E N H A N C E D    E N Z Y M E    A C T I V I T Y
 
 
 
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Introduction  
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Enzymes assume many conformations. However, the native structure is the most energetically preferred. Any alteration to the native structure may lead to loss of an enzyme's activity, a phenomenon called denaturation. Since the native structure is maintained mostly by the weak forces of hydrogen bonding and electrostatic and hydrophobic interactions, enzymes can easily be denatured during purification, storage, transport and use.  

A novel method uses cyclodextrins (CDs) without detergents to renature or refold a solution of denatured, inactive enzymes. The technology is expected to provide enzymes in higher specific activity.  

Research Corporation Technologies (RCT) applied for patent protection and manages the technology on behalf of Central Michigan University and the inventors, Drs. Ajit Sharma and Nadarajah Karuppiah.  

RCT seeks to commercialize the method through exclusive licenses by enzyme type. Examples of economically significant enzymes produced in medium-to-high purity are lactase, glucose oxidase, penicillin G acylase and thermolysin. 
 
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Inventive concept  
 		 CDs are water-soluble, doughnut-shaped molecules that possess a water-like outer surface and an oil-like inner surface. Some amino-acid side chains (residues) of enzymes have affinities for the inner surface of CDs. Once an enzyme is completely unfolded into a nearly linear chain of amino acids, these residues contact the inner surface of the CDs. The remaining amino acids interact with the water-like properties of the CD's outer lip; this causes the enzyme to collapse upon itself, facilitating the natural folding mechanism.  

No chemical bond forms between the enzyme and the CD, so the interaction is reproduced in solution, and over time the enzyme equilibrates into its low-energy active state. 
 
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Benefits Cyclodextrins provide four main advantages over in-vitro folding aids or renaturants such as sugars, surfactants, polyethylene glycol and polyamino acids:  
  •  CDs provide higher yields of renatured enzymes (>90%) than other methods and additives (<40~). 
  •  CDs can inhibit aggregation without interfering with protein refolding. Therefore, CD renaturation can be performed at high enzyme concentrations (0.5 to 1.0 mg/mL). This lowers large-scale process costs by reducing process volumes. 
  •  Alpha-CD (cyclohexaamylose) is commercially available in bulk quantities. CDs are priced competitively ($33 per pound in bulk) to other specialty chemicals used in the industry. 
  •  CDs can be separated from renatured enzymes by methods commonly used in the industry such as ultrafiltration or dialysis. This allows CDs to be recycled, further enhancing the economics of the process. 
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Typical procedure A sample containing inactive enzyme is denatured at a high concentration (50 mg/mL) using denaturants such as guanidinium chloride or urea. The enzyme can be renatured by rapid dilution into a solution of 5-10% buffered a-CD.  

Alternatively, active enzyme can be obtained by dialysis of the denatured solution against a 5-10% buffered a-CD solution. The guanidinium chloride or urea in the spent CD solution can be removed by filtration, thereby recycling the CD solution.  

Finished enzyme purity can be monitored, since literature references have reported common HPLC methods to detect cyclodextrins in protein matrices at sensitivities of <10 ppb.  
 
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Refolding of inactive enzyme in commercial samples The renaturation of commercially available carbonic anhydrase provides an impressive demonstration of this technology. The inventors denatured the sample, then renatured it to 144% of the original activity using CDs (Figure 1).  
  
  
  
  
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  Flgure 1. A vial of carbonic anhydrase solid (lyophilized) was obtained (Sigma #C2522). Buffer (50 mM Tris-sulfate, pH 8.5) was added and the amount of active carbonic anhydrase was determined by assaying for the enzyme by its esterase activity. This is given a value of 100%. GuHCI was then added so that the final protein concentration was 6.2 mg/mL. The vial was incubated overnight at room temperature to completely unfold all the active and inactive enzyme.  

The unfolded enzyme was refolded in the presence of buffered a-CD (10% w/v a-CD in 50mM Tris-sulfate buffer, pH 8.5). Protein concentration during refolding was 0.3 mg/mL.  

The final GuHCI concentration was 0.3 M. The vial was again incubated for 30 hours at room temperature. The amount of active enzyme was again determined by its esterase activity and compared to that obtained initially.  
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 Examples of enzyme sources that could undergo reactivation include finished enzyme products, chromatographic tailings, enzyme aggregates from ultrafiltration processes, and wastes from salt precipitations.  
 
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Refolding with a-CD compared to PEG The refolding of a-amylase with a-CD was compared with a well-known protein-folding aid, polyethylene glycol (3% PEG in 50 mM Tris sulfate buffer, pH 8.5). Renaturation was performed at a protein concentration of 0.5 mg/mL using 50 mM Tris sulfate buffer, pH 8.5 (Figure 2).   
  
Figure 2
 
Folding aid 
Enzyme activity (U/L)
Control (no folding aid)
 65
PEG (molecular weight 8000)
70
PEG (molecular weight 6000) 
123
a -CD (10%) 
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2025
  
The unoptimized results demonstrate that a-CD is a much better folding agent than PEG. Similar results were obtained with carbonic anhydrase.  
  
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High protein concentrations during refolding means lower processing costs  Processing costs to renature enzymes are minimized sine CD renaturation can be performed at high enzyme concentrations. Smaller process volumes are required to practice the technology compared to using other refolding aids. This results in lower costs for capital equipment, labor and raw materials.  

To illustrate, a commercial sample of a-amylase was renatured at variable enzyme concentrations with and without ? -CD (Figure 3). Higher enzyme activity was achieved at higher enzyme concentrations.  

The graph shows the recovery of native protein (U/L) obtained after 2 hr refolding with renaturation buffer (0 M Tris-Cl, pH 8.0) with and without a-CD (10%) at room temperature. Protein concentration during refolding was 0.0125 to 0.2 mg/mL.  

An explanation for obtaining higher renatured activity at higher enzyme concentrations may be provided by the results of the experiment described below.  
  

  
      Figure 3. Alpha-amylase from B. Iicheniformis (Sigma #A4551) was denatured in a urealacid mixture (8 M urea, 0.1 M HCI) for 18 hours. Unfolding was monitored by fluorescence spectra and enzyme activity (3, 5-dinitrosalicyclic method of Rick and Stegbauer in Methods of EnzymaticAnalysis, H.U. Bergmeyer, ea., Vol. 2, p. 885).
Carbonic anhydrase was submitted to four renaturing processes: a-, b-, g- and without CD. In each experiment, the amount of enzyme aggregation was measured over time (Figure 4).  
  
  
         
         
         
      Figure 4. Native enzyme (33 mM) was denatured overnight in 6.8 M GuHCI. In each case, protein was diluted to 1.7 pM and 0.034 M GuHCI, with renaturation buffer, containing 50 mM CD.  

      When denatured CAB in GuHCI was rapidly diluted with 50 mM Tris-sulfate buffer at pH 8.5, aggregation was observed immediately and monitored by light scattering at 400nm. Aggregation increased with time and then stabilized after about 5 min.  

      When the denatured protein was renatured in the presence of CDs under the same conditions, light scattering due to aggregation was significantly reduced.  
        
       

The ability of cyclodextrins to inhibit CAB aggregation was on the order of a-CD > hydroxypropyl p-CD > y-CD. Inhibition of CAB aggregation was enhanced with increasing concentration of CD in the renaturation buffer (data not shown).  
 
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Reactivation kinetics in the presence of cyclodextrins The CD-assisted reactivation of enzymes is a relatively fast process. The reaction kinetics observed in the reactivation of carbonic anhydrase (CAB) are typical for this technology.  

Reactivation kinetics of CAB at aggregating conditions in the presence of a, ,6 and y CDs and no CDs are shown in Figure 5. Alpha CD at l00mM provided over 80% recovery in less than an hour and then gradually reached a plateau of greater than 90% recovery within five hours of renaturation.  
  

      Figure 5. Denatured CAB (333 pM) was diluted to 17 pM protein and 0.34 M GuHCI with 50mM Tris-sulfate buffer, pH 8.5, containing 1 00 mM CD.  

      Recovery of esterase activity (compared to the native enzyme) was then measured as a function of time after dilution.

The highest recovery of activity was achieved with a-CD, followed by hydroxypropyl b-CD and g-CD. In the absence of cyclodextrin, the recovery of enzyme activity is only about 40%.  
 
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Effect of  
temperature and pH on cyclodextrin-assisted enzyme refolding  
 
 		 No complicated renaturation process conditions are necessary. Neutral or alkaline pH and ambient temperature can be used. Figures 6 and 7 demonstrate the effect of temperature and pH on the CD-assisted refolding of carbonic anhydrase. Optimal yield of active protein was observed between 25°C and 37°C and between pH 7-9.  
  
  
         

         

         
         
ABOUT RCT Research Corporation Technologies is the premier provider of technology transfer services to North American universities. Based on revenues and distributions, RCT is the world's most successful independent technology management organization.  

RCT identifies, appraises, protects and commercializes inventions developed at research organizations. Its scientists, lawyers, and experts in marketing, communications, and information retrieval work with industrial contacts to bring inventions to market.  

RCT licenses technologies, invests in invention development, forms new companies and participates in joint ventures.  

Project revenues for 1994 totaled $59.8 million, and RCT distributed $38.2 million to inventors and institutions. The company appraised 671 invention disclosures in 1994 from 144 research institutions. 
 
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