Effective extraction of elastase from Bacillus sp. fermentation broth using aqueous two-phase system

Xu, Ying; He, Guoqing; Li, Jingjun

doi:10.1631/jzus.2005.b1087

Cited by 19 publications

(15 citation statements)

References 29 publications

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“…Elastase hydrolyzes insoluble elastin much more efficiently than other proteases and has been widely applied in many aspects, such as a medicine to cure hyperlipidemia and arteriosclerosis (8), and producing soluble elastin which could be used in cosmetics (9) and as a meat tenderizer in the food industry (10,II).…”

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Cloning and Overexpression of Extracellular Elastase from Pseudomonas Aeruginosa

et al. 2013

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This study was an attempt to overexpress the extracellular elastase from Pseuodomonas aeruginosa in Escherichia coli and characterize the level of purified enzymes of recombinant bacterium. The gene encoding an elastase natively produced by Pseudomonas aeruginosa was cloned and overexpressed in Escherichia coli using pET-32a system and the resultant recombinant elastase was purified and compared with the native elastase gene. The 1497 bp gene was amplified and sub cloned in pET-32a and subsequently transformed into E. coli BL21. The media assay, SDS-PAGE and Western blotting were carried out to analyze the results, and the extracellular enzyme was purified to detect enzyme activity of recombinant E. coli. Nucleotide sequencing of the DNA insert from the clone revealed that the protease activity corresponded to an open reading frame consisting of 1497 bp coding for a 53.69-kDa protein.The clear zones around the recombinant colonies on skim milk agar as well as sharp band on 53-kD size on SDS-PAGE and Western blotting confirm the correct expression of elastase enzyme. Bacterial culture containing pET-32a-IasB showed high enzyme activity around 670 J.lg elastase ml". The results showed that elastase has potential to be produced industrially and be applied in medicine, food, etc. divisions.

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confidence: 99%

“…The reaction has been found to be a complex process of at least three steps. However, the mechanism still remains unknown (10).…”

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Cloning and Overexpression of Extracellular Elastase from Pseudomonas Aeruginosa

et al. 2013

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“…That means, the output responses are situated in the vicinity of the optimal experimental region, thus the significant factors can be optimized by RSM in the next DoE step [7,14]. On the other hand, when the output responses are far away from the optimal experimental region no difference or curvature is existing, and therefore steepest ascent [16,18,23,24] or steepest descent [25] experiments have to be carried out in order to increase or decrease the output responses, respectively, thus reaching the proximity of the optimal experimental region and investigate the experimental direction [7,9]. These steeptest ascent/descent experiments are initially performed at the center-point of the significant factors and each factor level is increased or decreased according to the magnitude of the main effects [7].…”

Section: Steepest Ascent / Descent Methodsmentioning

confidence: 99%

“…Moreover, FFD and fFD usually include 3-5 replicated center-point experiments which are carried out for an evaluation of the pure experimental error [9,13,[15][16][17][18]. These replicated experiments are called center-points because they are performed in the center of the experimental region located between the high and low levels, thus increasing the factor levels, at which the center level is denoted by 0 in a design matrix [13].…”

Section: Screening Of Significant Factorsmentioning

confidence: 99%

Optimization of PEG–salt aqueous two-phase systems by design of experiments

Glyk

Solle

Scheper

et al. 2015

Chemometrics and Intelligent Laboratory Systems

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Since the mechanism governing the partitioning behavior of biomolecules, such as proteins and enzymes, in polyethylene glycol (PEG)-salt aqueous two-phase systems (ATPS) is complex and not easily predictable, many laborious experiments have to be performed for an optimization of these systems, causing increased overall cost. However, the multivariate statistical design of experiments (DoE) methodology is representing a promising and efficient optimization technique which can overcome the limitations of traditional optimization methods. Therefore, DoE has emerged as a powerful and efficient optimization tool for PEGsalt ATPS, since it is faster, more efficient and cost-effective, allowing a simultaneous and rigorous evaluation of process/system parameters. In the present review, different DoE process steps are represented to highlight the feasibility of this approach to operate as a promising and efficient optimization tool, thus facilitating the evaluation of the partitioning behavior, recovery and purification of different proteins and enzymes in PEG-salt ATPS. In this context, several experimental designs, such as factorial and response surface designs, have been discussed and evaluated by statistical regression analysis and analysis of variance (ANOVA), as well as various applications of PEG-salt ATPS using DoE have been outlined which may further promote the optimization of these systems.

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“…[18] The selection of salt employed in the extraction system is also one of the key points of this technique. [19,20] To ensure the efficiency of the extraction, the partitioning of lipase from R. microsporus was carried out by employing various phase-forming salts, namely Na-citrate, MgSO 4 , Na 2 HPO 4 and (NH 4 ) 2 SO 4 with 12% PEG 4000 Da. Salts are often used in ATPS to facilitate the partitioning of the target molecules between the phases.…”

Section: Selection Of Phase-forming Saltmentioning

confidence: 99%

Extraction of lipase fromRhizopus microsporusfermentation culture by aqueous two-phase partitioning

Anvari

2015

Biotechnology & Biotechnological Equipment

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The purification of a desired protein from the complex mixture of biomolecules in a fermentation broth is one of the main challenges in industrial-scale biotechnological processes. For partial purification of industrial enzymes, one possible approach is to use aqueous two-phase extraction (APTE) as a primary downstream processing step. In the present study, the feasibility of utilizing ATPE for the purification of lipase from Rhizopus microsporus fermentation culture was investigated. The effects of the phase composition and salt type, molecular weight of the polyethylene glycol (PEG), pH of the system, sample loading and addition of neutral salts on lipase partitioning were investigated at 24 C. In most of the examined aqueous two-phase systems (ATPSs), lipase showed affinity to the top phase. Optimum conditions for lipase purification were obtained in 20% PEG 2000/12% (NH 4 ) 2 SO 4 , with 5% Na 2 CO 3 addition at pH D 8 for 30% crude load. For the optimized ATPS, the recovery yield and purification factor in the top phase were determined to be 92.3% and 19.8, respectively.

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Effective extraction of elastase from Bacillus sp. fermentation broth using aqueous two-phase system

Cited by 19 publications

References 29 publications

Cloning and Overexpression of Extracellular Elastase from Pseudomonas Aeruginosa

Cloning and Overexpression of Extracellular Elastase from Pseudomonas Aeruginosa

Optimization of PEG–salt aqueous two-phase systems by design of experiments

Extraction of lipase fromRhizopus microsporusfermentation culture by aqueous two-phase partitioning

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