Isolation and characterization of a newly identified type II restriction endonuclease from a local streptomyces sp. in Taiwan

Streptomyces chusanensis ZS-2, isolated from a soil sample in Chusan in Taiwan, was found to produce a new Type II restriction endonuclease. This restriction enzyme was designated as SchI. The purified enzyme was characterized as having a subunit mol wt of 28 kDa, and was apparently free from exonuclease activities. It cleaves the phosphodiester bond between the fourth C and the fifth G on the 5’-CCGCGG-3’ sequence of DNAs, leaving a 2-nucleotide protruding end at its 3’ site. This data suggests that SchI is an isoschizomer of SacII. In addition, based on the comparison between SchI and SacII regarding reaction parameters, it seems that SchI is a better choice of restriction enzyme for genetic analysis and mapping.     

A high-swelling reagent scaffold suitable for use in aqueous and organic solvents

A polymeric scaffold with excellent swelling properties in organic and aqueous environments is highly desirable for the medicinal chemist. Here, we demonstrate that a cross-linked polyacrylamide hydrogel that displays large swelling properties in both organic solvent and water can serve as a scaffold for the photosensitizer hematoporphyrin. Upon exposure to light, the resulting resin efficiently generates singlet oxygen which can then react with appropriate substrates.     

DISSOCIATION AND RECONSTITUTION OF IMMOBILIZED RIBULOSE-1, 5-BISPHOS PHATE CARBOXYLASE//OXYGENASE FROM TOBACCO

Ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco covalently coupled to CNBr-activated Sepharose 4B was treated with urea. Analysis by electrophoresis showed that the small subunit was dissociated at 2—2.5 mol/L urea, while the large subunit was still bound to matrix. The large subunit core, L_8, was further dissociated into monomer at 3 mol/L urea. It is suggested that RuBPCase is coupled to Sepharose by virtue of ε-NH_2 on a large subunit. The activity of the immobilized enzyme was inversely proportional to the amount of small subunit dissociated by urea. The dissociated small subunits were almost completely bound back to the S-depleted immobilized RuBPCase, if the urea concentration was diluted to 0.5 mol/L. The enzyme activity could be recovered nearly to 100％. The activity of the S-depleted enzyme was linearly correlated on the concentration of small subunits in solution. These results indicate that the small subunit plays an important role in the maintenance of RuBPCase activity.     

Studies on the immobilization of glucuronidase (part 1)

beta-Glucuronidase (EC 3.2.1.31) was immobilized on various organic and inorganic carriers by different methods. Optimum coupling conditions have been worked out. The immobilization were characterized and compared to each other. Parameters resulting in most stable preparations with high activities are discussed.     

Development of monolithic enzymatic reactors in glass microchips for the quantitative determination of enzyme substrates using the example of glucose determination via immobilized glucose oxidase

A one-step procedure for the immobilization of glucose oxidase in fused-silica capillaries and in glass microchips was developed based on enzyme entrapment in a polyacrylamide-based monolithic column. The inner capillary surface was silanized with gamma-methacryloxypropyltrimethoxysilane (gamma-MAPS) to allow covalent binding of the gel to the surface. The composition of the polymer was optimized to prevent the formation of bubbles, allow liquid transportation by electroosmotic flow and to maintain the enzymatic activity. These requirements resulted in the addition of polyethylene glycol and poly(acrylic acid) to the acrylamide mixture. The gel containing the enzyme was formed in situ in the capillaries, respectively, in one channel of the microchip. In the microchip, it was limited to the sample injection channel by accordingly controlled silanization of the inner capillary surface. Glucose was detected via the amperometric determination of hydrogen peroxide. A linear correlation between signals and glucose concentration was observed from 0.05 to 1.1 mM glucose with a correlation coefficient of 0.999. The enzymatic monolithic microreactor showed no loss of activity during 8 h of continuous use and during storage in the running buffer at 4 degrees C for about 2 months. Interferents, such as ascorbic acid, were separated from the analyte electrophoretically, so that glucose could be quantified in diluted juices.     

Shaping and Mechanical Reinforcement of Silica Aerogel Biocatalysts with Ceramic Fiber Felts

The synthesis of silica aerogels reinforced with either carbon or silica fibre felts and which encapsulate the lipase PS of Amano (LPS AB025407) obtained from Burkholderia cepacia is described. The materials were further shaped by moulding them in Teflon^® tubes. The silica aerogels were synthesized with various ratios of hydrophobic groups and dried according to the supercritical CO₂ method. Both types of reinforcements improve the catalytic activity of the material per mass of lipase. The fibre felts reinforcements also enable the encapsulation of higher concentrations of lipase. The materials were shaped into small moulded monoliths, which were readily washed and recycled without significant mechanical deterioration or loss of catalytic activity. In addition, hydrophobic carbon felts reinforce more efficiently silica aerogels that incorporate a high ratio of hydrophobic groups, while silica felts strengthen those aerogels that carry a low proportion of hydrophobic groups.