Optimization of phenoxazinone synthase production by response surface methodology and its application in Congo red decolourization

Background: Enzymatic decolourization has been recently proposed as a promising and eco-friendly method for treatment of synthetic dye-contaminated wastewaters. However, the processes require large quantities of enzymes, attracting significant attention in developing efficient methods for mass production of multifunctional enzymes. Several methods such as response surface methodology (RSM) and orthogonal experiment have been applied to optimize the parameters in bioprocesses for enzyme production. Results: In the present study, a laccase-like enzyme, phenoxazinone synthase (PHS) originated from Streptomyces antibioticus was recombinantly expressed in Escherichia coli BL21 (DE3). The production of PHS in E. coli BL21 was optimized by response surface methodology based on Box-Behnken design. A full third-order polynomial model was generated by data analysis with Statistica 8.0 in which the optimal conditions for PHS production were calculated to be 1.525 mM CuSO4 and 16.096 hrs induction at temperature of 29.88ºC. The highest PHS production under optimal conditions was calculated to be 4098.51 U/l using the established model. Average PHS production obtained from actual production processes carried out under the calculated optimal conditions was 4052.00 U/l, very close to the value predicted by the model. Crude PHS was subsequently tested in Congo red decolourization which exhibited a low decolourization rate of 27% without mediator. Several mediators were found to improve PHS-catalyzed Congo red decolourization, with the highest rate of 73.89% obtained with 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as mediator under optimized conditions of 4000 U/l PHS activity, 10 μM ABTS, 100 μM Congo red, and 8 hrs reaction time. Conclusion: Our results indicated that PHS recombinantly produced in E. coli BL21 was a prospective enzyme for decolorizing reactive dye Congo red.

Primary cutaneous amyloidosis: A clinico-pathological study with emphasis on polarized microscopy.

Background: Primary localized cutaneous amyloidosis (PCA) is a relatively rare condition characterized by amyloid deposition in dermis without systemic involvement. Although, histopathological examination of the lesion reveals amorphous eosinophilic deposits in papillary dermis examination of congo red stained slides under polarized light will give definitive diagnosis Aims: To study the clinicopathological features of cutaneous amyloidosis emphasizing the utility of polarized light in diagnosis. Materials and Methods : A clinicopathological study of primary cutaneous amyloidosis over a period of 8 years was undertaken. All the cases, clinically diagnosed and histopathologically proven as cutaneous amyloidosis were stained with congo red and studied under polarized light. Results and Conclusions: Of the 45 cases of clinically suspected amyloidosis, 32 cases were proven histopathologically as primary cutaneous amyloidosis and confirmed by congo red stain under polarized light which showed apple green birefringence. Among the two types of PCA, lichen amyloidosis was the most common variant accounting to 65.63% with pure cases of macular amyloidosis accounting for only 15.63%. Biphasic amyloidosis was seen in 18.75%. Knee was the commonest site of involvement with pruritis being the most common symptom. Histopathologically, the most common findings were hyperkeratosis, irregular acanthosis and expansion of dermal papillae by amyloid deposits showing apple green birefringence under polarized microscope with congo red staining. Although, H and E stain gives a clue for the diagnosis of amyloid nevertheless congo red staining under polarized light forms a very sensitive and definitive method for confirmation.

Primary amyloidosis presenting as intrahepatic cholestasis.

Hepatic involvement in primary amyloidosis is not rare but is often clinically silent. A mild elevation of the serum alkaline phosphatase level and hepatomegaly are the most common findings. We report a case of primary amyloidosis in an adult male presenting with intrahepatic cholestasis where viral, drug, alcohol, and autoimmune etiologies were all excluded and the definite diagnosis was established by liver biopsy with Congo red staining. Subsequently the patient was found to have monoclonal light chain disorder.