure, temperature, enzyme concentration, and contact time are found to impose a great impact on the equilibrium IFTs, i.e., a high temperature, a high enzyme concentration, and a long contact time reduce the equilibrium IFTs. All three enzyme flooding tests with different enzyme concentrations show the superior recovery performance in comparison to that of pure waterflooding. In addition to the IFT reduction, modification of relative permeability curves is found to be the main reason responsible for further mobilizing the residual heavy oil. A large slug size of enzyme solution usually leads to a high recovery factor, although its incremental oil production is gradually decreased. Plus, temperature is found to have a great effect on the recovery factor of enzyme flooding likely owing to reduction of both oil viscosity and IFT.

Gastric Enzyme Supplementation Inhibits Food Allergy in a BALB/c Mouse Model

Impaired gastric digestion due to suppressed gastric acidity enhances the risk for food allergy development. In the current study, we aimed to evaluate the impact of a supported gastric digestion via application of a pharmaceutical gastric enzyme solution (GES) on food allergy development and allergic reactions in a BALB/c mouse model. The ability of the GES to restore hypoacidic conditions was tested in mice treated with gastric acid suppression medication. To evaluate the impact on allergic symptoms, mice were orally sensitized with ovalbumin (OVA) under gastric acid suppression and subjected to oral challenges with or without GES. The immune response was evaluated by measurement of antibody titers, cytokine levels, mucosal allergy effector cell influx and regulatory T-cell counts. Clinical response was objectified by core body temperature measurements after oral OVA challenge. Supplementation of GES transiently restored physiological pH levels in the stomach after pharmaceutical gastric acid suppression. During oral sensitization, supplementation of gastric enzymes significantly reduced systemic IgE, IgG1 and IgG2a levels and allergic symptoms. In food allergic mice, clinical symptoms were reduced by co-administration of the gastric enzyme solution. Support of gastric digestion efficiently prevents food allergy induction and alleviates clinical symptoms in our food allergy model.