Altering the activation mechanism in Thermomyces lanuginosus lipase

Jakob Skjold-Jørgensen; Jesper Vind; Allan Svendsen; Morten Jannik Bjerrum

doi:10.1021/bi500233h

Altering the activation mechanism in Thermomyces lanuginosus lipase

Jakob Skjold-Jørgensen, Jesper Vind, Allan Svendsen, Morten Jannik Bjerrum

Kemisk Institut

34 Citationer (Scopus)

Abstract

It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.

Originalsprog	Engelsk
Tidsskrift	Biochemistry
Vol/bind	53
Udgave nummer	25
Sider (fra-til)	4152-4160
Antal sider	9
ISSN	0006-2960
DOI	https://doi.org/10.1021/bi500233h
Status	Udgivet - 1 jul. 2014

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10.1021/bi500233h

Citationsformater

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title = "Altering the activation mechanism in Thermomyces lanuginosus lipase",

abstract = "It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.",

keywords = "Amino Acid Sequence, Aspergillus, Butyrates, Carboxylic Ester Hydrolases, Decanoates, Enzyme Activation, Eurotiales, Fungal Proteins, Hydrolysis, Hydrophobic and Hydrophilic Interactions, Lipase, Models, Molecular, Molecular Sequence Data, Mutation, Nitrophenols, Protein Conformation",

author = "Jakob Skjold-J{\o}rgensen and Jesper Vind and Allan Svendsen and Bjerrum, {Morten Jannik}",

year = "2014",

month = jul,

day = "1",

doi = "10.1021/bi500233h",

language = "English",

volume = "53",

pages = "4152--4160",

journal = "Biochemistry",

issn = "0006-2960",

publisher = "American Chemical Society",

number = "25",

}

TY - JOUR

T1 - Altering the activation mechanism in Thermomyces lanuginosus lipase

AU - Skjold-Jørgensen, Jakob

AU - Vind, Jesper

AU - Svendsen, Allan

AU - Bjerrum, Morten Jannik

PY - 2014/7/1

Y1 - 2014/7/1

N2 - It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.

AB - It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.

KW - Amino Acid Sequence

KW - Aspergillus

KW - Butyrates

KW - Carboxylic Ester Hydrolases

KW - Decanoates

KW - Enzyme Activation

KW - Eurotiales

KW - Fungal Proteins

KW - Hydrolysis

KW - Hydrophobic and Hydrophilic Interactions

KW - Lipase

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutation

KW - Nitrophenols

KW - Protein Conformation

U2 - 10.1021/bi500233h

DO - 10.1021/bi500233h

M3 - Journal article

C2 - 24870718

SN - 0006-2960

VL - 53

SP - 4152

EP - 4160

JO - Biochemistry

JF - Biochemistry

IS - 25

ER -

Altering the activation mechanism in Thermomyces lanuginosus lipase

Abstract

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