Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii
The gene encoding a thermostable β-glucosidase ( cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kD...
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| Veröffentlicht in: | Protein expression and purification 2004-12, Vol.38 (2), p.248-257 |
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| creator | Murray, Patrick Aro, Nina Collins, Catherine Grassick, Alice Penttilä, Merja Saloheimo, Markku Tuohy, Maria |
| description | The gene encoding a thermostable β-glucosidase (
cel3a) was isolated from the thermophilic fungus
Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus
Trichoderma reesei. The
cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59
kDa.
Tal. emersonii β-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank
AAP57755) from
T. reesei, and a β-glucosidase from
Aspergillus Niger (GenBank
CAB75696), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130
kDa subunits with 17 potential N-glycosylation sites and a previously unreported β-glucosidase activity produced extracellularly by
Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5
°C and half life of 62
min at 65
°C and was a specific β-glucosidase with no β-galactosidase side activity. Cel3a had a high specific activity against
p-nitrophenyl-β-
d-glucopyranoside (
V
max, 512
IU/mg) and was competitively inhibited by glucose (
k
i, 0.254
mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose. |
| doi_str_mv | 10.1016/j.pep.2004.08.006 |
| format | Article |
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cel3a) was isolated from the thermophilic fungus
Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus
Trichoderma reesei. The
cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59
kDa.
Tal. emersonii β-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank
AAP57755) from
T. reesei, and a β-glucosidase from
Aspergillus Niger (GenBank
CAB75696), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130
kDa subunits with 17 potential N-glycosylation sites and a previously unreported β-glucosidase activity produced extracellularly by
Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5
°C and half life of 62
min at 65
°C and was a specific β-glucosidase with no β-galactosidase side activity. Cel3a had a high specific activity against
p-nitrophenyl-β-
d-glucopyranoside (
V
max, 512
IU/mg) and was competitively inhibited by glucose (
k
i, 0.254
mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.</description><identifier>ISSN: 1046-5928</identifier><identifier>EISSN: 1096-0279</identifier><identifier>DOI: 10.1016/j.pep.2004.08.006</identifier><identifier>PMID: 15555940</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; beta-Glucosidase - chemistry ; beta-Glucosidase - genetics ; beta-Glucosidase - isolation & purification ; Cloning, Molecular ; Enzyme Stability - physiology ; Fungus ; Gene cloning ; Gene Expression Regulation, Enzymologic ; Genetic Vectors - genetics ; Glycosyl hydrolase ; Heterologous expression ; Molecular Sequence Data ; Molecular Weight ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - isolation & purification ; Secondary structure prediction ; Sequence Alignment ; Sequence Homology, Amino Acid ; Talaromyces - enzymology ; Talaromyces - genetics ; Talaromyces - growth & development ; Temperature ; Thermostable ; Trichoderma - genetics ; β-glucosidase</subject><ispartof>Protein expression and purification, 2004-12, Vol.38 (2), p.248-257</ispartof><rights>2004 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c349t-94371503c2d5af93f0d7f5ef5b4f18c12dc28d3f1e4e1af40e3671b204d25b433</citedby><cites>FETCH-LOGICAL-c349t-94371503c2d5af93f0d7f5ef5b4f18c12dc28d3f1e4e1af40e3671b204d25b433</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.pep.2004.08.006$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3548,27923,27924,45994</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15555940$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Murray, Patrick</creatorcontrib><creatorcontrib>Aro, Nina</creatorcontrib><creatorcontrib>Collins, Catherine</creatorcontrib><creatorcontrib>Grassick, Alice</creatorcontrib><creatorcontrib>Penttilä, Merja</creatorcontrib><creatorcontrib>Saloheimo, Markku</creatorcontrib><creatorcontrib>Tuohy, Maria</creatorcontrib><title>Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii</title><title>Protein expression and purification</title><addtitle>Protein Expr Purif</addtitle><description>The gene encoding a thermostable β-glucosidase (
cel3a) was isolated from the thermophilic fungus
Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus
Trichoderma reesei. The
cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59
kDa.
Tal. emersonii β-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank
AAP57755) from
T. reesei, and a β-glucosidase from
Aspergillus Niger (GenBank
CAB75696), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130
kDa subunits with 17 potential N-glycosylation sites and a previously unreported β-glucosidase activity produced extracellularly by
Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5
°C and half life of 62
min at 65
°C and was a specific β-glucosidase with no β-galactosidase side activity. Cel3a had a high specific activity against
p-nitrophenyl-β-
d-glucopyranoside (
V
max, 512
IU/mg) and was competitively inhibited by glucose (
k
i, 0.254
mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.</description><subject>Amino Acid Sequence</subject><subject>beta-Glucosidase - chemistry</subject><subject>beta-Glucosidase - genetics</subject><subject>beta-Glucosidase - isolation & purification</subject><subject>Cloning, Molecular</subject><subject>Enzyme Stability - physiology</subject><subject>Fungus</subject><subject>Gene cloning</subject><subject>Gene Expression Regulation, Enzymologic</subject><subject>Genetic Vectors - genetics</subject><subject>Glycosyl hydrolase</subject><subject>Heterologous expression</subject><subject>Molecular Sequence Data</subject><subject>Molecular Weight</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Secondary structure prediction</subject><subject>Sequence Alignment</subject><subject>Sequence Homology, Amino Acid</subject><subject>Talaromyces - enzymology</subject><subject>Talaromyces - genetics</subject><subject>Talaromyces - growth & development</subject><subject>Temperature</subject><subject>Thermostable</subject><subject>Trichoderma - genetics</subject><subject>β-glucosidase</subject><issn>1046-5928</issn><issn>1096-0279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc-KFDEQh4Mo7rr6AF4kJ2_dVjr9L3iSZdWFBS_jOWSSyk6G7k6b6hbnYXwJH8RnMs0MeNu6pAjf7wfFx9hbAaUA0X44ljPOZQVQl9CXAO0zdi1AtQVUnXq-7XVbNKrqr9groiOAEC00L9mVaPKoGq7Z77tfc0KiECceJr5LwR6iwzQanhAJAzeT4_ZgkrELpkBm2dDoueHLIXORFrMfkHszhuHEJf_7p3gcVhspOEP5P8VxI_m41ZoFM3QOzocwBMv9Oj2uxHdmMBk9WSSOIyaKUwiv2QtvBsI3l_eGff98t7v9Wjx8-3J_--mhsLJWS6Fq2YkGpK1cY7ySHlznG_TNvvait6Jytuqd9AJrFMbXgLLtxL6C2lWZkfKGvT_3zin-WJEWPQayOAxmwriSbjtQsu1VBsUZtCkSJfR6TmE06aQF6M2JPursRG9ONPQ6O8mZd5fydT-i-5-4SMjAxzOA-cSfAZMmG3Cy6EJCu2gXwxP1_wAPmqHC</recordid><startdate>20041201</startdate><enddate>20041201</enddate><creator>Murray, Patrick</creator><creator>Aro, Nina</creator><creator>Collins, Catherine</creator><creator>Grassick, Alice</creator><creator>Penttilä, Merja</creator><creator>Saloheimo, Markku</creator><creator>Tuohy, Maria</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20041201</creationdate><title>Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii</title><author>Murray, Patrick ; Aro, Nina ; Collins, Catherine ; Grassick, Alice ; Penttilä, Merja ; Saloheimo, Markku ; Tuohy, Maria</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c349t-94371503c2d5af93f0d7f5ef5b4f18c12dc28d3f1e4e1af40e3671b204d25b433</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Amino Acid Sequence</topic><topic>beta-Glucosidase - chemistry</topic><topic>beta-Glucosidase - genetics</topic><topic>beta-Glucosidase - isolation & purification</topic><topic>Cloning, Molecular</topic><topic>Enzyme Stability - physiology</topic><topic>Fungus</topic><topic>Gene cloning</topic><topic>Gene Expression Regulation, Enzymologic</topic><topic>Genetic Vectors - genetics</topic><topic>Glycosyl hydrolase</topic><topic>Heterologous expression</topic><topic>Molecular Sequence Data</topic><topic>Molecular Weight</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - isolation & purification</topic><topic>Secondary structure prediction</topic><topic>Sequence Alignment</topic><topic>Sequence Homology, Amino Acid</topic><topic>Talaromyces - enzymology</topic><topic>Talaromyces - genetics</topic><topic>Talaromyces - growth & development</topic><topic>Temperature</topic><topic>Thermostable</topic><topic>Trichoderma - genetics</topic><topic>β-glucosidase</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Murray, Patrick</creatorcontrib><creatorcontrib>Aro, Nina</creatorcontrib><creatorcontrib>Collins, Catherine</creatorcontrib><creatorcontrib>Grassick, Alice</creatorcontrib><creatorcontrib>Penttilä, Merja</creatorcontrib><creatorcontrib>Saloheimo, Markku</creatorcontrib><creatorcontrib>Tuohy, Maria</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Protein expression and purification</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Murray, Patrick</au><au>Aro, Nina</au><au>Collins, Catherine</au><au>Grassick, Alice</au><au>Penttilä, Merja</au><au>Saloheimo, Markku</au><au>Tuohy, Maria</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii</atitle><jtitle>Protein expression and purification</jtitle><addtitle>Protein Expr Purif</addtitle><date>2004-12-01</date><risdate>2004</risdate><volume>38</volume><issue>2</issue><spage>248</spage><epage>257</epage><pages>248-257</pages><issn>1046-5928</issn><eissn>1096-0279</eissn><abstract>The gene encoding a thermostable β-glucosidase (
cel3a) was isolated from the thermophilic fungus
Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus
Trichoderma reesei. The
cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59
kDa.
Tal. emersonii β-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank
AAP57755) from
T. reesei, and a β-glucosidase from
Aspergillus Niger (GenBank
CAB75696), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130
kDa subunits with 17 potential N-glycosylation sites and a previously unreported β-glucosidase activity produced extracellularly by
Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5
°C and half life of 62
min at 65
°C and was a specific β-glucosidase with no β-galactosidase side activity. Cel3a had a high specific activity against
p-nitrophenyl-β-
d-glucopyranoside (
V
max, 512
IU/mg) and was competitively inhibited by glucose (
k
i, 0.254
mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>15555940</pmid><doi>10.1016/j.pep.2004.08.006</doi><tpages>10</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1046-5928 |
| ispartof | Protein expression and purification, 2004-12, Vol.38 (2), p.248-257 |
| issn | 1046-5928 1096-0279 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_67093689 |
| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Amino Acid Sequence beta-Glucosidase - chemistry beta-Glucosidase - genetics beta-Glucosidase - isolation & purification Cloning, Molecular Enzyme Stability - physiology Fungus Gene cloning Gene Expression Regulation, Enzymologic Genetic Vectors - genetics Glycosyl hydrolase Heterologous expression Molecular Sequence Data Molecular Weight Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Secondary structure prediction Sequence Alignment Sequence Homology, Amino Acid Talaromyces - enzymology Talaromyces - genetics Talaromyces - growth & development Temperature Thermostable Trichoderma - genetics β-glucosidase |
| title | Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii |
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