Large scale method for purification of high purity heparinase from flavobacterium heparinum

The present invention is an improved process for purification of active heparinase and heparinase like enzymes from Gram negative organisms, in particular, Flavobacterium heparinum. The primary advantage of the process is the fact that it allows large scale processing and high yield of heparinase. The heparinase is released from the periplasmic space of the organism by osmotic shock treatment, first into an osmotically stabilized medium, secondly into a non-stabilized medium having a pH of approximately pH 6.0 and 8.6 with subsequent release into a second non-stabilized medium containing approximately 0.15 M sodium chloride, followed by fractionation by cation exchange chromatography, and, optionally, electropheresis or gel filtration chromatography. Two proteins having heparinase activity have been isolated, one having a molecular weight of approximately 42,000 Daltons and the other having a molecular weight of 65,000 to 75,000 Daltons. Also described is the construction of a library for screening for the genes encoding the proteins having heparinase activity and two assay for detecting organisms producing heparinase, either F. heparinum or genetically engineered organisms.

BACKGROUND OF THE INVENTION 
This invention is a method for the purification of heparinase and other 
eliminases from F. heparinum. 
Heparinase is an eliminase which cleaves heparin at alphaglycosidic 
linkages in heparin's major repeating unit: -&gt;4)-2-deoxy-2-sulfamino- 
-D-glucopyranose 6-sulfate-(1-&gt;4)-alpha-L-idopyranosyluronic acid 
2-sulfate-(1-&gt;. Heparin is used clinically, both in vitro and in vivo, to 
inhibit blood coagulation. A mucopolysacchride with a wide range of 
molecular weights of up to 20,000, average molecular weight 13,500, 
heparin works by directly inhibiting thrombin and activated Factor X as 
well as other serine esterases in the blood. 
The anticoagulant effect of heparin is neutralized clinically either by 
precipitation with protamine or as described in U.S. Ser. No. 044,245 
entitled "Extracorporeal Reactors Containing Immobilized Species" filed 
May 22, 1987 by Howard Bernstein, et al., and U.S. Ser. No. 044,340 
entitled "Bioreactor Containing Suspended, Immobilized Species" filed Jun. 
6, 1987, by Lisa E. Freed, et al., reactors containing immobilized 
heparinase. The heparinase is immobilized to prevent leaching of the 
heparinase into the body via the blood passing through the reactor. 
Sulfatase free heparinase, also designated catalytic grade heparinase, is 
required to completely remove the anticoagulant properties of heparin by 
enzymatic degradation. As described in U.S. Pat. No. 4,341,869 to Langer, 
et al., heparinase is produced by bacteria such as Flavobacterium 
heparinum. The organism is grown, the cells lysed, debris removed by 
centrifugation, and the cell extract passed through a hydroxylapatite, 
3Ca.sub.3 (PO.sub.4).sub.2 or Ca(OH).sub.2 or Ca.sub.10 (PO.sub.4).sub.6 
(OH).sub.2 column. A hydroxylapatite column can provide 10 to 100 fold 
enzyme enrichment when the protein is eluted from the column at high salt 
concentrations in a step-wise fashion. As described, higher yield of the 
enzyme is obtained by step-wise elution of the heparinase using a 
phosphate buffer solution of increasing sodium chloride concentration, 
ranging from 0.01M sodium phosphate pH 6.8 up to 0.10M sodium phosphate 
0.19M sodium chloride pH 6.8. 
This purification process was greatly improved by combining the 
hydroxylapatite chromatography with repeated gel filtration chromatography 
and chromatofocusing, as described by Yang, et al. in "Purification and 
Characterization of Heparinase from Flavobacterium heparinum" J.Biol.Chem. 
260(3), 1849-1857 (1985). 
The purified heparinase, a protein, has a molecular weight of 
42,900.+-.1000 Daltons with a pI value of 8.5. 
Although these methods are useful in preparing laboratory reagent 
quantities and characterizing the enzyme, they are inadequate for 
preparing heparinase in the quantity and the purity required for large 
scale clinical application. Additionally, the purification scheme outlined 
would be difficult to adapt to large scale recovery of the enzyme. 
Other methods which have been used to extract proteins from the periplasmic 
space of Gram negative bacteria include osmotic shock treatment as the 
initial step. Typically these procedures include an initial disruption in 
osmotically stabilizing medium followed by selective release in 
non-stabilizing medium. The composition of these media (pH, protective 
agent) and the disruption methods used (chloroform, lysozyme, EDTA, 
sonication) vary among specific procedures reported. None of these has as 
yet been successfully applied to the purification of catalytic grade 
heparinase. 
It is therefore an object of the present invention to provide a method for 
preparing highly pure heparinase in large quantities for use in commercial 
and clinical applications. 
It is another object of the present invention to provide a method for 
isolation of other eliminases from F. heparinum. 
It is a still further object of the invention to provide large quantities 
of purified, enzymatically active heparinase and other eliminases. 
SUMMARY OF THE INVENTION 
F. heparinum cells, concentrated by ultrafiltration, are subjected to an 
osmotic shock treatment to release active heparinase from the periplasmic 
space. In the preferred embodiment, disruption of the cell envelope is 
induced by exposing the cells to an osmotically stabilized medium (20% 
sucrose), with or without EDTA, followed by an initial release of 
periplasmic material into a non-stabilized medium (10 mM phosphate, at a 
pH between 6.0 and 8.6) with the subsequent release of heparinase and 
other eliminase activity into a second non-stabilized medium (10 mM 
phosphate, 150 mM sodium chloride, at a pH between 6.0 and 8.6). This 
three step process allows an initial five to ten-fold purification with a 
yield of up to 75% activity. In particular, the impurities proving most 
difficult to remove by previously reported procedures are removed during 
the first two steps of the osmotic shock treatment. 
Following the removal of sodium ions by diafiltration, the concentrated 
material is fractionated by cation exchange chromatography, preferably 
using a FPLC Mono S column. Heparinase activity is present in two 
proteins, one approximately 42,000-43,000 Dalton protein and one 
65,000-75,000 protein. Overall yield is typically 25% with a 200-300 fold 
increase in purity. 
The effectiveness of the osmotic shock treatment may be improved by varying 
the pH and ionic strength of the two release media. Furthermore, a 
scale-up of this process may be carried out by employing mass flow ion 
exchange devices. 
A method for construction of a gene library and methods of screening for 
organisms producing heparinase are also described.

DETAILED DESCRIPTION OF THE INVENTION 
The preferred method of the present invention for large scale heparinase 
purification from bacteria is to produce heparinase in a fermentation 
reactor containing an organism such as Flavobacterium heparinum or any 
other Gram negative organism which has been engineered or mutated to 
produce heparinase, to remove the culture medium and concentrate the cells 
by a method such as ultrafiltration or centrifugation, to subject the 
concentrated cells to a three-step osmotic shock, to separate out the 
cells and non-specific periplasmic material, to concentrate the remaining 
periplasmic material by diafiltration using a membrane having a 10,000 
molecular weight cutoff to remove water and salts, to separate out the 
heparinase by ion exchange chromatography of the concentrated solution 
containing 10 mM NaCl solution (preferably on a cation exchange fast 
protein liquid chromatography column), and to further purify the material 
eluted from the column having heparinase activity by gel electrophoresis 
or gel filtration. 
SELECTIVE PERIPLASMIC PROTEIN RELEASE 
F. heparinum cells obtained from Alfred Linker, Veterans Administration 
Hospital, Salt Lake City, Utah, were grown at 30.degree. C. in 2.8 L 
shaker flasks containing 500 ml defined medium (3 g K.sub.2 HPO.sub.4 /L, 
1.5 g KH.sub.2 PO.sub.4 -H.sub.2 O/L, 0.5 g NaCl/L, 1.0 g NH.sub.4 Cl/L, 2 
mM MgSO.sub.4 -7H.sub.2 O, 0.2 g L-histidine/L, 0.2 g L-methionine/L, 8 g 
glucose/L, 1 g heparin/L, and 10.sup.-4 mM each of NaMoO.sub.4 -2H.sub.2 
O, CoCl.sub.2 -6H.sub.2 O, MnSO.sub.4 -H.sub.2 O, CuSO.sub.4 -5H.sub.2 O, 
FeSO.sub.4 -7H.sub.2 O and CaCl.sub.2). The organism can be stored for up 
to two weeks on agar plates containing 1% Difco agar in defined medium, 
containing 4 g heparin/L as the sole carbon source or indefinitely in 10% 
DMSO at -80.degree. C. 
Heparinase activity is assayed by observing the metachromatic shift of 
azure A from blue to red in the presence of heparin according to the 
procedure of Galliher, et al., Appl. Environ. Microbiol.41,360-365 (1981). 
The change in absorbance is measured at 620 nm in the linear range of the 
assay and compared with a standard curve of 0 to 8 mg/ml heparin in assay 
buffer (0.25M Na Acetate, 0.0025M Ca Acetate, pH 7.0). One unit of 
activity by this assay corresponds to the amount of enzyme which degrades 
1 mg of heparin/h. 
Beta galactosidase activity is measured by the method of Miller, 
Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold 
Spring Harbor, N.Y. Protein concentrations are measured by the Bio-Rad 
protein assay. Growth of the organism is monitored by measuring the 
absorbance of cell suspensions 600 mn. Viable cell counts are determined 
by plating appropriate dilutions on defined medium agar plates. 
The osmotic shock procedure is as follows. Cells are first suspended in an 
osmotically stabilizing medium, for example, a protective medium 
containing 20% sucrose, 10 mM sodium phosphate, pH 7.0. Following this 
treatment, the cells are resuspended consecutively into two 
non-stabilizing (recovery) media: 1) 10 mM sodium phosphate, pH 7.0 (a low 
ionic strength buffered solution) and 2) 10 mM sodium phosphate containing 
150 mM sodium chloride, pH 7.0 (a buffered salt solution). The cells are 
initially harvested and subsequently removed from each solution by 
centrifugation at 7000 g, 10 min, in a Sorval RC-2B refrigerated 
centrifuge. Unless otherwise stated, all procedures are carried out at pH 
7.0 and 4.degree. C. at cell concentration of 5.times.10.sup.10 cells/ml. 
An aliquot of cells, not subjected to osmotic shock, is sonicated with a 
Branson W-350 sonifier, 15 min, 50% pulsed, #6) and used as a 100% 
control. Supernatants from the osmotic release solutions and the sonicated 
cells are dialyzed in 10 mM phosphate, 150 mM NaCl prior to evaluation for 
enzymatic activity and protein content. 
As shown in Table 1, the higher specific activity of the released enzyme as 
compared to the specific activity of the whole cell (sonicate control) 
indicates that heparinase is preferentially released into the 
non-stabilizing media. Maximal release of heparinase is achieved when 
disrupted cells are first washed with the low salt solution follwed by a 
wash in high salt solution. Furthermore, only a small quantity of 
betagalactosidase activity is detected in the release supernatants 
indicating that cytoplasmic material is not released to any great extent 
by this procedure. EDTA, SDS, lysozyme, toluene or chloroform can be added 
to the non-stabilizing medium to aid in disruption of the cell and release 
of the enzyme from the periplasmic space or the cells can be subjected to 
freezing and thawing. Sonication will also selectively release the 
periplasmic proteins but is not readily controllable. None of these latter 
additives nor sonication are more effective than sucrose alone, however. 
TABLE 1 
______________________________________ 
Specific release of heparinase from F. heparinum by 
three step osmotic shock treatment. 
HEI- .beta.-GALAC- 
NASE TOSIDASE 
Ac- Ac- PROTEIN 
tivity tivity Total 
SAM- (U/ Sp. (U/ (mg/ 
PLE.sup.a 
ml) % act. ml) % ml) % 
______________________________________ 
SONI- 29.22 100.0 29.2 1.50 100.0 1.00 100.0 
CATE 
SU- 0.00 0.0 0.0 0.02 1.3 0.02 2.0 
CROSE 
LOW 1.31 4.5 26.2 0.01 0.7 0.05 5.0 
SALT 
HIGH 11.63 39.8 83.2 0.01 0.7 0.14 14.0 
SALT 
______________________________________ 
.sup.a all samples contained 5 .times. 10.sup.10 cells/ml 
Table 2 demonstrates the dependence of the enzymatic release on the ionic 
strength of the recovery solutions. Osmotically stabilized cells were 
divided into two equal batches and resuspended separately in low and high 
salt solutions. After the initial treatment, the cells were divided again 
into two batches and resuspended separately in the two non-stabilizing 
solutions. All supernatants were collected and assayed for heparinase 
activity and protein content. The results show the importance of using all 
three solutions: 20% sucrose, low salt, and high salt, in that order. 
TABLE 2 
______________________________________ 
Dependence of enzymatic release on the ionic strength 
of recovery solutions. 
HEINASE PROTEIN 
Activity Sp. Total 
SAMPLE (U/ml) % act. (mg/ml) 
% 
______________________________________ 
SONICATE 50.74 100.0 26.0 1.95 100.0 
I: low salt 6.47 12.8 38.1 0.17 8.7 
II: low salt 4.58 9.0 65.4 0.07 3.6 
high salt 
21.66 42.7 216.6 0.10 5.1 
I: high salt 
0.72 1.4 24.0 0.03 1.5 
II: low salt 1.52 3.0 76.0 0.02 1.0 
high salt 
2.33 4.6 233.0 0.01 0.5 
______________________________________ 
Table 3 demonstrates the effects of EDTA and pH on the 
release of heparinase from F. heparinum using the three step osmotic shock 
process. The presence or amount of EDTA does not seem to alter the release 
of the heparinase. However, the amount of heparinase released to the low 
salt solution increases with increasing pH over pH 6.0 to pH 8.7. The 
greatest overall recovery for either low salt or high salt fractions is at 
pH 7.5. 
TABLE 3 
______________________________________ 
Effects of EDTA and pH on the release of heparinase 
from F. heparinum by three step osmotic shock. 
HEINASE 
EDTA.sup.a Activity 
SAMPLE (mM) pH.sup.b (U/ml) % 
______________________________________ 
Effect of EDTA: 
SONICATE 7.0 22.6 100.0 
low salt 0.0 7.0 4.03 17.8 
high salt 10.25 45.2 
low salt 1.0 7.0 3.59 15.8 
high salt 10.87 48.0 
low salt 2.0 7.0 2.26 10.0 
high salt 10.50 46.3 
low salt 5.0 7.0 2.57 11.3 
high salt 11.80 52.1 
low salt 10.0 7.0 3.51 15.5 
high salt 10.80 47.7 
low salt 20.0 7.0 5.61 24.8 
high salt 11.07 48.9 
Effect of pH: 
SONICATE 7.0 24.44 100.0 
low salt 0.0 6.0 1.29 5.3 
high salt 5.07 20.7 
low salt 0.0 6.7 3.35 13.7 
high salt 10.95 44.8 
low salt 0.0 7.5 4.29 17.6 
high salt 19.75 80.8 
low salt 0.0 8.6 6.18 25.3 
high salt 16.76 68.6 
______________________________________ 
.sup.a amount of EDTA added in the first stage 
.sup.b pH of recovery solutions 
The stage of cell growth has an effect on the extent of recovery. The 
maximal recovery occurs from samples taken during mid to late exponential 
phase, at the maximal growth rate of 0.21.sup.-1 for F. heparinum. The 
specific activity of heparinase released increases throughout exponential 
growth while the total amount of protein released remains relatively 
constant. The decrease in recovery of heparinase during stationary growth 
phase appears to be related to a decrease in the amount of protein 
released rather than a decline in specific activity. 
In general, the amount of protein released into each of the recovery 
solutions is approximately equal, 5-8% of the total cell protein. At pH 
7.0, heparinase is preferentially released to the high salt solution which 
contains 65-80% of the total released activity. 
Using these methods, the conditions for optimum recovery of heparinase 
using a three step osmotic shock treatment can be determined. Based on the 
data in Tables 1, 2, and 3, an improved initial purification step was 
designed in which heparinase is selectively released from F. heparinum and 
simultaneously separated from other periplasmic components by varying the 
salt concentration in release media. 
While the protein content in each recovery solution is approximately equal, 
5 to 8% of total cell protein, approximately 75% of the heparinase activity 
released is found in the high salt recovery fraction with a typical 
ten-fold increase in specific activity. Exposing osmotically stabilized 
cells immediately to a high salt solution results in a poor release of 
protein. Additionally, replacing the third step of the procedure with a 
low salt solution wash fails to release heparinase activity comparable to 
that released into a high salt solution. 
ION EXCHANGE CHROMATROGRAPHY AND ELECTROPHORESIS 
Heparinase, isolated from F. heparinum by osmotic release, can be further 
purified by cation exchange chromatography, preferably using a fast 
protein liquid chromatography (FPLC) apparatus (Mono S, Pharmacia Fine 
Chemicals, Piscataway, N.J.). Samples are dialyzed and loaded in a 10 mM 
phosphate buffer at pH 7.0, and eluted with a linear salt gradient ranging 
from 0.0 to 0.3M NaCl at a flow rate of 1 ml/min. 
More than 70% of the total protein applied is not absorbed to the column. 
Activity is recovered in two fractions containing less than one percent of 
the total protein, as shown in FIG. 1. The protein eluting at 150 mM NaCl 
has a molecular weight of 42,900 Daltons. The specific activity of this 
fraction is in the range of 2000-3000 U/mg protein. A second enzyme is 
eluted at 75 mM NaCl. The heparinase activity of the material eluting at 
75 mM NaCl appears to be sensitive to freezing, however, greater than 90% 
activity is retained for as long as seven days in 10 mM phosphate, 0.1M 
NaCl, pH 7.0, .+-.20% glycerol, at -20.degree. C. 
Enzyme preparations can be further purified and analyzed by gel filtration 
on a molecular sieve such as Sephadex G100 or SDS-PAGE using the procedure 
of Laemmli, Nature 227,680-685 (1970)(12.5% acrylamide resolving gels). The 
42,900 Dalton protein contains three other major contaminants are removed 
by electrophoresis. The material eluting at 75 mM NaCl can be further 
purified by chromatography using a FPLC apparatus with a gel filtration 
matrix such as Superose 12, Pharmacia Fine Chemicals, Piscataway, N.J. The 
sample is loaded directly from the cation exchange chromatography and 
eluted with 10 mM sodium phosphate, 0.1M NaCl, pH 7.0 at a flow rate of 
0.1 ml/min. Heparinase activity is detected in the fraction having a 
molecular weight in the range of 65,000 to 75,000 Daltons. When analyzed 
by SDS-PAGE, the material having the greatest activity has a molecular 
weight of 70,000, even under reducing conditions. 
LARGE SCALE PRODUCTION BY FERMENTATION 
Following the two major purification steps, osmotic release and FPLC, two 
concentration steps, ultrafiltration and diafiltration, are used to 
facilitate the handling of larger amounts of material, the results of 
which are shown in Table 4 for a ten liter fermentation of F. heparinum 
grown to 2 g/L DCW and concentrated by microfiltration using a 0.1.mu. 
Romicon hollow fiber membrane device to one liter. Material released to 
the high salt solution was concentrated by diafiltration using a 10,000 
Dalton cutoff ultrafiltration membrane and fractionated by FPLC cation 
exchange chromatography. Typically, 20-25% of the heparinase activity is 
recovered with a 200 to 300 fold increase in specific activity. 
TABLE 4 
______________________________________ 
Recovery of heparinase from a 10 liter fermentation of 
F. heparinum. 
Recovery 
Total of Total 
activity 
activity Sp. act. 
protein 
STEP (units) (%) (U/mg) (mg) 
______________________________________ 
FERMENTATION 13350 100 4.6 2930 
ULTRAFILTRATION 
12700 95 ND ND 
OSMOTIC SHOCK 7000 52 27.4 255 
DIAFILTRATION 6750 51 ND ND 
FPLC 3200 24 2100 1.5 
______________________________________ 
PRODUCTION OF ANTIBODIES AND HYBRIDIZATION PROBES FOR CLONING 
The purified heparinase proteins obtained by gel electrophoresis or gel 
filtration can be used to produce antibody using methods known to those 
skilled in the art. For example, antibodies can be generated by injection 
of a protein in a suitable adjuvant such as Freund's incomplete adjuvant 
into an animal like a rabbit or goat. Alternatively, monoclonal antibodies 
can be prepared by immunizing a mouse and fusing the spleen cells with 
hybridoma cells following elicitation of the antibody. 
The material eluted from the SDS-PAGE was of sufficient purity to allow 
sequencing using methods and equipment available to those skilled in the 
art. The results in Table 5 show a similar composition profile for both 
proteins although some discrepancies are evident, most notably 
glutamine/glutamate, lysine and methionine. The sequence for the 42,000 
Dalton protein is modified at the N-terminus, as determined by inhibition 
of the Edmund degradation technique. The nucleotide and amino acid 
sequences can be used in the preparation of hybridization probes and other 
means for obtaining nucleic acid sequences encoding heparinase, for 
subsequent use in genetically engineering organisms for increased 
production of heparinase or production under external control. 
TABLE 5 
______________________________________ 
Amino acid composition of proteins from F. heparinum 
displaying heparinase activity. 
42,000 D protein 
70,000 D protein 
residues/ residues/ 
amino acid mole % 400 mole % 720 
______________________________________ 
glutamine/glutamate 
14.6 58 10.3 77 
asparagine/aspartate 
17.3 69 14.6 106 
serine 7.6 30 5.3 38 
glycine 6.9 28 7.1 51 
histidine 1.6 6 2.2 16 
arginine 3.0 12 3.7 27 
threonine 6.4 26 5.8 42 
alanine 11.9 48 10.1 73 
proline 3.0 12 4.8 35 
tyrosine 3.4 14 3.6 26 
valine 5.3 21 5.2 38 
methionine 1.0 4 2.1 15 
isoleucine 3.5 14 3.6 99 
leucine 4.4 18 6.5 47 
phenylalanine 
2.9 12 2.9 21 
lysine 7.4 30 12.1 88 
______________________________________ 
METHODS FOR SCREENING OF EXPRESSION LIBRARY FOR HEINASE GENES 
Assays were developed for screening large populations of genetically 
engineered organisms for heparinase production. Previous attempts to 
screen using antibodies to heparinase have been unsuccessful due to 
extensive cross reactivity with several other F. heparinum proteins. The 
assays and methods for screening can be used to isolate and characterize 
the gene for either the 42,000 Dalton heparinase or the 65,000-75,000 
Dalton protein with heparinase activity. 
An agar plate assay was developed based on the precipitation of heparin 
from human blood by electrostatic association with protamine sulfate. 
Heparinase assay plates consisting of 0.25M sodium acetate, 0.0025M 
CaCl.sub.2, 1.0 g heparin from porcine intestinal mucosa (Hepar 
Industries, Franklin, OH)/liter, and 1.5% agarose (BRL), pH 7.0 are 
prepared. Plates are innoculated with the cells to be screened for 
heparinase production. As a control, heparinase is isolated by the method 
described above and applied in various quantities, 0.0, 0.01, 0.10, and 
1.00 U, in 10 .mu.l of 10 mM sodium phosphate, 150 mM NaCl, pH 7.0 to a 
plate which is then incubated at 37.degree. C. for 1 h. A 2% protamine 
sulfate (salmon, Sigma Chemical Co, St. Louis, Mo.) solution is poured 
over the surface of the plates. A white precipitate forms over a 1 to 2 h 
period leaving clearing zones of increasing intensity at the areas where 
increasing amounts of heparinase are added or where a bacterial colony is 
producing heparinase. For example, clearing zones were formed around F. 
heparinum colonies grown on LB agar plates containing 1.0 g heparin/l but 
not around E. coli JM83 grown on the same plates. 
Detection of a constitutive producing strain of F. heparinum requires 
growth of the organism on medium without heparin. An assay was developed 
where F. heparinum are grown in minimal medium containing 1 mM MgSO.sub.4 
(repressing conditions) and plated out onto two minimal medium agar plates 
one of which is supplemented with 1.0 g/l heparin (inducing conditions). 
The plates are incubated at 30.degree. C. for 36 h and the colonies 
transferred to nitrocellulose (NC) paper. F. heparinum colonies adhere to 
the NC paper and are lysed by exposure to chloroform vapors for 20 min. 
The NC paper is then overlayed onto heparinase assay plates (described 
above) and incubated at 37.degree. C. for 1 h. The NC papers are discarded 
and the plates developed with 2% protamine sulfate. Clear zones appear on 
the plate corresponding to the cells grown under inducing conditions 
(heparin supplemented plate) while no zones can be detected on the plate 
corresponding to the cells grown under sulfate repressing conditons. 
The plate assay is sufficient for detecting heparinase activity and does 
not require the presence of other heparin catabolic enzymes. This feature 
represents an improvement over previously reported methods and may 
therefore prove useful in screening E. coli expression gene banks for the 
cloned heparinase gene. Additionally the ability to differentiate F. 
heparinum grown under repressed and induced conditions, using NC paper, 
increases the usefulness of this technique in identifying constitutive 
mutants. 
The heparinase assay based on the metachromatic shift of Azure A from blue 
to red in the presence of heparin was used in the development of a 
microculture assay to identify cells producing heparinase, particularly 
cells which normally do not produce heparinase such as E. coli which have 
been genetically engineered. Previous attempts to use Azure A in 
microculture assays were stymied by background effects, presumably due to 
a media component, rendering color differences in samples with or without 
heparin undetectable. Sodium chloride was identified as a component 
responsible for this background effect by adding B-broth containing 0.0 
g/l heparin and varying amount of NaCl to the wells of 96-well 
microculture plates, then adding an equal volume of 0.04 g Azure A/l to 
each well and measuring the absorbance at 605 nm measured with a Titertek 
Multiscan plate reader. Keeping the concentration of NaCl below one g/l 
sufficiently reduces background effects. 
The assay is as follows: modified B-broth containing bacto tryptone, 10 
g/l; NaCl, 1.0 g/l; heparin 0.02 g/l; and supplemented with methionine, 
proline, histidine and thiamine is filter sterilized and added to 
microculture wells (150 .mu.l/well). Entire rows of wells are either left 
uninoculated, inoculated with E. coli JM83 or inoculated with F. 
heparinum. One row contains modified B-broth without heparin. The plate is 
incubated at 30.degree. C. for 36 h and subsequently frozen and thawed. The 
thawed plate is incubated at 37.degree. C. for 3 h prior to the addition of 
150 .mu.l 0.04 mg Azure A/ml to each well and measuring the absorbance at 
605 nm. Furthermore, the difference in color among the different sets of 
wells: uninoculated, E. coli JM83 cultures and F. heparinum cultures is 
detectable by simple visual observation. 
SCREENING OF EXPRESSION LIBRARY FOR HEINASE GENES 
A F. heparinum chromosomal gene bank was constructed in E. coli using the 
plasmid expression vector pUC18. F. heparinum chromosomal DNA was treated 
with light sonication prior to the addition of BamHl linkers and ligation 
into the dephosphorylated BamHl site of pUC18. 50,000 independent 
transformants were isolated having an average chromosomal DNA insert size 
of 6 kbp. The use of sonicated DNA in this construction enhances the 
randomness of generated fragments over those obtained by restriction 
enzyme digestion which cleave at specific sites, potentially located 
within the structural gene. This pUC18 gene bank is therefore more 
appropriate for use with screening techniques which rely on the expression 
of active protein for detection. Both assay techniques described above are 
being used to screen candidates from this gene bank. 
The difficulties encountered in obtaining sufficiently pure preparations of 
heparinase could be resolved by expression of the heparinase gene in an 
organism such as E. coli. E. coli would provide an environment for 
biosynthesis, free of the contaminating background enzymes; sulfatases, 
glycuronidase, etc. which are present in F. heparinum, greatly simplifying 
purification processes for catalytic grade heparinase, required for blood 
deheparinization. Additionally one could expect an increase in product 
titers in a recombinant system over those displayed by F. heparinum 
fermentations. Improvement in the overall production process is necessary 
for the economic feasibility of an industrial scale production process. A 
rudimentary analysis suggests that the economic breakeven point is at a 
production level of 1.times.10.sup.6 U pure enzyme/liter fermentation 
broth. Using the method of the present invention, 2.times.10.sup.4 U pure 
enzyme/ liter can be obtained from F. heparinum fermentations, assuming a 
20% yield. 
The present invention has been described with reference to specific 
embodiments. Variations and modifications of these methods will be obvious 
to those skilled in the art from the foregoing detailed description of the 
invention. Such modifications and variations are intended to come within 
the scope of the appended claims.