Innovative biocatalyst synthesis of pectinolytic enzymes by cross-linking strategy: Potentially immobilised pectinases for the production of pectic-oligosaccharides from pectin.

Pectinases are outstanding multienzymes, which have the potential to produce new emerging pectic-oligosaccharides (POS) via enzymatic hydrolysis of pectin. However, free pectinase is unable to undergo repeated reaction for the production of POS. This study proposed a sustainable biocatalyst of pectinases known as cross-linked pectinase aggregates (CLPA). Pectinase from Aspergillus aculeatus was successfully precipitated using 2 mg/mL pectinase and 60 % acetone for 20 min at 20 °C, which remained 36.3 % of its initial activity. The prepared CLPA showed the highest activity recovery (85.0 %), under the optimised conditions (0.3 % (v/v) starch and glutaraldehyde mixture (St/Ga), 1.5: 1 of St/Ga, 25 °C, 1.5 h). Furthermore, pectin-degrading enzymes from various sources were used to produce different CLPA. The alteration of pectinase secondary structure gave high stability in acidic condition (pH 4), thermostability, deactivation energy and half-life, and improved storage stability at 4 °C for 30 days. Similarly to their free counterpart, the CLPA exhibited comparable enzymatic reaction kinetics and could be reused eight times with approximately 20 % of its initial activity. The developed CLPA does not only efficaciously produced POS from pectin as their free form, but also exhibited better operational stability and reusability, making it more suitable for POS production.

Molecular Modeling and Simulation of Transketolase from Orthosiphon stamineus.

BACKGROUND: Orthosiphon stamineus is a traditional medicinal plant in Southeast Asia countries with various well-known pharmacological activities such as antidiabetic, diuretics and antitumor activities. Transketolase is one of the proteins identified in the leaves of the plant and transketolase is believed able to lower blood sugar level in human through non-pancreatic mechanism. In order to understand the protein behavioral properties, 3D model of transketolase and analysis of protein structure are of obvious interest. METHODS: In the present study, 3D model of transketolase was constructed and its atomic characteristics revealed. Besides, molecular dynamic simulation of the protein at 310 K and 368 K deciphered transketolase may be a thermophilic protein as the structure does not distort even at elevated temperature. This study also used the protein at 310 K and 368 K resimulated back at 310 K environment. RESULTS: The results revealed that the protein is stable at all condition which suggest that it has high capacity to adapt at different environment not only at high temperature but also from high temperature condition to low temperature where the structure remains unchanged while retaining protein function. CONCLUSION: The thermostability properties of transketolase is beneficial for pharmaceutical industries as most of the drug making processes are at high temperature condition.

Plant Genomic DNA Extraction for Selected Herbs and Sequencing their Internal Transcribed Spacer Regions Amplified by Specific Primers.

This study was focused on plant genomic DNA extraction and sequencing from five commonly used medicinal herbs, namely Impatiens balsamina, Ficus deltoidea, Centella asiatica, Andrographis paniculata and Orthosiphon aristatus. This molecular technique is another highly reliable alternative for plant species identification besides phytochemical profiling. Three cetyl hexadecyltrimethylammonium bromide (CTAB) based methods with slight modification on incubation time, salt content and other additives were used for DNA extraction. The CTAB method of Doyle and Doyle produced higher DNA concentration from L balsamina, most probably due to the presence of ammonium acetate in the washing buffer and longer incubation time (2 h). The CTAB based method was suitable for A. paniculata because a high DNA concentration of acceptable quality was obtained for all the modified methods. However, Ο. aristatus was likely to have a lower DNA concentration (33-87 µg/g) and quality, probably due to the high concentration of phenolic compounds, particularly rosmarinic acid. The extracted genomic DNA was effectively amplified by a polymerase chain reaction using a universal primer of internal transcribed spacer (ITS), particularly AB 101 and AB 102 at the optimum annealing temperature of 48°C. The DNA sequences were analyzed by phenetic analysis and it was found that they have high similarity with the nucleotide sequences of ITS regions for similar plant species in the GenBank database of the National Center for Biotechnology Information.