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 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 6.2×109 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.

[1]  Alle Bruggink,et al.  Concepts of Nature in Organic Synthesis: Cascade Catalysis and Multistep Conversions in Concert , 2003 .

[2]  Jian-Jiang Zhong,et al.  Biofuel production by in vitro synthetic enzymatic pathway biotransformation. , 2010, Current opinion in biotechnology.

[3]  Joseph A. Rollin,et al.  High-yield production of dihydrogen from xylose by using a synthetic enzyme cascade in a cell-free system. , 2013, Angewandte Chemie.

[4]  Zhiguang Zhu,et al.  Toward low-cost biomanufacturing through in vitro synthetic biology: bottom-up design , 2011 .

[5]  George M Church,et al.  Synthetic biology projects in vitro. , 2006, Genome research.

[6]  Y.‐H.P. Zhang,et al.  Fructose-1,6-bisphosphatase from a hyper-thermophilic bacterium Thermotoga maritima: Characterization, metabolite stability, and its implications , 2010 .

[7]  Andreas S Bommarius,et al.  Utilizing Simple Biochemical Measurements to Predict Lifetime Output of Biocatalysts in Continuous Isothermal Processes. , 2010, Chemical engineering science.

[8]  M S Thakur,et al.  Enhancement of operational stability of an enzyme biosensor for glucose and sucrose using protein based stabilizing agents. , 2002, Biosensors & bioelectronics.

[9]  L. Bachas,et al.  Cloning, expression, and characterization of the gsdA gene encoding thermophilic glucose-6-phosphate dehydrogenase from Aquifex aeolicus , 2002, Extremophiles.

[10]  Zhiguang Zhu,et al.  Biohydrogenation from biomass sugar mediated by in vitro synthetic enzymatic pathways. , 2011, Chemistry & biology.

[11]  C. You,et al.  Facilitated substrate channeling in a self-assembled trifunctional enzyme complex. , 2012, Angewandte Chemie.

[12]  Y. Wang,et al.  A highly active phosphoglucomutase from Clostridium thermocellum: cloning, purification, characterization and enhanced thermostability , 2010, Journal of applied microbiology.

[13]  Fangfang Sun,et al.  Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases. , 2012, Biosensors & bioelectronics.

[14]  Xueyan Zhao,et al.  Enzymatic synthesis of L-lactic acid from carbon dioxide and ethanol with an inherent cofactor regeneration cycle. , 2011, Biotechnology and bioengineering.

[15]  Liang Feng,et al.  Simple bioseparations using self-cleaving elastin-like polypeptide tags , 2005, Nature Methods.

[16]  R. Ellis.  Macromolecular crowding : obvious but underappreciated , 2022 .

[17]  A thermostable recombinant transaldolase with high activity over a broad pH range , 2012, Applied Microbiology and Biotechnology.

[18]  Kenji Okano,et al.  Direct conversion of glucose to malate by synthetic metabolic engineering. , 2013, Journal of biotechnology.

[19]  Xiao‐Zhou Zhang,et al.  Ultra‐stable phosphoglucose isomerase through immobilization of cellulose‐binding module‐tagged thermophilic enzyme on low‐cost high‐capacity cellulosic adsorbent , 2011, Biotechnology progress.

[20]  Takeshi Omasa,et al.  Synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway , 2012, Microbial Cell Factories.

[21]  Volker Sieber,et al.  Cell-free metabolic engineering: production of chemicals by minimized reaction cascades. , 2012, ChemSusChem.

[22]  M. Adams,et al.  Finding and using hyperthermophilic enzymes. , 1998, Trends in biotechnology.

[23]  Sven Panke,et al.  Bioengineering novel in vitro metabolic pathways using synthetic biology. , 2007, Current opinion in microbiology.

[24]  Y.‐H.P. Zhang,et al.  Substrate channeling and enzyme complexes for biotechnological applications. , 2011, Biotechnology advances.

[25]  Y.‐H.P. Zhang,et al.  Constructing the electricity-carbohydrate-hydrogen cycle for a sustainability revolution. , 2012, Trends in biotechnology.

[26]  J. Mielenz,et al.  Spontaneous high-yield production of hydrogen from cellulosic materials and water catalyzed by enzyme cocktails. , 2009, ChemSusChem.

[27]  F. Kargı,et al.  Bioprocess Engineering: Basic Concepts , 1991 .

[28]  C. You,et al.  Self-assembly of synthetic metabolons through synthetic protein scaffolds: one-step purification, co-immobilization, and substrate channeling. , 2013, ACS synthetic biology.

[29]  James R. Swartz,et al.  Transforming biochemical engineering with cell-free biology , 2012 .

[30]  Y.‐H.P. Zhang,et al.  Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations: challenges and opportunities. , 2010, Biotechnology and bioengineering.

[31]  Fangfang Sun,et al.  Thermophilic Thermotoga maritima ribose-5-phosphate isomerase RpiB: optimized heat treatment purification and basic characterization. , 2012, Protein expression and purification.

[32]  Y.‐H.P. Zhang,et al.  Microbial Cell Factories Overexpression and Simple Purification of the Thermotoga Maritima 6-phosphogluconate Dehydrogenase in Escherichia Coli and Its Application for Nadph Regeneration , 2009 .

[33]  Chang Bs,et al.  Enzyme thermostabilization by bovine serum albumin and other proteins: evidence for hydrophobic interactions , 1995 .

[34]  J. Mielenz,et al.  High-Yield Hydrogen Production from Starch and Water by a Synthetic Enzymatic Pathway , 2007, PloS one.

[35]  Karen M Polizzi.  What is synthetic biology? , 2013, Methods in molecular biology.

[36]  Shelley D. Minteer,et al.  Enzymatic Biofuel Cell for Oxidation of Glucose to CO2 , 2012 .

[37]  Roger A Sheldon,et al.  Immobilised enzymes: carrier-bound or carrier-free? , 2003, Current opinion in biotechnology.