Purification Of Cutinase Research Articles

Optimization of Cutinase Purification using a Hydrophobic Interaction Membrane Chromatographic Process by Response Surface Methodology

Several methods have been demonstrated in the recovery and purification of cutinase; however, hydrophobic interaction membrane chromatography (HIMC) has yet to be implemented for this purpose. In this study, two factors, that is, buffer pH and ammonium sulphate concentration for HIMC operation, were optimized using response surface methodology (RSM), aiming to obtain the highest cutinase purification factor and recovery possible. The experimental design used was a central composite design (CCD) comprised of 13 experimental runs. The analysis of variance (ANOVA) indicated that a quadratic model best represented the system. Based on the R 2 values, it can be concluded that around 90% of the variability in the system can be explained by the fitted models. The optimized HIMC conditions were buffer pH 6.0 and 1.3 M ammonium sulphate, which was expected to provide 27-fold cutinase purification factor and 85% recovery. However, the actual values deviated from the predicted values with 13% and 18% error, respectively. The statistical analysis showed that only the ammonium sulphate concentration had a great influence on cutinase purification factor. Meanwhile, for cutinase recovery, both parameters were important in determining the optimal condition for cutinase adsorption and desorption to the chromatographic media.

Comparison of Cutinase Separation in Different Chromatographic Media

Cutinase is a hydrolytic enzyme that has both properties of lipase and esterase, thus finding its use in many areas. Previous studies have investigated both upstream and downstream processes for cutinase production from microbial source. However, no study has yet to address the use of membrane chromatography for cutinase purification, which is more favourable in terms of process resolution and product throughput as compared to the conventional gel chromatography. Hydrophobic interaction was chosen as the separation mechanism for cutinase purification in this study. The optimisation of cutinase purification in two different types of chromatographic media; conventional packed-gel and membrane matrix, were pre-determined by the best compromise between the recovery and purity of the purified cutinase. It was found that the optimised condition were of pH 4.0 and 1.0 M ammonium sulfate for the conventional column (50% recovery, 4.8-fold purity) and pH 6.0 with 1.5 M ammonium sulfate for the membrane–matrix column(87% recovery, 30-fold purity). Preferential interaction analysis was used to describe the protein chromatographic behaviour in each chromatographic media. Graph of natural algorithm of protein retention data to the function of salt concentration at pH 4.0 and 6.0 for each column were plotted. It was found that at the optimum pH condition for gel-packed column, a small amount of ammonium sulfate was sufficient to achieve maximum cutinase recovery and purity since the effect of salt at that particular pH were less significant. Consequently, the number of released water molecules were calculated and it was observed that for membrane column, larger number of water was released at pH 6.0 illustrating more protein were bounded to the stationary phase, thus explaining the optimum pH condition of the particular column.

Influence of tryptophan tags on the purification of cutinase, secreted by a recombinant Saccharomyces cerevisiae, using cationic expanded bed adsorption and hydrophobic interaction chromatography

During cationic bed adsorption (EBA), with cutinase with varying length tryptophan tags (WP)(2)and (WP)(4), 33% and 10% of adsorption capacity and 80% and 32% eluted specific activity were observed in relation to wild type (wt)-cutinase in the conventional process. Therefore, as the hydrophobicity of the protein increases, it is important to integrate the EBA step with a hydrophobic interaction chromatography (HIC) process. As the length of the hydrophobic tag-(WP) increases from n = 2 to n = 4, the purification factor obtained by HIC was 1.8 and 2.2-fold higher than wt-cutinase. However, the recovery yield obtained in HIC decreases substantially as the length of hydrophobic tag increases (97%, 84% and 70% for wt-cutinase, cutinase-(WP)(2) and cutinase-(WP)(4)). The integration of two purification steps, EBA followed by HIC, resulted in the highest overall purity level for cutinase-(WP)(2), and the highest overall recovery yield for wt-cutinase. When optimizing the design of a hydrophobic tag fused to a protein secreted by Saccharomyces cerevisiae it must be considered that the cultivation parameters could impair the downstream process, and consequently the optimum tag is not necessarily the one that presents the highest purification factor in HIC.

Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strain

By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock. However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step. Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process. From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose. This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose. Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose. The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth. Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture.

The search for a peptide ligand targeting the lipolytic enzyme cutinase

A constrained nonapeptide phage display library was evaluated as a potential source of affinity ligand(s) for purification of cutinase, a lipolytic enzyme. After seven cycles of biopanning, 500 clones were isolated and individually tested for their capability to interact with cutinase. Three out of six sequenced clones carrying a cutinase-specific constrained peptide showed the same insert sequence (CRLHHWRYC). Sequences of two of the other clones highlighted a LXXW motif as a critical determinant in the make-up of a cutinase-specific sequence. Although the affinity of the most commonly found peptide for cutinase is low, we suggest that LXXW motif may be a suitable starting point in the development of affinity peptides suitable for use in the study and purification of cutinase.

Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinase

AbstractThe effect of culture media composition, and fermentation conditions and strategy on the growth and cutinase production of recombinant Saccharomyces cerevisiae and subsequent cutinase purification by expanded bed adsorption (EBA) was studied. The reduction in the amount of yeast extract used as nitrogen source from 20 g dm−3 to 10 g dm−3 in batch cultures led to a 29% decrease in the heterologous cutinase production, while the 5% cutinase dynamic adsorption capacity (q5%) on the cation Streamline SP XL was increased 6.7‐fold. By dilution of the whole fermentation broth, performed with the lowest yeast extract content, which reduces conductivity, the q5% was additionally increased by 1.9‐fold. After implementation of a fed‐batch strategy the cutinase concentration, cutinase yield on carbon source, cutinase yield on nitrogen source and productivity were increased by 10.8‐, 2.9‐, 5.3‐ and 6.4‐fold, respectively, in relation to the previously‐mentioned batch fermentation. However, the increased cutinase production was compromised by heterologous protein loss during the EBA recovery operation and the cutinase dynamic adsorption capacity and purification productivity decreased by 90% and 75%, respectively. Thus, target protein production by S cerevisiae fermentation and a downstream process with EBA cannot be considered as separate entities, where the understanding of the factors that affect the interactions among them are crucial towards optimization of the overall production process of heterologous proteins.© 2002 Society of Chemical Industry

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH 4) 2SO 4], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (p I) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.

Hydrolysis of plant cuticle by plant pathogens. Purification, amino acid composition, and molecular weight of two isozymes of cutinase and a nonspecific esterase from Fusarium solani f. pisi.

The extracellular fluid of the plant pathogen, Fusarium solani f. pisi, grown on the plant cuticular polymer, cutin, was shown to contain cutinase and p-nitrophenyl palmitate hydrolase activities (R.E. Purdy and P.E. Kolattukudy (1973), Arch. Biochem. Biophys. 159, 61). From this extracellular fluid two isozymes of cutinase and a nonspecific esterase (p-nitrophenyl palmitate hydrolase) were isolated using Sephedex G-100 gel filtration, QAE-Sephadex chromatography, and SE-Sephedex chromatography. Phenolics contained in the extracellular fluid were found to be associated with the cutinase but not with the nonspecific esterase, and the phenolic materials were removed from cutinase at the QAE-Sephedex step. A 34-fold purification of the nonspecific esterase and a 6.5-fold purification of cutinase were achieved by the procedure described. The two isozymes of cutinase (I and II) and the nonspecific esterase were homogeneous as judged by polyacrylamide disc gel electrophoresis and sedimentation equilibrium centrifugation. Molecular weights of cutinase I, cutinase II, and the nonspecific esterase were determined by Sephedex G-100 gel filtration, sedimentation equilibrium centrifugation, amino acid composition, and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. The values obtained with these techniques agreed with each other and were about 22,000 for both cutinases and 52,000 for the nonspecific esterase. The dodecyl sulfate gel electrophoresis indicated that a small portion of cutinase II contained proteolylic clips, near the middle of the polypeptide chain, and that the nonspecific esterase might also have undergone some proteolylic modification. The amino acid composition of cutinase I was similar to that of cutinase II except for the presence of a larger number of tryptophan residues in the latter, while the amino acid composition of the nonspecific esterase showed more differences from that of either cutinase.