A new organic solvent-tolerant strain Bacillus megaterium AU02 which secretes an organic solvent-tolerant protease was isolated from milk industry waste. Statistical methods were employed to achieve optimum protease production of 43.6 U/ml in shake flask cultures. The productivity of the protease was increased to 53 U/ml when cultivated under controlled conditions in a 7-L fermentor. The protease was purified to homogeneity by a three-step process with 24 % yield and specific activity of 5,375 U/mg. The molecular mass of the protease was found to be 59 kDa. The enzyme was active over a wide range of pH (6.0–9.0), with an optimum activity at pH 7.0 and temperature from 40 to 70 °C having an optimum activity at 50 °C. The thermal stability of the enzyme increased significantly in the presence of CaCl2, and it retained 90 % activity at 50 °C for 3 h. The Km and Vmax values were determined as 0.722 mg/ml and 0.018 U/mg respectively. The metalloprotease exhibited significant stability in the presence of organic solvents with log P values more than 2.5, nonionic detergents and oxidising agent. An attempt was made to test the synthesis of aspartame precursor (Cbz-Asp-Phe-NH2) which was catalysed by AU02 protease in the presence of 50 % DMSO. These properties of AU02 protease make it an ideal choice for enzymatic peptide synthesis in organic media.

The activity of six hydrolytic enzymescarboxyl esterase, acid phosphatase, alkaline phosphatase, b-galactosidase, b-glucosidase and b-hexosaminidase, were studied in different regions of the normal human brain tissue obtained at autopsy. Protein estimation and activities of the hydrolytic enzymes with respective substrates were assayed by spectrophotometric and spectroflourometric methods. Amongst the eight regions of the brain-frontal, parietal, occipital, temporal, thalamus, cerebellum and hippocampus, the pineal gland showed highest activity for all hydrolytic enzymes studied except for carboxyl esterase. Among six hydrolases studied, hexosaminidase exhibited highest activity in all regions of the human brain while alkaline phosphatase activity was the least amongst all regions studied. A majority of the enzymes studied showed higher activity in gray matter as compared to the white matter except acid phosphatase and b-glucosidase which exhibited higher activity in the white matter. The most significant finding in the present study was the high activity of all hydrolytic enzymes noted in the pineal gland as compared to all other regions of the human brain. Such a finding has not been hitherto reported earlier in human brain tissue samples. If the specific activities of these enzymes are to be considered as any functional index, then pineal gland may be more metabolically active tissue with respect to the hydrolytic function as compared to the other regions of the brain.

A bacterial strain producing both a protease and an esterase was isolated from the seed of Tamarindus indica. The isolate was identified as Lysinibacillus fusiformis AU01 by phylogenetic analysis of the 16S rDNA sequence. The isolate produced an extracellular protease and an intracellular esterase simultaneously under the same culture conditions. Statistical methods, i.e., Plackett-Burman followed by response surface methodology (RSM), were applied to optimize media components and culture conditions in 50 mL shake flask cultures. Culturing in shake flasks with the optimized medium resulted in a 6-fold and a 3.5-fold increase in protease and esterase production, respectively, when compared to nutrient medium. The productivity of the protease and esterase was increased to 75 U/mL and 370 U/mL when cultivated under controlled conditions in a 3-L bioreactor. The extracellular protease was purified to 34.6 fold with 38.8 % recovery and the molecular mass of the enzyme was found to be 48 kDa. The partial amino acid sequence of the protease was determined by MALDI-TOF-MS analysis. The optimum temperature and pH for protease activity were found to be pH 9.0 and 40 °C. The inhibition of purified protease by EDTA and PMSF confirmed that the enzyme belonged to the family of serine metalloproteases. The enzyme was found to be stable in the presence of some hydrophobic and hydrophilic solvents. The ability of AU01 protease to dissociate monolayer cells for subculturing adherent cell lines was also investigated.

Activity staining or zymography is a technique to detect enzymes based on their function/activity toward a specific substrate. Multiple enzyme-producing microbes secrete enzymes along with other proteins at varying time points during fermentation. The technique of zymography can be used to detect functionality of enzymes in complex protein/other enzyme mixtures. The protein bands corresponding to specific enzyme among other enzymes/proteins can be located by polyacrylamide gel electrophoresis (PAGE) followed by zymogram analysis. This can be employed to locate the secretion pattern of protein/enzyme from intracellular region to extracellular medium. Here we describe simple method for detection and cellular localization of esterases and protease secreted by single microbial strain in one PAGE gel.