Non-complexed four cascade enzyme mixture: simple purification and synergetic co-stabilization
Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabil...
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| description | Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9) mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment. |
| doi_str_mv | 10.1371/journal.pone.0061500 |
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However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9) mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0061500</identifier><identifier>PMID: 23585905</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Access control ; Albumin ; Aldolase ; Ammonia ; Bacterial Proteins - chemistry ; Bacterial Proteins - isolation & purification ; Biocatalysis ; Biological products ; Biology ; Bovine serum albumin ; Cellulose ; Cloning ; Clostridium thermocellum ; Clostridium thermocellum - chemistry ; Clostridium thermocellum - enzymology ; Dehydrogenases ; E coli ; Engineering ; Enzyme Assays ; Enzyme Stability ; Enzymes ; Fructose ; Fructose-Bisphosphatase - chemistry ; Fructose-Bisphosphatase - isolation & purification ; Fructose-Bisphosphate Aldolase - chemistry ; Fructose-Bisphosphate Aldolase - isolation & purification ; Glucose ; Glucose-6-Phosphate Isomerase - chemistry ; Glucose-6-Phosphate Isomerase - isolation & purification ; Half-Life ; Heat treatment ; Kinetics ; Macromolecules ; Microorganisms ; Phosphoglucose isomerase ; Plasmids ; Precipitation ; Precipitation (Meteorology) ; Protein expression ; Proteins ; Serum albumin ; Serum Albumin, Bovine - chemistry ; Stability ; Stabilization ; Sulfates ; Synthetic biology ; Temperature ; Thermophiles ; Thermophilic microorganisms ; Thermotoga maritima - chemistry ; Thermotoga maritima - enzymology ; Thermus thermophilus - chemistry ; Thermus thermophilus - enzymology ; Trends ; Triose-phosphate isomerase ; Triose-Phosphate Isomerase - chemistry ; Triose-Phosphate Isomerase - isolation & purification</subject><ispartof>PloS one, 2013-04, Vol.8 (4), p.e61500-e61500</ispartof><rights>COPYRIGHT 2013 Public Library of Science</rights><rights>2013 Myung and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9) mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.</description><subject>Access control</subject><subject>Albumin</subject><subject>Aldolase</subject><subject>Ammonia</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - isolation & purification</subject><subject>Biocatalysis</subject><subject>Biological products</subject><subject>Biology</subject><subject>Bovine serum albumin</subject><subject>Cellulose</subject><subject>Cloning</subject><subject>Clostridium thermocellum</subject><subject>Clostridium thermocellum - chemistry</subject><subject>Clostridium thermocellum - enzymology</subject><subject>Dehydrogenases</subject><subject>E coli</subject><subject>Engineering</subject><subject>Enzyme Assays</subject><subject>Enzyme Stability</subject><subject>Enzymes</subject><subject>Fructose</subject><subject>Fructose-Bisphosphatase - 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chemistry</subject><subject>Thermotoga maritima - enzymology</subject><subject>Thermus thermophilus - chemistry</subject><subject>Thermus thermophilus - enzymology</subject><subject>Trends</subject><subject>Triose-phosphate isomerase</subject><subject>Triose-Phosphate Isomerase - chemistry</subject><subject>Triose-Phosphate Isomerase - isolation & purification</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk0tv1DAQxyMEoqXwDRBEQkJwyOJH_AgHpKrisVJFJV5HLMexd71K4tRO0G4_Pc5uWm1QD5xszfzmP57xTJI8h2ABMYPvNm7wrawXnWv1AgAKCQAPklNYYJRRBPDDo_tJ8iSEDQAEc0ofJycIE04KQE6T319dmynXdLXe6io1UTRVMihZ6VS3N7tGp43d9oPX79NgRyztBm-NVbK3rk1lW6Vh12q_0r1VqXJZ6GVpa3uz9z9NHhlZB_1sOs-Sn58-_rj4kl1efV5enF9mihaoz1BZ5kWOcsxMRZXkgORYk6rChSEqGmlJmOG8rJjJsTKGlZAgUxBEI65Nhc-SlwfdrnZBTK0JAmIMeIEpRpFYHojKyY3ovG2k3wknrdgbnF8J6WMJtRas4FwxhYApdc4QKXOOOKEFwaiC0RG1PkzZhrLRldJt72U9E517WrsWK_dHYIog3z_mzSTg3fWgQy8aG5Sua9lqN4zvRowhzjiN6Kt_0Purm6iVjAXY1riYV42i4jxnHFMIGYjU4h5Kjp_dWBXnyNhonwW8nQVEptfbfiWHEMTy-7f_Z69-zdnXR-xay7pfB1cP48iEOZgfQOVdCF6buyZDIMY1uO2GGNdATGsQw14cf9Bd0O3c47-x6AM9</recordid><startdate>20130409</startdate><enddate>20130409</enddate><creator>Myung, Suwan</creator><creator>Zhang, Y-H Percival</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</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>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20130409</creationdate><title>Non-complexed four cascade enzyme mixture: simple purification and synergetic co-stabilization</title><author>Myung, Suwan ; Zhang, Y-H Percival</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-2bb4942437fd6ca80543e5dd39f5c37f6b57f88bd7f43cff7b152f9526d6cefd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Access control</topic><topic>Albumin</topic><topic>Aldolase</topic><topic>Ammonia</topic><topic>Bacterial Proteins - 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However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9) mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23585905</pmid><doi>10.1371/journal.pone.0061500</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Access control Albumin Aldolase Ammonia Bacterial Proteins - chemistry Bacterial Proteins - isolation & purification Biocatalysis Biological products Biology Bovine serum albumin Cellulose Cloning Clostridium thermocellum Clostridium thermocellum - chemistry Clostridium thermocellum - enzymology Dehydrogenases E coli Engineering Enzyme Assays Enzyme Stability Enzymes Fructose Fructose-Bisphosphatase - chemistry Fructose-Bisphosphatase - isolation & purification Fructose-Bisphosphate Aldolase - chemistry Fructose-Bisphosphate Aldolase - isolation & purification Glucose Glucose-6-Phosphate Isomerase - chemistry Glucose-6-Phosphate Isomerase - isolation & purification Half-Life Heat treatment Kinetics Macromolecules Microorganisms Phosphoglucose isomerase Plasmids Precipitation Precipitation (Meteorology) Protein expression Proteins Serum albumin Serum Albumin, Bovine - chemistry Stability Stabilization Sulfates Synthetic biology Temperature Thermophiles Thermophilic microorganisms Thermotoga maritima - chemistry Thermotoga maritima - enzymology Thermus thermophilus - chemistry Thermus thermophilus - enzymology Trends Triose-phosphate isomerase Triose-Phosphate Isomerase - chemistry Triose-Phosphate Isomerase - isolation & purification |
| title | Non-complexed four cascade enzyme mixture: simple purification and synergetic co-stabilization |
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