Abstract
BackgroundToo many different protein and enzyme purification techniques have been reported, especially, chromatographic techniques. Apart from low recovery, these multi-step methods are complicated, time consuming, high operating cost. So, alternative beneficially methods are still required. Since, the outstanding advantages of aqueous two phase system (ATPS) such as simple, low cost, high recovery and scalable, ATPS have been used to purify various enzymes. To improve purification efficiency, parameters affected to enzyme recovery or purity was investigated. The objectives of the present study were to optimize of alkaline protease recovery from giant catfish fish viscera by using ATPS and to study of hydrolytic patterns against gelatin.ResultsUsing 70% (w/w) crude enzyme extract (CE) in system (15% PEG2000-15% sodium citrate) provided the highest recovery, PF and KE. At unmodified pH (8.5) gave the best recovery and PF with compare to other pHs of the system. The addition of 1% (w/w) NaCl showed the recovery (64.18%), 3.33-fold and 15.09 of KE compared to the system without NaCl. After addition of 10% (w/w) sodium citrate in the second ATPS cycle, the highest protease recovery (365.53%) and PF (11.60-fold) were obtained. Thus, the top phase from the system was subjected to further studied. The protein bands with molecular weights (MWs) of 20, 24, 27, 36, 94 and 130 kDa appeared on the protein stained gel and also exhibited clear zone on casein-substrate gel electrophoresis. The β, α1, α2 of skin gelatin extensively degraded into small molecules when treated with 10 units of the extracted alkaline protease compared to those of the level of 0.21 units of Flavourzyme.ConclusionsRepetitive ATPS is the alternative strategy to increase both recovery and purity of the alkaline protease from farmed giant catfish viscera. Extracted alkaline protease exposed very high effectiveness in gelatin hydrolysis. It is suggested that the alkaline protease from this fish viscera can further be used in protein hydrolysate production.
Highlights
Too many different protein and enzyme purification techniques have been reported, especially, chromatographic techniques
The loaded crude enzyme extract (CE) in the enzyme partitioning had played a major role in aqueous two phase system (ATPS)
It illustrated that the KE, recovery, and purification fold (PF) of extracted protease were gradually increased for six different amounts of CE manipulated
Summary
Too many different protein and enzyme purification techniques have been reported, especially, chromatographic techniques. The outstanding advantages of aqueous two phase system (ATPS) such as simple, low cost, high recovery and scalable, ATPS have been used to purify various enzymes. Most previous works reported that to purify protease from fish digestive organs involved several methods, including ammonium sulphate precipitation [3], size exclusion chromatography [4], ion-exchange chromatography [5], Aqueous two phase system (ATPS) could be an efficient method for the recovery of protease due to the ease and lower cost [8]. ATPS have several advantages in comparison with conventional methods for the isolation and purification of proteins such as low cost, nontoxic, the possibility of application on a large scale and the short time required for reaching equilibrium [9]. The pH value and the presence of electrolytes in the system have a pronounced effect on the partitioning of proteins between the two phases [12]
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