Purification and In Situ Immobilization of Papain with Aqueous Two-Phase System

Papain was purified from spray-dried Carica papaya latex using aqueous two-phase system (ATPS). Then it was recovered from PEG phase by in situ immobilization or preparing cross-linked enzyme aggregates (CLEAs). The Plackett-Burman design and the central composite design (CCD) together with the response surface methodology (RSM) were used to optimize the APTS processes. The highly purified papain (96–100%) was achieved under the optimized conditions: 40% (w/w) 15 mg/ml enzyme solution, 14.33–17.65% (w/w) PEG 6000, 14.27–14.42% (w/w) NaH2PO4/K2HPO4 and pH 5.77–6.30 at 20°C. An in situ enzyme immobilization approach, carried out by directly dispersing aminated supports and chitosan beads into the PEG phase, was investigated to recover papain, in which a high immobilization yield (>90%) and activity recovery (>40%) was obtained. Moreover, CLEAs were successfully used in recovering papain from PEG phase with a hydrolytic activity hundreds times higher than the carrier-bound immobilized papain

<it>In vivo</it> functional expression of a screened <it>P. aeruginosa</it> chaperone-dependent lipase in <it>E. coli</it>

<p>Abstract</p> <p>Background</p> <p>Microbial lipases particularly <it>Pseudomonas</it> lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the <it>Pseudomonas</it> lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and its chaperone (LipB) from a screened strain named AB which belongs to <it>Pseudomonas aeruginosa</it> were overexpressed in <it>E. coli</it> with two dual expression plasmid systems to enhance the production of the active lipase LipA without <it>in vitro</it> refolding process.</p> <p>Results</p> <p>In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as <it>P. aeruginosa</it> on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene <it>lipA</it> (936 bp) and lipase specific foldase gene <it>lipB</it> (1023 bp). One single expression plasmid system <it>E. coli</it> BL21/pET28a-<it>lipAB</it> and two dual expression plasmid systems <it>E. coli</it> BL21/pETDuet-<it>lipA</it>-<it>lipB</it> and <it>E. coli</it> BL21/pACYCDuet-<it>lipA</it>-<it>lipB</it> were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by <it>E. coli</it> BL21/pET28a-<it>lipAB</it> and <it>E. coli</it> BL21/pETDuet-<it>lipA</it>-<it>lipB</it> were 1300 U/L and 3200 U/L, respectively, while the activity of the lipase expressed by <it>E. coli</it> BL21/pACYCDuet-<it>lipA</it>-<it>lipB</it> was up to 8500 U/L. The lipase LipA had an optimal temperature of 30°C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively.</p> <p>Conclusions</p> <p>The effect of different plasmid system on the active LipA expression was significantly different. pACYCDuet-<it>lipA</it>-<it>lipB</it> was more suitable for the expression of active LipA than pET28a-<it>lipAB</it> and pETDuet-<it>lipA</it>-<it>lipB</it>. The LipA showed obvious esterification activity and thus had potential biocatalytic applications. The expression method reported here can give reference for the expression of those enzymes that require chaperones.</p