A process for purifying laccase from an ectomycorrhizal fruiting body is disclosed. The process includes steps of homogenization, sonication, centrifugation, filtration, affinity chromatography, ion exchange chromatography, and gel filtration. Purified laccase can also be separated into isomers

Cullings, Kenneth W.

DeSimone, Julia C.

Paavola, Chad D.

NASA Technical Reports Server

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(12) United States Patent 
Cullings et al. 
(54) METHODS FOR PURIFYING ENZYMES FOR 
MYCOREMEDIATION 
(71) Applicant: The United States of America as 
Represented by the Administrator of 
the National Aeronautics & Space 
Administration (NASA), Washington, 
DC (US) 
(72) Inventors: Kenneth W. Cullings, Ventura, CA 
(US); Julia C. DeSimone, San Jose, CA 
(US); Chad D. Paavola, Carmel, IN 
(US) 
(73) Assignee: The United States of America as 
Represented by the Administrator of 
the National Aeronautics & Space 
Administration (NASA), Washington, 
DC (US) 
(*) Notice: 	 Subject to any disclaimer, the term of this 
patent is extended or adjusted under 35 
U.S.C. 154(b) by 0 days. 
(21) Appl. No.: 13/854,620 
(22) Filed: 	 Apr. 1, 2013 
Related U.S. Application Data 
(63) Continuation-in-part of application No. 13/438,793, 
filed on Apr. 3, 2012. 
(60) Provisional application No. 61/619,906, filed on Apr. 
3, 2012, provisional application No. 61/471,605, filed 
on Apr. 4, 2011. 
(51) 	 Int. Cl. 
C12N 9198 (2006.01) 
C12N 9102 (2006.01) 
C12N 9100 (2006.01) 
(1o) Patent No.: 	 US 8,759,057 B1 
(45) Date of Patent: 	 Jun. 24, 2014 
(52) U.S. Cl. 
CPC ................ C12N 9100 (2013.01); C12N 910004 
(2013.01); C12N 9198 (2013.01) 
USPC ............................ 435/183; 435/187; 435/189 
(58) Field of Classification Search 
None 
See application file for complete search history. 
(56) 	 References Cited 
FOREIGN PATENT DOCUMENTS 
EP 	 2078755 Al * 7/2009 ................ C12P 1/02 
OTHER PUBLICATIONS 
Wang, H.X. et al. 2006. Purification of a laccase from fruiting bodies 
of the mushroom Pleurotus eryngii. Applied Microbiology and 
Biotechnology 69:521-525.* 
Cuttings, et al., Effects of Artificial Defoliation of Pines on the 
Structure and Physiology of the Soil .... Applied and Environmental 
Microbiology, Apr. 2005, 1996-2000. 
Wang, et al., Purification of a laccase from fruiting bodies of the 
mushroom Pleurotus eryngii, App Microbol Biotechnol 2006, 
69:521-525. 
* cited by examiner 
Primary Examiner John S Brusca 
Assistant Examiner Sharon M Papciak 
(74) Attorney, Agent, or Firm Christopher J. Menke; 
Robert M. Padilla 
(57) ABSTRACT 
A process for purifying laccase from an ectomycorrhizal 
fruiting body is disclosed. The process includes steps of 
homogenization, sonication, centrifugation, filtration, affin-
ity chromatography, ion exchange chromatography, and gel 
filtration. Purified laccase can also be separated into isomers. 
7 Claims, 1 Drawing Sheet 
https://ntrs.nasa.gov/search.jsp?R=20150003316 2019-08-31T11:21:54+00:00Z
U.S. Patent 	 Jun. 24, 2014 	 US 8,759,057 B1 
US 8,759,057 B1 
METHODS FOR PURIFYING ENZYMES FOR 
MYCOREMEDIATION 
CROSS-REFERENCE TO RELATED 
APPLICATIONS 
This application claims the benefit of U.S. Provisional 
Application No. 61/619,906, filed Apr. 3, 2012 and is a con-
tinuation-in-part of U.S. application Ser. No. 13/438,793, 
filed Apr. 3, 2012, which claims the benefit of U.S. Provi-
sional Application No. 61/471,605, filed Apr. 4, 2011, all 
three of which are incorporated by reference in their entirety 
herein. 
ORIGIN OF THE INVENTION 
The invention described herein was made in part by 
employees of the United States Government and may be 
manufactured and used by or for the Government of the 
United States of America for governmental purposes without 
the payment of any royalties thereon or therefor. 
BACKGROUND OF THE INVENTION 
This invention relates to bioremediation of contaminants in 
the environment. More particularly, this invention relates to 
using fungi to degrade or sequester contaminants in the envi-
ronment. 
Bioremediation is emerging as a cost effective and efficient 
way to remediate environmental hazards. The work is per-
formed naturally by microbes (bacteria and fungi) that are 
capable of breaking down the long- and short-chain organic 
backbones of solvents and fuels such as diesel. Fungi are 
capable of enhancing the natural process by as much as 
10-fold through synergistic action with natural bacteria, and 
they are also capable of rapid remediation of solvents in their 
own right. 
The fungi that perform this function are wood-rotting and 
ectomycorrhizal fungi. They naturally possess the enzymes 
necessary to break down the carbon chains found in many 
types of contamination. Studies of model white rot fungi 
(e.g., Pleurotus ostreatus) indicate that lignin-degrading 
fungi will degrade lignin and can reduce hydrocarbon con-
tamination in soils by as much as 40% in as little as one 
month. Much of the work in these reductions is performed by 
fungal laccases. 
Laccases are polyphenol oxidases that utilize a wide array 
of phenolic substrates that include lignin in wood and soil 
humic compounds, polycyclic aromatic hydrocarbons 
(PAH), and polychlorinated biphenyls (PCB's). PAH and 
PCB's are widespread soil and water contaminants of human 
origin that can accumulate via several sources, including 
diesel spills. Laccases are easily inducible, are involved in 
lignin beak-down, utilize a broad range of substrates, are 
often extracellular, and reduce toxicity of these compounds 
via immobilization to humic substances, thus lowering their 
bioavailability. Hence, laccases are considered excellent can-
didates for use in remediation. 
In one experiment known in the art, a plot of soil contami-
nated with diesel was inoculated with mycelia of oyster 
mushrooms; traditional bioremediation techniques (i.e., 
inoculation with bacteria) were used on control plots. After 
about a month, much of the polycyclic aromatic hydrocar-
bons (PAH) had reportedly been reduced to non-toxic com-
ponents in the mycelia-inoculated plots. It appears the natural 
microbial community participates with the fungi to break 
down contaminants, eventually into carbon dioxide and 
2 
water. Also known in the art, white-rot or wood-decay fungi 
have been found to be effective in breaking down aromatic 
pollutants (toxic components of petroleum), as well as chlo-
rinated compounds (certain persistent pesticides). 
5 	 H. X. Wang & T. B. Ng, Purification of a laccase from 
fruiting bodies of the mushroom Pleurotus eryngii, 69 Appl. 
Microbiol. Biotechnol. 521-525 (2006), describes a basic 
laccase purification method for the mushroom, Pleurotus 
eryngii. The disclosed method is insufficient for use in puri- 
10 fying laccases from fungi that contain more fleshy structures 
and fails to provide for separation of laccase isozymes. There-
fore, an improved, broadened, and expanded method that 
would apply to many more types of fungi is needed. 
While prior art methods of laccase purification are known 
15 and are generally suitable for their limited purposes, they 
possess certain inherent deficiencies that detract from their 
overall utility in environmental bioremediation. For example, 
they are not generally applicable to mushroom-forming 
fungi. They are generally suitable for use with some fruiting 
20 bodies, but they are not well suited for use with fleshier fungal 
species. As a further example, they do not provide for sepa-
ration of enzyme isomers and purification of such isomers. 
Bio-prospectors continue to search for new forms of 
mycoremediation and new methods of decontaminating the 
25 environment because prior art products and methods, though 
generally suitable for their limited purposes, possess certain 
inherent deficiencies that detract from their overall utility in 
environmental bioremediation. 
In view of the foregoing, it will be appreciated that provid-
30 ing improved products and methods for environmental biore-
mediation would be a significant advancement in the art. 
SUMMARY OF THE INVENTION 
35 	 An illustrative embodiment of a process according to the 
present invention for purifying laccase from an ectomycor-
rhizal fruiting body comprises: 
(A) homogenizing the fruiting body in a buffer of about pH 
7 to obtain a slurry; 
40 	 (B) sonicating the slurry to obtain a sonicated slurry; 
(C) centrifuging the sonicated slurry at a relative centrifu-
gal force of about 15,000 to about 20,000 and collecting a 
resulting first supernatant; 
(D) sonicating the first supernatant to obtain a sonicated 
45 first supernatant; 
(E) ultracentrifuging the sonicated first supernatant such 
that a resulting second supernatant separates into top and 
bottom supernatant layers, and collecting the bottom super-
natant layer; 
50 	 (F) filtering the bottom supernatant layer through a bacte- 
ria-retaining filter and collecting the filtrate; 
(G) subjecting the filtrate to DEAE cellulose chromatog-
raphy and eluting retained proteins with a first sodium chlo-
ride gradient; 
55 	 (H) removing the sodium chloride from the eluted, retained 
proteins to obtain desalted proteins, subjecting the desalted 
proteins to ion exchange chromatography, desorbing 
adsorbed proteins with a second sodium chloride gradient, 
and collecting the first desorbed peak; and 
60 	 (I) fractionating the first desorbed peak into fractions by 
gel filtration. 
BRIEF DESCRIPTION OF THE DRAWING 
65 	 FIG. 1 shows enzymatic activity of a laccase purified 
according to an illustrative embodiment of the present inven-
tion (column 6); a positive control is shown in column 9. 
US 8,759,057 B1 
3 
DETAILED DESCRIPTION OF THE INVENTION 
Before the processes of the presently claimed invention are 
disclosed and described, it is to be understood that this inven-
tion is not limited to the particular configurations, process 
steps, and materials disclosed herein as such configurations, 
process steps, and materials may vary somewhat. It is also to 
be understood that the terminology employed herein is used 
for the purpose of describing particular embodiments only 
and is not intended to be limiting since the scope of the 
present invention will be limited only by the appended claims 
and equivalents thereof. 
The publications and other reference materials referred to 
herein to describe the background of the invention and to 
provide additional detail regarding its practice are hereby 
incorporated by reference. The references discussed herein 
are provided solely for their disclosure prior to the filing date 
of the present application. Nothing herein is to be construed 
as an admission that the inventors are not entitled to antedate 
such disclosure by virtue of prior invention. 
It must be noted that, as used in this specification and the 
appended claims, the singular forms "a," "an," and "the" 
include plural referents unless the context clearly dictates 
otherwise. Thus, for example, reference to "a laccase" 
includes reference to a mixture of two or more laccases, 
reference to "an ectomycorrhizal fruiting body" includes ref-
erence to one or more of such fruiting bodies, and reference to 
"the product" includes reference to a mixture of two or more 
of such products. 
Unless defined otherwise, all technical and scientific terms 
used herein have the same meanings as commonly under-
stood by one of ordinary skill in the art to which this invention 
belongs. 
In describing and claiming the present invention, the fol-
lowing terminology will be used in accordance with the defi-
nitions set out below. 
As used herein, "comprising," "including," "containing," 
"characterized by," and grammatical equivalents thereof are 
inclusive or open-ended terms that do not exclude additional, 
unrecited elements or method steps. "Comprising" is to be 
interpreted as including the more restrictive terms "consisting 
of and "consisting essentially of." 
As mentioned above, illustrative embodiments of the 
present invention include processes for purifying laccase 
from ectomycorrhizal fruiting bodies and processes for sepa-
rating purified laccase into isomers. These processes will be 
further described in the examples that follow. The scope of the 
invention, however, will be limited only by the claims. 
Example 1 
Prior Art 
H. X. Wang & T. B. Ng, Purification of a laccase from 
fruiting bodies of the mushroom Pleurotus eryngii, 69 Appl. 
Microbiol. Biotechnol. 521-525 (2006), describes the purifi-
cation of laccase as follows: 
The fruiting bodies of Pleurotus eryngii (2 kg) were 
extracted by homogenization in distilled water (2 ml/g). 
The homogenate was filtered through cheesecloth 
before centrifugation. The resulting supernatant was 
applied to a 5x20 cm diethylaminoethyl (DEAF) cellu-
lose (Sigma) column in 10 mM Tris HCl buffer (ph 7.3). 
Following removal of unadsorbed proteins, the column 
was eluted with 0.8 M NaCl added to the Tris HCl buffer. 
The adsorbed fraction was dialyzed prior to loading on a 
2.5x20 cm column of carboxymethyl (CM) cellulose 
4 
(Sigma) in 10 mM NH4OAc buffer (ph 5.1). After 
removal of unadsorbed proteins, adsorbed proteins were 
desorbed by addition of 1 M NaCl to the elution buffer. 
The unadsorbed fraction was subsequently subjected to 
5 ion exchange chromatography on a 1.5x20 cm 
Q-SEPHAROSE® column (Amersham Biosciences) in 
10 mM NH4OAc buffer (ph 5.1). Unadsorbed proteins 
were eluted with the same buffer. Adsorbed proteins 
were desorbed with a linear 0-1 M NaCl gradient in 10 
to mM NH4OAc buffer (ph 5.1). The first adsorbed peak 
obtained was fractionated by gel filtration on a SUPER-
DEX® 75 HR 10/30 column (Amersham Biosciences) 
by fast protein liquid chromatography (FPLC). 
15 	 Example 2 
Improved Laccase Purification 
The method of Example 1 was carried out, with the follow- 
20 ing changes. Fruiting bodies of Suilllus granulatus were 
homogenized in 10 mM Tris-HCl buffer, pH 7.3, instead of in 
water. Filtration through cheesecloth was omitted, because 
the homogenate was too thick to pass readily through the 
cheesecloth filter. Instead, the slurry was sonicated, then cen- 
25 trifuged at 10,500 rpm (relative centrifugal force (RCF) of 
18,093). The supernatant was collected, sonicated, and ultra-
centrifuged at 45,000 rpm. This treatment caused the super-
natant to separate into two layers. The bottom supernatant 
layer was collected and then filtered using a 0.45 Fm syringe 
30 filter (polyethersulfone, PES). The filtrate was loaded onto a 
DEAE Sepharose column and eluted using an NaCl gradient. 
Dialysis was performed on the active fractions as described in 
Example 1. The CM cellulose chromatography step was 
omitted, but the ion exchange chromatography on 
35 Q-SEPHAROSE® and gel filtration on SUPERDEX® 75 
were carried out as described in Example 1. 
Example 3 
40 	 Laccase purified according to the procedure of Example 2 
was separated into isomers as follows. Active fractions were 
concentrated to <2 ml using a VivaSpin centrifugal filter 
(PES; 10,000 MWCO), and assayed for concentration and 
specific activity, then loaded onto a Phenyl Fast-Flow (low 
45 substitution) HIC (hydrophobic interaction chromatography) 
column. This treatment resulted in five peaks, the most active 
being of interest. Active fractions after HIC chromatography 
were concentrated to <1 ml using a VivaSpin (Vivaproducts, 
Inc., Littleton, Mass.) centrifugal filter (PES; 10,000 
50 MWCO), and assayed for concentration and specific activity, 
resulting in separation and the obtaining of pure individual 
enzyme. 
Example 4 
55 
Laccase purified according to the procedure of Example 2 
was assayed for phenol oxidation activity in an ABTS (2,2'-
azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay 
according to procedures well known in the art. The sample to 
6o be assayed was placed in 100 mM sodium tartrate buffer (pH 
4.3) containing ABTS. The increase in absorbance at 405 mu 
was recorded over 15 min. 
FIG.1 shows the results of such an assay. Laccase purified 
from a Russula species is shown in the wells in column 6. 
65 Trametes versicolor laccase purchased from Sigma Chemical 
Co. (St. Louis, Mo.) was a positive control, as shown in 
column 9. 
US 8,759,057 B1 
5 
These results demonstrate that laccase purified according 
to the present invention contains phenol oxidation activity, 
which is consistent with known laccase enzymes. 
What is claimed is: 
1. A process for purifying laccase from an ectomycorrhizal 
fruiting body, the process comprising: 
(A) homogenizing the fruiting body in a buffer of about pH 
7 to obtain a slurry; 
(B) sonicating the slurry to obtain a sonicated slurry; 
(C) centrifuging the sonicated slurry at a relative centrifu-
gal force of about 15,000 to about 20,000 and collecting 
a resulting first supernatant; 
(D) sonicating the first supernatant to obtain a sonicated 
first supernatant; 
(E) ultracentrifuging the sonicated first supernatant such 
that a resulting second supernatant separates into top and 
bottom supernatant layers, and collecting the bottom 
supernatant layer; 
(F) filtering the bottom supernatant layer through a bacte-
ria-retaining filter and collecting the filtrate; 
(G) subjecting the filtrate to DEAE cellulose chromatog-
raphy and eluting retained proteins with a first sodium 
chloride gradient; 
6 
(H) removing the sodium chloride from the eluted, retained 
proteins to obtain desalted proteins, subjecting the 
desalted proteins to ion exchange chromatography, des-
orbing adsorbed proteins with a second sodium chloride 
5 	 gradient, and collecting the first desorbed peak; and 
(I) fractionating the first desorbed peak into fractions by 
gel filtration. 
2. The process of claim 1, wherein the ultracentrifuging is 
at about 45,000 rpm. 
10 	 3. The process of claim 1, wherein the ion exchange chro- 
matography of step (H) is carried out on a column. 
4. The process of claim 1, wherein the gel filtration of step 
(I) is carried out on a column by fast protein liquid chroma-
tography. 
15 	 5. The process of claim 1, further comprising assaying the 
fractions of step (I) for phenol oxidation activity. 
6. The process of claim 5, wherein phenol oxidation activ-
ity is assayed by (2,2'-azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid) (` ABTS") assay. 
20 	 7. The process of claim 1, further comprising separating 
fractions from the gel filtration step of step (I) into laccase 
isomers by hydrophobic interaction chromatography. 


Methods for Purifying Enzymes for Mycoremediation

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