Understanding the Biocatalytic Potential of Lipase from Rhizopus chinensis

Lipases occupy the third position in the world market for the sale of the most industrially used enzymes, being one of the most versatile and promising classes, highlighting the genus Rhizopus chinensis, which is exceptionally useful as biocatalysts for short-chain fatty acid esterification reactions with ethanol, biodiesel, and n-heptane when used directly in the solvent-free system and in potential critical applications. In this context, the present work presents the first complete general review of the lipases of the genus Rhizopus chinensis focusing on their industrial applications, in addition to the different immobilization techniques and the main reactions as biocatalysts, since these are an excellent alternative due to their advantages in economic accessibility and respect for the environment. In addition, they have high specificity catalytic activity for esterification and transesterification reactions in the presence or absence of organic solvents; they can also have great thermophilicity and moderate pressure resistance, with their catalytic behavior modulated by changes in these conditions. This review showed that Rhizopus chinensis lipase has ample potential for many other biotechnological applications with the appropriate chemical modifications and immobilization strategy.

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Functional expression of a novel methanol-stable esterase from Geobacillus subterraneus DSM13552 for biocatalytic synthesis of cinnamyl acetate in a solvent-free system

10.21203/rs.2.15615/v4 ◽

2020 ◽

Author(s):

Xianghai Cai ◽

Lin Lin ◽

Yaling Shen ◽

wei wei ◽

Dong-zhi Wei

Keyword(s):

Elevated Temperatures ◽

Specific Activity ◽

Acyl Chain ◽

Industrial Applications ◽

Catalytic Triad ◽

Solvent Free ◽

Original Activity ◽

Free System ◽

Potential Value ◽

Solvent Free System

Abstract Background: Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing.Results: The gene estGSU753 from Geobacillus subterraneus DSM13552 was cloned, sequenced and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp and encodes 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196 and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of pNP-esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate pNP-caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60°C respectively. The resulting EstGSU753 showed remarkable stability against methanol. After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 35 h, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M).Conclusions: This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.

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Functional expression of a novel methanol-stable esterase by Geobacillus subterraneus DSM13552 for biocatalytic synthesis of cinnamyl acetate in a solvent-free system

10.21203/rs.2.15615/v2 ◽

2019 ◽

Author(s):

Xianghai Cai ◽

Lin Lin ◽

Yaling Shen ◽

wei wei ◽

Dong-zhi Wei

Keyword(s):

Elevated Temperatures ◽

Specific Activity ◽

Acyl Chain ◽

Industrial Applications ◽

Catalytic Triad ◽

Solvent Free ◽

Original Activity ◽

Free System ◽

Potential Value ◽

Solvent Free System

Abstract Background: Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing. Results: The gene estGSU753 from Geobacillus subterraneus DSM13552 was cloned, sequenced, and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp, and encoding 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196, and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate p-nitrophenyl caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60°C respectively. The resulting EstGSU753 showed remarkable stability against methanol. After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 2100 minutes, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M). Conclusions: This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.

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Functional expression of a novel methanol-stable esterase by Geobacillus subterraneus DSM13552 for biocatalytic synthesis of cinnamyl acetate in a solvent-free system

10.21203/rs.2.15615/v5 ◽

2020 ◽

Author(s):

Xianghai Cai ◽

Lin Lin ◽

Yaling Shen ◽

Wei Wei ◽

Dong-zhi Wei

Keyword(s):

Elevated Temperatures ◽

Specific Activity ◽

Acyl Chain ◽

Industrial Applications ◽

Catalytic Triad ◽

Solvent Free ◽

Original Activity ◽

Free System ◽

Potential Value ◽

Solvent Free System

Abstract Background: Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing. Results: The gene estGSU 753 from Geobacillus subterraneus DSM13552 was cloned, sequenced and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp and encodes 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196 and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of p NP-esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate p NP-caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60 °C respectively. The resulting EstGSU753 showed remarkable stability against methanol . After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 35 h, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M). Conclusions: This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.

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Functional expression of a novel methanol-stable esterase by Geobacillus subterraneus DSM13552 for biocatalytic synthesis of cinnamyl acetate in a solvent-free system

10.21203/rs.2.15615/v1 ◽

2019 ◽

Author(s):

Xianghai Cai ◽

Lin Lin ◽

Yaling Shen ◽

Wei Wei ◽

Dong-zhi Wei

Keyword(s):

Elevated Temperatures ◽

Specific Activity ◽

Acyl Chain ◽

Industrial Applications ◽

Catalytic Triad ◽

Solvent Free ◽

Original Activity ◽

Free System ◽

Potential Value ◽

Solvent Free System

Abstract Background: Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing. Results: The gene estGSU753 from Geobacillus subterraneus DSM13552 was cloned, sequenced, and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp, and encoding 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196, and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate p-nitrophenyl caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60°C respectively. The resulting EstGSU753 showed remarkable stability against methanol. After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 2100 minutes, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M). Conclusions: This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.

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Functional expression of a novel methanol-stable esterase by Geobacillus subterraneus DSM13552 for biocatalytic synthesis of cinnamyl acetate in a solvent-free system

10.21203/rs.2.15615/v3 ◽

2020 ◽

Author(s):

Xianghai Cai ◽

Lin Lin ◽

Yaling Shen ◽

wei wei ◽

Dong-zhi Wei

Keyword(s):

Elevated Temperatures ◽

Specific Activity ◽

Acyl Chain ◽

Industrial Applications ◽

Catalytic Triad ◽

Solvent Free ◽

Original Activity ◽

Free System ◽

Potential Value ◽

Solvent Free System

Abstract Background: Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing. Results: The gene estGSU753 from Geobacillus subterraneus DSM13552 was cloned, sequenced and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp and encodes 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196 and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of pNP-esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate pNP-caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60°C respectively. The resulting EstGSU753 showed remarkable stability against methanol. After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 35 h, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M). Conclusions: This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.

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LIPASE-CATALYZED SYNTHESIS OF OCTYL ACETATE IN SOLVENT-FREE SYSTEM

Catalysis in Green Chemistry and Engineering ◽

10.1615/catalgreenchemeng.2020034461 ◽

2020 ◽

Vol 3 (1) ◽

pp. 33-43

Author(s):

Meera T. Sose ◽

Virendra K. Rathod

Keyword(s):

Solvent Free ◽

Free System ◽

Octyl Acetate ◽

Solvent Free System

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Enzyme Promiscuous Activity: How to Define it and its Evolutionary Aspects

Protein and Peptide Letters ◽

10.2174/0929866527666191223141205 ◽

2020 ◽

Vol 27 (5) ◽

pp. 400-410

Author(s):

Valentina De Luca ◽

Luigi Mandrich

Keyword(s):

Gene Evolution ◽

Sequence Divergence ◽

High Specificity ◽

Industrial Applications ◽

Point Of View ◽

Enzyme Promiscuity ◽

Primary Activity ◽

Starting Point ◽

Main Activity

: Enzymes are among the most studied biological molecules because better understanding enzymes structure and activity will shed more light on their biological processes and regulation; from a biotechnological point of view there are many examples of enzymes used with the aim to obtain new products and/or to make industrial processes less invasive towards the environment. Enzymes are known for their high specificity in the recognition of a substrate but considering the particular features of an increasing number of enzymes this is not completely true, in fact, many enzymes are active on different substrates: this ability is called enzyme promiscuity. Usually, promiscuous activities have significantly lower kinetic parameters than to that of primary activity, but they have a crucial role in gene evolution. It is accepted that gene duplication followed by sequence divergence is considered a key evolutionary mechanism to generate new enzyme functions. In this way, promiscuous activities are the starting point to increase a secondary activity in the main activity and then get a new enzyme. The primary activity can be lost or reduced to a promiscuous activity. In this review we describe the differences between substrate and enzyme promiscuity, and its rule in gene evolution. From a practical point of view the knowledge of promiscuity can facilitate the in vitro progress of proteins engineering, both for biomedical and industrial applications. In particular, we report cases regarding esterases, phosphotriesterases and cytochrome P450.

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