Production and characterization of keratinolytic protease from new wool-degrading Bacillus species isolated from Egyptian ecosystem.

Novel keratin-degrading bacteria were isolated from sand soil samples collected from Minia Governorate, Egypt. In this study, the isolates were identified as Bacillus amyloliquefaciens MA20 and Bacillus subtilis MA21 based on morphological and biochemical characteristics as well as 16S rRNA gene sequencing. B. amyloliquefaciens MA20 and B. subtilis MA21 produced alkaline keratinolytic serine protease when cultivated in mineral medium containing 1% of wool straight off sheep as sole carbon and nitrogen source. The two strains were observed to degrade wool completely to powder at pH 7 and 37°C within 5 days. Under these conditions the maximum activity of proteases produced by B. amyloliquefaciens MA20 and B. subtilis MA21 was 922 and 814 U/ml, respectively. The proteases exhibited optimum temperature and pH at 60°C and 9, respectively. However, the keratinolytic proteases were stable in broad range of temperature and pH values towards casein Hammerstein. Furthermore the protease inhibitor studies indicated that the produced proteases belong to serine protease because of their sensitivity to PMSF while they were inhibited partially in presence of EDTA. The two proteases are stable in most of the used organic solvents and enhanced by metals suggesting their potential use in biotechnological applications such as wool industry.

Purification and characterization of camel (Camelus dromedarius) milk amylase.

Skimmed camel milk contains 59,900 U/L amylase, which is 39,363 times less than serum and plasma amylase. Camel milk beta-amylase was purified as a 61 KDa band using DEAE-Sepharose and Sephadex G-100 and yielded 561 U/mg. The optimum working pH, Km and temperature were 7.0, 13.6 mg/Lstarch, 30-40 degrees C, respectively. The enzyme has been shown higher affinity toward amylose and soluble starch than glycogen, amylopectin, dextrin, or pullulan. Magnesium chloride, CaCl(2) and NaCl activated the amylase, while EDTA and EGTA decreased its activity. While its activity was increased in the presence of Triton X-100 and Triton X-114. Phenylmethanesulfonyl fluoride did not show any effect on enzyme activity. However, the enzyme activity was inhibited by urea, SDS, DTNB, iodoacetamide, N-ethylmalimide, aprotinin, and trypsin inhibitor. It worked on starch to yield a maltose. Scanning electron microscope images demonstrated a nano-degrading ability on starch granules from various sources (potato, corn, cassava, and rice).

Synthesis of the human insulin gene: protein expression, scaling up and bioactivity.

Optimized Synthetic human insulin gene was preferred to easy of cloning, plasmid stability, and protein expression away from the native sequence and its rare codons. Two steps to obtain the insulin, so we assembled the gene of 293 bp using a battery of overlapped synthetic oligos, then cloned into pET101directional TOPO expression vector downstream to the T7 promoter. The proinsulin products were produced as inclusion bodies in E. coli at a level of 10%. The batch cultivation of the strain yielded 6 g/L, while the high cell density of fed-batch cultivation yielded 46 g/L. The proinsulin purification yielded 110 mg/gram cell weight, and 1.3 mg/gram of a bioactive insulin. The native insulin was generated by enzymatic conversion of chemically processed proinsulin. The produced insulin was matched with that of a commercial aqueous version at a level of enzyme immunoassys, SDS-PAGE, RP-HPLC, and bioactivity. The present results showed that the produced insulin has a comparable biochemical and potency similar to that of commercial one.