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Optimising biocatalyst design for obtaining high transesterification activity by alpha-chymotrypsin in non-aqueous media.

Solanki K, Gupta MN - Chem Cent J (2008)

Bottom Line: Presence of trehalose resulted in EPRPs of smaller sizes.The two different forms of enzymes (EPRP and PCMC) known to show higher activity in organic solvents were found to be different only in the way the low molecular weight additive was present along with the protein.This effect disappeared where the reaction media was polar as the polar solvent (acetonitrile) is more effective in stripping off the water from the enzyme; (b) reduction in particle size as revealed by SEM.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India. kusumsolanki@gmail.com

ABSTRACT

Background: Enzymes are often used in organic solvents for catalyzing organic synthesis. Two enzyme preparations, EPRP (enzyme precipitated and rinsed with n-propanol) and PCMC (protein coated microcrystals) show much higher activities than lyophilized powders in such systems. Both preparations involve precipitation by an organic solvent. The clear understanding of why these preparations show higher catalytic activity than lyophilized powders in organic solvents is not available.

Results: It was found that EPRPs of alpha-chymotrypsin prepared by precipitation with n-propanol in the presence of trehalose contained substantial amount of trehalose (even though trehalose alone at these lower concentrations was not precipitated by n-propanol). The presence of trehalose in these EPRPs resulted in much higher transesterification rates (45.2 nmoles mg(-1) min(-1)) as compared with EPRPs prepared in the absence of trehalose (16.6 nmoles mg(-1) min(-1)) in octane. Both kinds of EPRPs gave similar initial transesterification rates in acetonitrile. Use of higher concentrations of trehalose (when trehalose alone also precipitates out), resulted in the formation of PCMCs, which showed higher transesterification rates in both octane and acetonitrile. SEM analysis showed the relative sizes of various preparations. Presence of trehalose resulted in EPRPs of smaller sizes.

Conclusion: The two different forms of enzymes (EPRP and PCMC) known to show higher activity in organic solvents were found to be different only in the way the low molecular weight additive was present along with the protein. Therefore, the enhancement in the transesterification activity in EPRPs prepared in the presence of trehalose was due to: (a) better retention of essential water layer for catalysis due to the presence of the sugar. This effect disappeared where the reaction media was polar as the polar solvent (acetonitrile) is more effective in stripping off the water from the enzyme; (b) reduction in particle size as revealed by SEM. In the case of PCMC, the enhancement in the initial rates was due to an increase in the surface area of the biocatalyst since protein is coated over the core material (trehalose or salt).It is hoped that the insight gained in this work would help in a better understanding for designing high activity biocatalyst preparation of non-aqueous media.

No MeSH data available.


Related in: MedlinePlus

Scanning electron microscopy (SEM) images of (A) EPRP of α-chymotrypsin (0 percent trehalose) precipitated into n-propanol (magnification: 5000 ×); (B) EPRP of α-chymotrypsin (5 percent trehalose) precipitated into n-propanol (magnification: 5000 ×); (C) PCMC of α-chymotrypsin (40 percent trehalose) precipitated into n-propanol (magnification: 5000 ×). SEM was carried out on a Zeiss EVO50 scanning electron microscope. Samples were dried by rinsing with anhydrous propanol, placed on a sample holder, and coated with silver before being scanned in vacuo.
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Figure 2: Scanning electron microscopy (SEM) images of (A) EPRP of α-chymotrypsin (0 percent trehalose) precipitated into n-propanol (magnification: 5000 ×); (B) EPRP of α-chymotrypsin (5 percent trehalose) precipitated into n-propanol (magnification: 5000 ×); (C) PCMC of α-chymotrypsin (40 percent trehalose) precipitated into n-propanol (magnification: 5000 ×). SEM was carried out on a Zeiss EVO50 scanning electron microscope. Samples were dried by rinsing with anhydrous propanol, placed on a sample holder, and coated with silver before being scanned in vacuo.

Mentions: The results given in Table 2 and Table 3 also clearly establishes the superiority of PCMCs over EPRPs. PCMCs were reported to give higher activity than lyophilized powders as in PCMCs, "the organic solvent dehydrates the enzymes by mechanism that minimizes denaturation and appears to leave the majority of enzyme molecules in active conformation". That mechanism as per results obtained in the present work should apply equally well to EPRPs. However, EPRPs show much lower activity than PCMCs. The present work, by establishing close relationship between EPRPs and PCMCs, also indicates that additional mechanisms for higher activity established by PCMC should exist. Presumably, the enzyme spread as a coat over the inner microcrystal core offers larger catalytic surface area. This also explains why potassium sulphate works just as well as trehalose as the core matrix in PCMCs. Fig. 2 shows the SEMs of EPRP prepared without the presence of trehalose (Fig. 2A), EPRP prepared with presence of 5 percent trehalose (Fig. 2B) and PCMC (Fig. 2C). The range of particle sizes of EPRP prepared in the presence of trehalose was found to be substantially smaller. This raises the possibility that higher surface areas available with biocatalysts of smaller sizes also contributed to higher transesterification activity. PCMCs (Fig. 2C), of course looked crystalline in nature. PCMCs, though, of bigger size range have enzyme coated over the surface. Thus, the enhancement of biocatalyst surface is much larger than even EPRPs of smaller size range.


Optimising biocatalyst design for obtaining high transesterification activity by alpha-chymotrypsin in non-aqueous media.

Solanki K, Gupta MN - Chem Cent J (2008)

Scanning electron microscopy (SEM) images of (A) EPRP of α-chymotrypsin (0 percent trehalose) precipitated into n-propanol (magnification: 5000 ×); (B) EPRP of α-chymotrypsin (5 percent trehalose) precipitated into n-propanol (magnification: 5000 ×); (C) PCMC of α-chymotrypsin (40 percent trehalose) precipitated into n-propanol (magnification: 5000 ×). SEM was carried out on a Zeiss EVO50 scanning electron microscope. Samples were dried by rinsing with anhydrous propanol, placed on a sample holder, and coated with silver before being scanned in vacuo.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262082&req=5

Figure 2: Scanning electron microscopy (SEM) images of (A) EPRP of α-chymotrypsin (0 percent trehalose) precipitated into n-propanol (magnification: 5000 ×); (B) EPRP of α-chymotrypsin (5 percent trehalose) precipitated into n-propanol (magnification: 5000 ×); (C) PCMC of α-chymotrypsin (40 percent trehalose) precipitated into n-propanol (magnification: 5000 ×). SEM was carried out on a Zeiss EVO50 scanning electron microscope. Samples were dried by rinsing with anhydrous propanol, placed on a sample holder, and coated with silver before being scanned in vacuo.
Mentions: The results given in Table 2 and Table 3 also clearly establishes the superiority of PCMCs over EPRPs. PCMCs were reported to give higher activity than lyophilized powders as in PCMCs, "the organic solvent dehydrates the enzymes by mechanism that minimizes denaturation and appears to leave the majority of enzyme molecules in active conformation". That mechanism as per results obtained in the present work should apply equally well to EPRPs. However, EPRPs show much lower activity than PCMCs. The present work, by establishing close relationship between EPRPs and PCMCs, also indicates that additional mechanisms for higher activity established by PCMC should exist. Presumably, the enzyme spread as a coat over the inner microcrystal core offers larger catalytic surface area. This also explains why potassium sulphate works just as well as trehalose as the core matrix in PCMCs. Fig. 2 shows the SEMs of EPRP prepared without the presence of trehalose (Fig. 2A), EPRP prepared with presence of 5 percent trehalose (Fig. 2B) and PCMC (Fig. 2C). The range of particle sizes of EPRP prepared in the presence of trehalose was found to be substantially smaller. This raises the possibility that higher surface areas available with biocatalysts of smaller sizes also contributed to higher transesterification activity. PCMCs (Fig. 2C), of course looked crystalline in nature. PCMCs, though, of bigger size range have enzyme coated over the surface. Thus, the enhancement of biocatalyst surface is much larger than even EPRPs of smaller size range.

Bottom Line: Presence of trehalose resulted in EPRPs of smaller sizes.The two different forms of enzymes (EPRP and PCMC) known to show higher activity in organic solvents were found to be different only in the way the low molecular weight additive was present along with the protein.This effect disappeared where the reaction media was polar as the polar solvent (acetonitrile) is more effective in stripping off the water from the enzyme; (b) reduction in particle size as revealed by SEM.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India. kusumsolanki@gmail.com

ABSTRACT

Background: Enzymes are often used in organic solvents for catalyzing organic synthesis. Two enzyme preparations, EPRP (enzyme precipitated and rinsed with n-propanol) and PCMC (protein coated microcrystals) show much higher activities than lyophilized powders in such systems. Both preparations involve precipitation by an organic solvent. The clear understanding of why these preparations show higher catalytic activity than lyophilized powders in organic solvents is not available.

Results: It was found that EPRPs of alpha-chymotrypsin prepared by precipitation with n-propanol in the presence of trehalose contained substantial amount of trehalose (even though trehalose alone at these lower concentrations was not precipitated by n-propanol). The presence of trehalose in these EPRPs resulted in much higher transesterification rates (45.2 nmoles mg(-1) min(-1)) as compared with EPRPs prepared in the absence of trehalose (16.6 nmoles mg(-1) min(-1)) in octane. Both kinds of EPRPs gave similar initial transesterification rates in acetonitrile. Use of higher concentrations of trehalose (when trehalose alone also precipitates out), resulted in the formation of PCMCs, which showed higher transesterification rates in both octane and acetonitrile. SEM analysis showed the relative sizes of various preparations. Presence of trehalose resulted in EPRPs of smaller sizes.

Conclusion: The two different forms of enzymes (EPRP and PCMC) known to show higher activity in organic solvents were found to be different only in the way the low molecular weight additive was present along with the protein. Therefore, the enhancement in the transesterification activity in EPRPs prepared in the presence of trehalose was due to: (a) better retention of essential water layer for catalysis due to the presence of the sugar. This effect disappeared where the reaction media was polar as the polar solvent (acetonitrile) is more effective in stripping off the water from the enzyme; (b) reduction in particle size as revealed by SEM. In the case of PCMC, the enhancement in the initial rates was due to an increase in the surface area of the biocatalyst since protein is coated over the core material (trehalose or salt).It is hoped that the insight gained in this work would help in a better understanding for designing high activity biocatalyst preparation of non-aqueous media.

No MeSH data available.


Related in: MedlinePlus