Comparative study of 6-APA production by free and agar immobilized bacteria in nutrient broth culture.

In the present study different bacterial samples were isolated from soil of different places of Dibrugarh and screened for biotransformation ability to produce 6-Aminopenicillanic acid. Among ten isolated bacterial samples, three gram positive bacterial samples designated as AKDD-2, AKDD-4 and AKDD-6 showed the production of 6-APA from penicillin G. Assessment of production of 6-APA after incubation in penicillin G (2 mg/ml) by three different samples separately in free and agar immobilization state was done by HPLC analysis. Reusability of immobilized cells was found successful up to 14 days.

Improvement of penicillin G acylase expression in Escherichia coli through UV induced mutations

We used ultraviolet (UV) radiation to induce mutation in three locally isolated strains of Escherichia coli. Different dilutions of bacterial cultures were exposed to UV lamp of 254 nm wavelength for different time intervals at varied distances ranging from 5 to 210 sec and 5 to 100 cm. Viable colonies were screened for mutants with an increased production of penicillin G acylase (PGA) and a reduced production of β-lactamase, which are the desired properties of PGA producing industrial strains. A survival curve was made to get optimum exposure time and distance. The survival percentage for each exposure period was calculated and 1-5 percent survival was found useful for obtaining mutants with desired change. Screening for PGA and β-lactamase constitutive and/or deficient mutants was made by Serratia marcescens overlay test. A total of 100 survivors were selected of which 49 percent expressed PGA activity higher than the parent strain. Frequency of β-lactamase constitutive and deficient mutants was 48 and 52 percent, respectively. The best hyper-producing mutant (BDCS-N-M74), with almost negligible expression of β-lactamase, exhibited three-fold (22.5 mg 6-APA h-1 mg-1 wet cells) increase in PGA activity compared with that in the parent strain (6.7 mg 6-APA h-1 mg-1 wet cells). The results indicated the successful induction of UV mediated mutation in E. coli for PGA hyper-producing mutants lacking β-lactamase activity.

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading / Restricciones para la difusión de penicilina G acilasa inmovilizada en glicosil agarosa

Particle size and enzyme protein loading are design parameters of enzyme immobilization affecting biocatalyst performance that can be varied within broad margins. Their effect on mass transfer limitations at different bulk penicillin G concentrations has been studied with glyoxyl agarose immobilized penicillin G acylase biocatalysts of average particle size of 5·10-5m and 10·10-4m at protein loadings from 15 to 130 mg/g gel. Internal diffusional restrictions were evaluated for such biocatalysts: Thiele modulus varied from 1.17 for the small particles at the lower protein load to 5.84 for the large particles at the higher protein load. Effectiveness factors at different bulk substrate concentrations were determined for all biocatalysts, values ranging from 0.78 for small particle size at 25 mM penicillin G to 0.15 for large particle size at 2 mM penicillin G. Enzyme protein loading had a strong impact on the effectiveness factors of immobilized penicillin G acylase, being it more pronounced in the case of large particle size biocatalysts. At conditions in which 6-aminopenicillanic acid is industrially produced, all biocatalysts tested were mass-transfer limited, being this information valuable for reactor design and performance evaluation.

Specific binding of folate conjugated PGA to FR-positive tumor cells / 中国药理学通报

Aim To investigate the specific binding of folate conjugated PGA to FR-positive tumor cells.Method Folate-PGA and PGA were radiolabeled with 125I by the Iodogen method to examine the binding of PGA to FR positive HeLa cells and SKOV3 cells, or FR negative A549 cells. Results 125I-folate-PGA showed specific bound to HeLa cells and SKOV3 cells; Scatchard analysis of the data estimated the Kd of binding to be 0.11 nmol?L-1 and 0.25 nmol?L-1 respectively. 125I-folate-PGA showed virtually little specific binding to A549 cells which lack folate receptors. Conclusions folate-PGA displayed high affinity and good targeting activities for FR-positive tumor cells and the data warranted further studies for enzyme prodrug therapy.