U.S. patent application number 13/810134 was filed with the patent office on 2013-08-15 for asparaginase from basidiomycetes.
This patent application is currently assigned to NESTEC S.A.. The applicant listed for this patent is Pieter Berends, Ralf Gunter Berger, Nadine Eisele, Diana Linke, Swen Rabe. Invention is credited to Pieter Berends, Ralf Gunter Berger, Nadine Eisele, Diana Linke, Swen Rabe.
Application Number | 20130209608 13/810134 |
Document ID | / |
Family ID | 44275917 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130209608 |
Kind Code |
A1 |
Berends; Pieter ; et
al. |
August 15, 2013 |
ASPARAGINASE FROM BASIDIOMYCETES
Abstract
An asparaginase enzyme derived from the fungi Basidiomycete, in
particular the Basidiomycete is Flammulina velutipes. A method for
hydrolysing at least one of L-asparagine or L-glutamine. A method
for reducing acrylamide formation in a substance comprising
L-asparagine is also described.
Inventors: |
Berends; Pieter;
(Zoznegg-muhlingen, DE) ; Rabe; Swen; (Muehlingen,
DE) ; Berger; Ralf Gunter; (Hannover, DE) ;
Linke; Diana; (Bad Rehburg, DE) ; Eisele; Nadine;
(Kufstein, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Berends; Pieter
Rabe; Swen
Berger; Ralf Gunter
Linke; Diana
Eisele; Nadine |
Zoznegg-muhlingen
Muehlingen
Hannover
Bad Rehburg
Kufstein |
|
DE
DE
DE
DE
AT |
|
|
Assignee: |
NESTEC S.A.
Vevey
CH
|
Family ID: |
44275917 |
Appl. No.: |
13/810134 |
Filed: |
April 6, 2011 |
PCT Filed: |
April 6, 2011 |
PCT NO: |
PCT/EP11/55375 |
371 Date: |
January 14, 2013 |
Current U.S.
Class: |
426/18 ; 426/52;
426/549; 426/637; 435/229 |
Current CPC
Class: |
A23L 5/25 20160801; A23K
10/10 20160501; A23V 2002/00 20130101; C12N 9/82 20130101 |
Class at
Publication: |
426/18 ; 435/229;
426/52; 426/549; 426/637 |
International
Class: |
C12N 9/82 20060101
C12N009/82; A23L 1/015 20060101 A23L001/015 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 14, 2010 |
EP |
10169405.7 |
Claims
1. An asparaginase enzyme obtainable from Basidiomycete.
2. The asparaginase enzyme of claim 1, wherein the Basidiomycete is
Basidiomycete Flammulina velutipes.
3. An asparaginase enzyme with the amino acid sequence:
TABLE-US-00001 MKSFALFVPL IVAAVVNSAV VTFSTGLGCN SVSQTYRGNG
NFCADPPGDW SSVGFSEIGG DNRVTVHNQN SCTPASQVGQ GFGPACWNQG ATKLRSAWVA
CPGQRLAENG TIVDDDGAFI DFA
4. The asparaginase enzyme of claim 1 in a form selected from the
group consisting of a solid, a liquid and an intermediate between a
solid and a liquid.
5. A combination of an asparaginase enzyme of obtainable from
Basidiomycete and an exipient selected from the group consisting of
lactose, glycerol, and albumin.
6. A method for hydrolysing at least one of L-asparagine or
L-glutamine comprising: treating a substance comprising at least
one of L-asparagine or L-glutamine with an asparaginase enzyme
obtainable from Basidiomycete.
7. The method of claim 6, wherein the substance comprising at least
one of L-asparagine or L-glutamine is at least one of a human
consumable product or an animal consumable product.
8. The method of claim 6, wherein the substance is selected from
the group consisting of beverages, cocoa beans, cheese, coffee
beans, confectionery, desserts, dough, dressings, French fries,
drinks, meat products, medical supplements, nutritional
supplements, pet food, potato chips, sauces, snacks and soups
9. A method for reducing acrylamide formation in a food substance
comprising L-asparagine comprising: applying to the food substance
comprising L-asparagine an asparaginase enzyme obtainable from
Basidiomycete; and heating the substance comprising
L-asparagine.
10. The method of claim 9, wherein the substance is heated to at
least 120.degree. C.
11. The method of claim 9, wherein the source of L-asparagine is at
least one of a human consumable product or an animal consumable
product.
12. A product obtainable by the method of claim 6.
13. A product obtainable by the method of claim 9.
Description
FIELD OF THE INVENTION
[0001] The field of the present invention relates to an
asparaginase enzyme obtainable from the fungi Basidiomycetes, esp.
Basidiomycetes Flammulina velutipes. A method for the hydrolysis of
L-asparagine and L-glutamine are also disclosed. A method for
reducing the formation of acrylamide in a substance comprising
L-asparagine is also disclosed.
BACKGROUND OF THE INVENTION
[0002] Applications of asparaginase enzymes in food technology
originate from the finding that a thermal treatment of food
converts asparagine in the presence of reducing carbohydrates
partly to acrylamide. Since carbohydrates are as ubiquitous as
amino acids in food, there is a permanent risk of generating the
cancerogenic and genotoxic acrylamide during the thermal treatment
of food. The thermal treatment is for example a baking, a roasting,
a barbecuing or a deep-fat frying of the food. The onset of
acrylamide formation during the thermal treatment of the food is
observed at temperatures exceeding 120.degree. C. The Joint FAO/WHO
Expert Committee on Food Additives (JECFA) has stated that dietary
exposure to acrylamide may indicate a human health concern given
its genotoxicity and carcinogenicity.
(See www.inchem.org/documents/jecfa/jeceval/jec.sub.--41.htm viewed
on 1 Jul. 2010.)
[0003] Particular concerns in the food industry arise for the
numerous varieties of for example breads, cookies, snacks,
biscuits, cereals, roasted seeds (such as cocoa, coffee), extruded
and cut potato products that need to be inherently thermally
treated.
[0004] The thermal treatment of the food is indispensible for a
quality of the food. For example the browning (Maillard) reaction
in the food forms the typical flavours, colours, and antioxidants
in the food. Furthermore microbial safety and extended shelf-life
of the food are achieved due to the thermal treatment of the
food.
[0005] It would be desirable to enable a selective removal of
L-asparagine prior to the thermal treatment of the food.
[0006] Genetic engineering of potato using an antisense asparagine
synthase gene and tuber specific promoters have been reported to
reduce, but not to eliminate asparagine from the potato tuber
(Rommens 2007); a full elimination of asparagine is supposedly
lethal for the plant.
[0007] Enzymes are ideal selective tools to modify a food
constituent without affecting other food constituents. A catalytic
action of enzymes on the food is distinguished by a high substrate
plus reaction specificity and by gentle physical conditions of
enzyme action. The enzyme action on the food is more
environmentally friendly as no organic solvents or heavy metals are
involved ("green chemistry"; "white biotechnology"). Enzymes used
to modify the food constituent allow changing a single food
constituent whilst avoiding any side-reactions which could
eventually result in the formation of toxic compounds in the
food.
[0008] However, no enzyme technology can be currently envisaged for
the selective hydrolysis of a protein bound amino acid, such as
asparagine, from a food protein, even less while maintaining the
typical structural and sensory properties of the respective food
material.
[0009] It would be desirable to hydrolyse e.g. free and mobile
asparagine in the food to aspartic acid. The asparagine cannot then
serve as a precursor molecule for acrylamide formation when the
food is thermally treated.
STATE OF THE ART
[0010] Asparaginase (EC 3.5.1.1; L-asparagine amidohydrolases) is
an enzyme that catalyses the hydrolysis of L-asparagine to aspartic
acid with the liberation of ammonia. By definition asparaginase
enzymes act on a nitrogen-carbon bond in linear amides, but not on
peptide bonds of the L-asparagine.
[0011] L-asparagine was the first amino acid detected (1806 in the
juice of Asparagus officinalis) and L-asparagine is ubiquitous in
all living cells. Accordingly, asparaginase enzymes occur
abundantly in nature from prokaryotic microorganisms to
vertebrates; see Halpern, Y. S. and Grossowicz, N., Hydrolysis of
amides by extracts from mycobacteria, Biochem. J. 65: 716-720
(1957); Ho, P. P. K., Frank, B. H. and Burck, P. J., Crystalline
L-asparaginase from Escherichia coli B., Science 165: 510-512
(1969); Suld, H. M. and Herbut, P. A., Guinea pig serum and liver
L-asparaginases--Comparison of serum and papain-digested liver
L-asparaginase. J. Biol. Chem. 245: 2797-2801 (1970). The tetramer
asparaginases from E. coli with 326 amino acids (Jackson, R. Ch.
and Handschumacher, R. E., Escherichia coli L-asparaginase.
Catalytic activity and subunit nature, Biochemistry, 1970, 9 (18),
pp 3585-3590) were the first to be examined in detail.
[0012] Until recently L-asparaginase is used as a cytostaticum in
cancer therapy to fight leukemia cells and mast cell tumors
(Herbert F. Oettgen, L-Asparaginase: Ein neues Prinzip in der
Chemotherapie maligner Neoplasien, Annals of Hematology, 1969,
19(6), 351-356).
[0013] More recently asparaginase enzymes were reported to be
derived from bacteria (Helicobacter pylori, Scotti et al. 2010;
Pyrococcus furiosus, Greiner-Stoeffele and Struhalla, 2008) and
from molds (Aspergillus niger, Van der Laan et al. 2008;
Aspergillus oryzae, Matsui et al. 2008). The addition of di- and
tri-valent cations and various amino acids and free thiols (Elder
et al. 2007), or of alpha-amylase (de Boer, 2006), or of calcium
chloride in conjunction with phosphoric or citric acid (Elder et
al. 2005) was claimed to support somehow the activity of the
asparaginase enzyme.
[0014] A Glutaminase enzyme is related to the asparaginase enzyme.
The glutaminase enzyme is typically derived from either lactic acid
bacteria as they, for example, occur in the chicken intestinal
flora (Thongsanit et al. 2008; Lactobacillus rhamnosus,
Weingand-Ziade et al. 2003), or from yeasts (Zygosaccharomyces
rouxii, Iyer and Singhal 2010), or from marine fungi (Beauveria
bassiana, Sabu et al. 2002), or again from Aspergillus molds
(Prasanth et al. 2009).
[0015] A concerted use of the asparaginase enzyme in food
technology is rather recent. In 2007 PreventAse (DSM) enzyme was
introduced on the European market. The PreventAse (DSM) enzyme is
produced by a recombinant mold, Aspergillus niger. A competing
asparaginase enzyme, called Acrylaway (Novozymes), has been
obtained from a related mold species, Aspergillus oryzea by using
submerged feed-batch fermentation of a genetically modified strain
carrying a gene coding for an asparaginase enzyme from Aspergillus
oryzae. Both Aspergilli (Aspergillus niger and Aspergillus oryzae)
are described as having a long history of safe industrial use,
being widely distributed in nature and being commonly used for
production of food-grade enzymes.
[0016] In baking applications, the asparaginase enzyme is typically
mixed with the dough before the thermal treatment of the food (for
example baking) to eliminate acrylamide formation. For French
fries, the dipping or spraying of potato pieces in or with a
solution of the asparaginase enzyme solution may be used. Such a
treatment may be very efficient. In potato chip manufacture,
Corrigan (2008) reported a decrease of acrylamide levels in the
finished product from 1688 .mu.g/kg down to 60 .mu.g/kg in
comparison to untreated potato chips. A reduction of the formation
of acrylamide by >99.9% was supposed to be feasible (Elder et
al. 2004).
[0017] Product safety in terms of the asparaginase enzyme applied
to food is not an issue, as the asparaginase enzyme will be
heat-inactivated by the thermal treatment of the food in the step
before packaging. Therefore the asparaginase enzyme will unlikely
come into contact with a consumer in its active form.
Enzymes from Basidiomycetes
[0018] Most of the around 1,000 edible fungi belong to the class of
Basidiomycota (Basidiomycetes). Basidiomycetes are often referred
to as higher fungi. Basidiomycetes reproduce by forming pillar-like
cells carrying four meiospores. The anatomy of Basidiomycetes was
name-giving (lat. Basidium=pillar). Basidiomycetes are appreciated
all over the world by their rich flavour, a high protein and a high
fiber content together with low energy. The Asian cultures
additionally assign distinct health protecting and healing
activities to many of the Basidiomycetes fungi.
[0019] Saprotrophic Basidiomycetes commonly inhabit forest
detritus, forest soils, leaf litter, and fallen trees. The
vegetative cells spread out in the sub-terranean sphere forming
long filamentous cells (hyphae). To survive on the most
recalcitrant organic material on earth, the three-dimensional
lignin network, they possess a remarkably potent set of
oxidoreductases. Among the oxidoreductases are lignin peroxidase,
manganese peroxidase, versatile peroxidase, H.sub.2O.sub.2
producing oxidases such as glucose oxidase, and phenol-oxidases of
the Laccase type. Glycosidases, such as cellulases, are also found
and help to degrade the cellulose portion of wood.
[0020] As deciduous and coniferous wood does not contain a
significant amount of protein and amino acids, an occurrence of a
asparaginase enzyme activity in a fungi growing in this particular
natural habitat would not be envisaged.
[0021] Cultivars of Flammulina velutipes from the Basidiomycetes
are also known as known as Enokitake, golden needle mushroom or
velvet foot. The Flammulina velutipes form long, thin white
fruiting bodies are used in Asian cuisines as versatile mushrooms.
The mushroom is traditionally used fresh, canned for soups, salads
and other dishes. The mushroom can be refrigerated for about one
week.
OBJECT OF THE INVENTION
[0022] An object of the present invention is to provide an
asparaginase enzyme with a high activity and a high operational
stability.
[0023] A further object of the present invention is to reduce the
formation of acrylamide in a food product by use of the
asparaginase enzyme.
SUMMARY OF THE INVENTION
[0024] In an aspect of the present invention, the invention relates
to an asparaginase enzyme obtainable from Basidiomycete. In
particular the Basidiomycete Flammulina velutipes.
[0025] In a further aspect the present invention relates to a
method for hydrolysing at least one of L-asparagine or L-glutamine.
The method comprises treating a substance comprising at least one
of L-asparagine or L-glutamine with the asparaginase enzyme
obtainable from Basidiomycete.
[0026] In a further aspect the present invention relates to a
method for reducing acrylamide formation in a substance that
comprises L-asparagine. The method comprises applying to the
substance that comprises the L-asparagine the asparaginase enzyme
obtainable from Basidiomycete. The method then comprises heating
the substance comprising the L-asparagine.
[0027] The substance comprising at least one of L-asparagine or
L-glutamine can be a food product.
[0028] The invention further relates to the products obtained by
the methods of the present invention.
FIGURES
[0029] The present invention is described hereinafter with
reference to some embodiments as shown in the following figures
wherein:
[0030] FIG. 1 shows a time course of intracellular formation of
asparaginase enzyme of Flammulina velutipes grown in a submerged
culture.
[0031] FIG. 2 shows a time course of extracellular formation of the
asparaginase enzyme of Flammulina velutipes grown in submerged
culture.
[0032] FIG. 3 shows a genomic (A) and coding (B) nucleotide
sequences and the amino acid sequence (C) of the asparaginase
enzyme of Flammulina velutipes. The first 19 amino acids were
identified as signal sequence.
[0033] FIG. 4 shows a salt tolerance of the asparaginase enzyme
from Flammulina velutipes, expressed in E. coli as a heterologous
host and used as a crude enzyme.
[0034] FIG. 5 shows a pH stability of the asparaginase enzyme of
Flammulina velutipes, expressed in E. coli as a heterologous host
and used as a crude enzyme.
[0035] FIG. 6 shows a pH-optimum of the asparaginase enzyme of
Flammulina velutipes.
[0036] FIG. 7 shows a temperature stability of the asparaginase
enzyme of Flammulina velutipes, expressed in E. coli as a
heterologous host and used as a crude enzyme.
[0037] FIG. 8 shows a) activity stained native polyacrylamide gel
electrophoresis (PAGE) and b) denaturing-PAGE separations of
asparaginase enzyme of Flammulina velutipes.
[0038] FIG. 9 shows a temperature optimum of the asparaginase
enzyme of Flammulina velutipes.
DETAILED DESCRIPTION OF THE INVENTION
[0039] For a complete understanding of the present invention and
the advantages thereof, reference is made to the detailed
description of the invention taken in conjunction with the
figures.
[0040] It should be appreciated that various aspects of the present
invention are merely illustrative of the specific ways to make and
use the invention and do not limit the scope of the invention when
taken into consideration with the claims and the following detailed
description.
[0041] In the present invention, the term asparaginase enzyme is
used to refer to an enzyme that is capable of hydrolysing both
L-asparagine and L-glutamine.
[0042] An aim of the present invention is to significantly reduce
the formation of carcinogenic acrylamide in thermally treated food
by a concerted enzymatic hydrolysis of the acrylamide precursor,
L-asparagine with the asparaginase enzyme.
[0043] In an embodiment of the present invention a method for the
manufacture of the asparaginase enzyme is disclosed. The
asparaginase enzyme possesses operational stability and is obtained
from mycelium of the Basidiomycetes Flammulina velutipes.
[0044] A strain of the Flammulina velutipes is commercially
available through culture collections, such as the DSMZ (Deutsche
Sammlung fur Mikroorganismen and Zellkulturen GmbH, Braunschweig,
Germany) or the CBS (Centraalbureau voor Schimmelcultures, Utrecht,
The Netherlands).
[0045] The use of mycelium of the Basidiomycetes Flammulina
velutipes offers great advantages in terms of ease of production
and cultivation, as the collection of fruiting bodies from the
wilderness is not required. As a result of the extensive use of
this species Basidiomycetes Flammulina velutipes as a foodstuff,
there are no visible health risks or safety concerns.
[0046] The fungus of the Basidiomycetes Flammulina velutipes can be
easily grown in a submerged culture with minimum demands for medium
supplements. An organic carbon source, a nitrogen source, and a
phosphorous source have to be present; these sources are typically
provided by natural mixtures such as a yeast extract or glucose
plus inorganic ammonium and phosphate salts. A mixture of minor and
trace elements, are recommended in all nutrient media of
micro-organisms, is added. The cultivation of the Basidiomycetes
Flammulina velutipes is preferably carried out in a submerged
culture for 3 to 20 days, preferably for 6 to 15 days. A
temperature during cultivation of the Basidiomycetes Flammulina
velutipes is typically in a range from 10 to 35.degree. C.,
preferably from 20 to 30.degree. C. A pH of about 4 to 8 is
typical, with a pH of about 5 to 7 being preferred. Furthermore
conditions of low light are typical of the method.
[0047] The method of biomass and asparaginase enzyme production
operates under mild conditions and is environmentally friendly in
contrast to the methods of the prior art.
[0048] The asparaginase enzyme activity is first accumulated
intra-cellularly as shown in FIG. 1 and then secreted into a
nutrient medium as shown in FIG. 2.
[0049] The nutrient medium facilitates a handling of the method as
well as asparaginase enzyme isolation and enrichment using
techniques known in the art. The techniques can be
ultra-filtration, precipitation or adsorption. A cell-free,
concentrated culture supernatant of asparaginase enzyme may thus be
obtained and further used for technical hydrolysis. Although the
asparaginase enzymes may be isolated by techniques known in the
art, it is not necessary to do so, and a crude mixture of the
asparaginase enzyme obtained may also be further used in the
present method.
[0050] In the course of a submerged cultivation of Flammulina
velutipes a peak of intracellular asparaginase enzyme activity was
found after approximately one week. An excretion into the
extracellular space started after 12 days and peaked after
approximately 14 days.
[0051] As seen in FIG. 8, activity staining on a native
poly-acrylamide gel confirmed the catalytic specificity and showed
active bands of the purified enzyme at 13 and 74 kDa indicating the
presence of an oligomer form besides the monomer.
[0052] If a maximum purity of the asparaginase enzyme is required,
a recombinant product from Bacillus subtilis may be used. To
develop recombinant strains, the full amino acid sequence of the
asparaginase enzyme needs to be known. The full amino acid sequence
of the asparaginase enzyme is shown in FIG. 3 which shows the full
sequence with all 123 amino acid moieties, as deduced from the full
372 base pair sequence of the structural gene. An 18 base pair
signal sequence precedes the coding region.
[0053] The asparaginase enzyme is added to a substrate. By adding
the asparaginase enzyme to the substrate it is intended that the
asparaginase enzyme contacts the substrate. This can include for
example spraying, dipping or coating the substrate with the
asparaginase enzyme. The substrate is preferably a food material
that comprises any one of L-asparagine or L-glutamine. The
asparaginase enzyme is usually applied to the substrate at
concentrations at a total level of 1 to 200 millimolar, preferably
10 to 20 millimolar depending on the specific activity. The
asparaginase enzyme can be added as the pure protein. Alternatively
the asparaginase enzyme can be tailored according to the intended
use by adding ingredients to the asparaginase enzyme, such as
lactose, glycerol or albumin to facilitate dosage. The manufactured
asparaginase enzyme or the tailored asparaginase enzyme can be in
the form of, an enzyme tablet, a granulate, a stabilized liquid or
a paste-like preparation.
[0054] A hydrolysis of the substrate is performed to obtain the
substrate with a significantly lower levels of asparagine or
glutamine as compared to the substrate prior to treatment. The
conditions which may be used for the hydrolysis are standard, and
can be easily determined by a person of skill in the art.
[0055] As the asparaginase enzyme activity is not affected by the
chemical environment in which it is present, the substrate to be
treated may be, for example: [0056] Beverages [0057] Cocoa beans
[0058] Cheese [0059] Coffee beans [0060] Confectionery [0061]
Desserts [0062] Doughs [0063] Dressings [0064] French fries [0065]
Fruit drinks [0066] Meat products [0067] Medical diets [0068]
Nutritional supplements [0069] Pet food [0070] Potato chips [0071]
Sauces [0072] Snacks [0073] Soups
[0074] In particular the substrate is any item consumable by a
human or an animal.
[0075] The degree of hydrolysis of the asparagine in the substrate
can be either assessed by measuring asparagine decrease, aspartic
acid or ammonia increase or, after processing the food, by
measuring a level of any residual acrylamide.
[0076] The advantage provided by the invention is that the
resulting novel asparaginase enzyme has a distinct affinity and
improved efficacy for the hydrolysis of L-asparagine.
[0077] Even more surprising is the excellent technical properties
of the asparaginase enzyme with regards to operational stability
that enables the use in processes with elevated temperature and
ionic strength and different conditions of pH (FIG. 4-7). No
additive or further co-substrates other than water are
necessary.
[0078] The novel asparaginase enzyme possesses good pH stability
and a broad pH-optimum between pH 5.5 and 9, see FIGS. 5 and 6. The
pH of most foods is found in this range.
[0079] An operational stability of the asparaginase enzyme is not
decreased even at temperatures as high as 55.degree. C., see FIG.
7.
[0080] An iso-electric point of the asparaginase enzyme monomer and
oligomer is near 5.2, as determined by isoelectric focussing gel
electrophoresis. The molecular masses of the asparaginase enzyme
monomer and oligomer are 12.8, as deduced from the full sequence
and around 74 for the aggregated form, as deduced from native
polyacrylamide gel electrophoresis (PAGE), see FIG. 8.
[0081] The unique sequence of the asparaginase enzyme as shown in
FIG. 3 was determined by ESI-MS analysis. The best homologies of
the initially found peptides were found to a
carboxylase/metallo-peptidase (E-value >30), a
lipase/esterase/deacetylase (E-value >100), and to a pepsin-like
aspartic/glycoside hydrolase (E-value >14). The generally
inhomogeneous results and poor E-values indicate that this
asparaginase enzyme is without precedent and novel indeed. This is
explained by the unique source, the basidiomycete species.
[0082] The present invention is described further herein by way of
illustration in the following non-limiting examples.
EXAMPLES
[0083] In the following examples, materials and methods were used
as outlined.
Material and Methods
[0084] Cultivation of Flammulina velutipes
[0085] All media and equipment were autoclaved prior to use and
standard sterilisation techniques were applied throughout the
procedure. Flammulina velutipes was maintained on standard agar
plates (30.0 g L.sup.-1 glucose-monohydrate; 4.5 g L.sup.-1
asparagine-monohydrate; 1.5 g L.sup.-1 KH.sub.2PO.sub.4; 0.5 g
L.sup.-1 MgSO.sub.4; 3.0 g L.sup.-1 yeast extract; 15.0 g L.sup.-1
agar agar; 1.0 mL L.sup.-1 trace metal solution containing 0.005 g
L.sup.-1 CuSO.sub.4.5H.sub.2O, 0.08 g L.sup.-1 FeCl.sub.3.
6H.sub.2O, 0.09 g L.sup.-1 ZnSO.sub.4.7H.sub.2O, 0.03 g L.sup.-1
MnSO.sub.4.H.sub.2O and 0.4 g L.sup.-1 EDTA (Ethylene diamine tetra
acetic acid). The pH of the medium was adjusted to a pH 6 with 1 M
NaOH prior to sterilisation.
[0086] Precultures were prepared by homogenisation of a 10.times.10
mm agar plug with mycelium of Flammulina velutipes in 100 mL of
sterile standard nutrition solution using an Ultra Turrax (Miccra
D-9, Art, Mullheim, Germany). Submerged cultures were maintained at
24.degree. C. and 150 rpm. After cultivation for 5 days, 50 ml
preculture were transferred into 250 ml main culture medium
consisting of minimal medium (1.5 g L.sup.-1 KH.sub.2PO.sub.4; 0.5
g L.sup.-1 MgSO.sub.4; 1.0 ml L.sup.-1 trace metal solution) and 40
g L.sup.-1 gluten or 10 mM glutamine, respectively.
Asparaginase Enzyme Preparation from Flammulina velutipes
[0087] After 18 days of cultivation, the culture was filtrated and
the extracellular asparaginase enzyme-containing supernatant (200
mL) was reversed foamed [1]. The retentate was concentrated using
ultra-filtration with a MWCO of 10,000 kDa (Millipore, Bedford,
Mass.) and separated via size exclusion chromatography at a
Superose 6 with 200 mM Tris/HCl pH 7.5.
Activity Test
[0088] 10 mM L-glutamine or 10 mM L-asparagine in 0.1 M potassium
phosphate pH 7.0, respectively, were preheated to 37.degree. C. The
resulting assay was started by addition of 50 .mu.l native or 10
.mu.l recombinant enzyme in a total reaction volume of 150 .mu.L
and stopped after 10-20 min by addition of 20 .mu.L 3% TCA or by
heating at 95.degree. C. for 10 min. A control experiment was
carried out without amino acids. Formation of product was followed
using HPLC. One unit of enzyme activity was calculated as the
amount of enzyme required to produce 1 .mu.M glutamic acid or
aspartic acid respectively, at 37.degree. C. per minute.
[0089] HPLC was performed using a C18 Nucleodur Pyramid, 5 .mu.m, 4
mm ID column, methanol as eluent A, 0.1 M sodium acetate plus
0.044% triethylamine (pH adjusted to 6.5 with HCl) as the eluent B,
o-phthaldialdehyde as the derivatisation reagent, and a
fluorescence detector.
Free Protein
[0090] The protein concentration in the hydrolysis supernatant was
estimated according to the Lowry-method using bovine serum albumin
as a standard.
Temperature and pH Optima
[0091] The determination of the temperature and pH optima of the
asparaginase enzyme was performed with enzyme solutions harvested
during the cultivation, or after the recombinant protein was
available in a soluble form. The pH optimum was examined in the
range of pH 4 to 9 (0.1 M sodium acetate pH 4, 5; 0.1 M potassium
phosphate pH 6, 7, 8; 0.1 M sodium carbonate pH 9) at 37.degree. C.
The optimal temperature determination ranged from 20 to 70.degree.
C. at optimal pH.
Temperature and pH Stability
[0092] To determine the pH stability 10 .mu.L of the recombinant
enzyme were incubated for 16 h at 37.degree. C. in 40 .mu.L of the
respective buffer above. 100 .mu.L of 10 mM glutamine in 0.1 M
potassium phosphate (pH 7) was added and the reaction was incubated
for 20 min at 37.degree. C. A control experiment was carried out
without substrate. The reaction was stopped at 95.degree. C. for 10
min. The generated glutamic acid was calculated after HPLC analysis
as described above.
[0093] For analysis of temperature stability 10 .mu.L of the
recombinant enzyme were incubated for 1 h at the respective
.degree. C. in 40 .mu.L of 0.1 M potassium phosphate buffer (pH 7).
Afterwards, 100 .mu.L of 10 mM glutamine in 0.1 M potassium
phosphate (pH 7) were added and the assay mixture was incubated for
20 min at 37.degree. C. A control experiment was carried out
without substrate. The reaction was stopped at 95.degree. C. for 10
min. The generated glutamic acid was calculated after HPLC analysis
as described above.
ESI-Tandem MS Analysis of Tryptic Peptides
[0094] The peptidase bands were excised from SDS polyacrylamide
gels, dried, and digested with trypsin. The resulting peptides were
extracted and purified according to standard protocols. A Qtof II
mass spectrometer (Micromass, U.K) equipped with a nanospray ion
source and gold-coated capillaries was used for electrospray
ionisation (ESI) MS of peptides. For collision-induced dissociation
experiments, multiple charged parent ions were selectively
transmitted from the quadrupole mass analyser into the collision
cell (25-30 eV). The resulting daughter ions were separated by an
orthogonal time-of-flight mass analyser. Peptide mass fingerprints
obtained from ESI-Tandem MS analysis were used for cross-species
protein identification in public protein primary sequence
databases.
Native-PAGE and Denaturing SDS-PAGE
[0095] SDS-PAGE analyses were performed on a 12% polyacrylamide
separation gel. Samples were prepared by mixing 20 .mu.L of
asparaginase enzyme solution and 20 .mu.L of loading buffer [0.1 M
Tris/HCl (pH 6.8), 0.2 M DTT, 4% SDS, 20% glycerol, 0.2%
bromophenol blue] and boiling for 15 min. After electrophoresis at
20 mA per gel, the gels were stained with silver or Coomassie
Brilliant Blue. For molecular determinations, marker proteins from
250 to 10 kDa (BioRad, Germany) were used.
[0096] Native PAGE was performed under non-denaturating conditions.
Samples were prepared by mixing 1:1 (v/v) with loading buffer [0.05
M Tris/HCL (pH 6.8), 2% SDS, 10% glycerol, 0.1% bromophenol blue].
After electrophoresis at 10 mA per gel and at 8.degree. C., gels
were washed 2 times in 2.5% Triton X-100. The staining procedure is
based on the deamidation of L-glutamine by glutaminase to produce
L-glutamate. The oxidation of the L-glutamate by glutamate
dehydrogenase is coupled to the reduction of a tetrazolium dye to
its colored insoluble formazan. The glutaminase-staining solution
contained 15 mM L-glutamine, 0.5 g mL.sup.-1 bovine liver glutamate
dehydrogenase, 0.1 M potassium phosphate pH 7, 2 mg mL.sup.-1 NAD,
0.04 mg mL.sup.-1 phenazine methosulfate, and 2 mg mL.sup.-1
nitroblue tetrazolium. Enzyme activity appeared after incubation at
37.degree. C. as violet bands.
Isoelectric Focusing
[0097] IEF polyacrylamide gel electrophoresis was performed on a
Multiphor II system (Pharmacia LKB, Sweden) using Servalyt.TM.
Precotes.TM. precast gels with an immobilised pH gradient from 3 to
10 (Serva, Germany) for 3500 V h (2000 V, 6 mA, 12 W). The
isoelectric points of asparaginase were estimated to be 5 using a
protein test mixture from pI 3.5 to 10.7 (Serva, Germany). Gels
were Coomassie, silver or activity stained as described above.
RNA-Preparation
[0098] RNA was prepared from 500 mg mycelium stored in
RNALater.RTM. (Invitrogen) using the NucleoSpin.RTM. RNA Plant Kit
(Macherey-Nagel, Duren, Germany).
cDNA-Synthesis
[0099] 5 .mu.g total RNA were mixed with 25 pmol 3'PCR
(ATTCTAGAGGCCGAGGCGGCCGACATG 30*T VN) and filled up to 11 .mu.l
with DEPC-treated H.sub.2O. The mixture was incubated at 70.degree.
C. for 5 min and then chilled on ice for 2 min. 4 .mu.l 5.times.
reaction buffer, 2 .mu.l dNTP mix (10 mM ea.), 0.5 .mu.l
RiboLock.TM. and 20 pmol SMART IIA (AAGCAGTGGTATCAACGCAGAGTACGCGGG)
were added, mixed and incubated at 37.degree. C. for 5 min. After
the addition of 200 U RevertAid.TM. H Minus M-MuLV Reverse
Transcriptase the mixture was incubated at 42.degree. C. for 60
min. Termination was carried out by heating at 70.degree. C. for 5
min.
[0100] Second strand synthesis was carried out by mixing 2.5 .mu.l
10.times. Long PCR buffer, 2 .mu.l dNTP mix (2.5 mM ea.), 25 pmol
5'PCR (AAGCAGTGGTATCAACGCAGAGT), 25 pmol 3'PCR, 1 .mu.l DMSO, 1 U
Long PCR Enzyme Mix, 3 .mu.l ss cDNA and ddH.sub.2O to 25
.mu.l.
[0101] The reaction mixture was incubated at 94.degree. C. for 5
min, followed by 30 cycles at 94.degree. C. for 20 s and 68.degree.
C. for 6 min, final elongation was carried out at 68.degree. C. for
20 min.
[0102] Enzymes and reagents were purchased from Fermentas, St.
Leon-Rot, Germany. Oligonucleotides were synthesized by Eurofins
MWG Operon, Ebersberg, Germany.
Sequence Fishing
[0103] Degenerated primers were deduced from peptide sequences.
PCRs were performed by mixing 2.5 .mu.l 10.times. TrueStart.TM.
Taq-buffer, 2 .mu.l dNTP mix (2.5 mM ea.), 2 .mu.l 25 mM
MgCl.sub.2, 25 pmol forward primer, 25 pmol reverse primer, 0.8
.mu.l DMSO, 0.625 U TrueStart.TM. Taq DNA Polymerase, 1 .mu.l ds
cDNA and ddH.sub.2O to 25 .mu.l.
[0104] Touchdown PCR [2] was performed by incubating the reaction
mixture at 95.degree. C. for 5 min, then for 12 cycles at
95.degree. C. for 30 s, (72.degree. C.-1.degree. C./cycle) for 60 s
and 72.degree. C. for 90 s. Another 25 cycles were carried out at
60.degree. C. annealing temperature. Final elongation was performed
at 72.degree. C. for 20 min.
[0105] PCRs were analyzed by agarose gel electrophoresis (1%
agarose (Serva, Heidelberg, Germany) cooked in TAE-buffer (40 mM
Tris, 20 mM acetic acid, 1 mM EDTA pH 8). For detection of DNA
0.05% SYBRSafe.TM. (Invitrogen) was added to the solution after it
cooled down to about 50.degree. C.
[0106] DNA extraction from agarose gels was carried out with the
NucleoSpin Extract II Kit (Macherey-Nagel).
[0107] DNA fragments were ligated into the pCR2.1.RTM. TA-Vector
(Invitrogen) by mixing 1 .mu.l vector, 1 .mu.l 10.times. T4 DNA
Ligase-buffer, 5 U T4 DNA Ligase, 0.5 .mu.l 5 mM ATP and 6.5 .mu.l
Insert-DNA. The reaction mixture was incubated at 25.degree. C. for
two hours.
[0108] For transformation 5 .mu.l ligation reaction were added to
50 .mu.l chemically competent E. coli TOP10 (Invitrogen), incubated
on ice for 20 min, heat shocked at 42.degree. C. for 45 s and
transferred back on ice. 500 .mu.l of SOC medium (Invitrogen) were
added immediately. The cells were shaked at 200 rpm and 37.degree.
C. for 60 min and then plated on LB-agar containing 50 .mu.g/ml
ampicillin and 20 .mu.g/ml X-Gal (Roth). Inoculated plates were
incubated at 37.degree. C. overnight. Selection of positive clones
was performed by colony PCR. The reaction mixture was composed as
stated above but primers M13 uni (-21) (TGTAAAACGACGGCCAGT) and M13
rev (-29) (CAGGAAACAGCTATGACC) were used. Template was added by
resuspending white colony material in the reaction mixture.
[0109] The reaction mixture was incubated at 95.degree. C. for 5
min, followed by 40 cycles at 95.degree. C. for 30 s, 55.degree. C.
for 1 min and 72.degree. C. for 1 min/kb. Final elongation was
performed at 72.degree. C. for 20 min.
[0110] Plasmid DNA was isolated with the NucleoSpin Plasmid DNA Kit
(Macherey-Nagel). Sequencing was performed by Eurofins MWG Operon
(Ebersberg, Germany).
[0111] In order to complete the sequence, specific primers were
derived from identified asparaginase DNA fragments and paired with
primers 5'PCR or 3'PCR, respectively. PCRs were carried out as
stated above with an annealing temperature of 55.degree. C. and an
elongation step of 1 min at 72.degree. C.
[0112] Amplification of the complete asparaginase sequence was
achieved with primers FvNase.sub.--5' (ATGAAATCTTTTGCCCTCTTCG) and
FvNase.sub.--3' (TCAAGCAAAGTGATGAAGG) at an annealing temperature
of 55.degree. C. and an elongation step of 1 min at 72.degree.
C.
[0113] To verify the sequence, genomic DNA was prepared from
mycelium by using the Genomic DNA Purification Kit (Fermentas). The
complete asparaginase sequence was amplified and sequenced.
Analysis of DNA and Amino Acid Sequence
[0114] Identification of an N-terminal signal sequence was carried
out by analysis with Signal P 3.0 [3]. Sequence homology was
investigated through a GenBank data base search using BLAST
[4].
[0115] Heterologous Expression in E. Coli
[0116] For cloning of asparaginase, the gene was amplified from the
plasmid DNA by PCR with flanking restriction sites EcoRI and BamHI
using the primers FvNase_EcoRI (ATAGAATTCATGAAATCTTTTGCCCTCTTC) and
FvNase_BamHI (ATAGGATCCTCAAGCAAAGTCGATGAA). The gene cassette was
digested and ligated into X-Zyme's pCTP2 expression vector to yield
the expression construct pCTP2-Aspa. The E. coli strains DH5alpha
and JM105 transformed with pCTP2-Aspa were grown in LB-medium at
37.degree. C. to an OD.sub.600 nm of 0.7, induced with 0.5 mM IPTG
and further cultured overnight. Cells were resuspended in
Tris-buffer pH 7.5, lysed with sonication and cell debris was
removed by centrifugation. Purification of asparaginase was
facilitated with ammonium sulphate.
Recombinant Asparaginase from Bacillus subtilis
[0117] The secretion of proteins from bacteria is an ATP-dependent
process which involves the translocation of a pre-protein and the
subsequent proteolytic cleavage of the pre-protein on the outside
surface of the membrane, into the mature enzyme. A signal sequence
contains all of the information necessary to target the protein to
the membrane for translocation.
[0118] Although secretion in Bacillus subtilis is not as well
understood as secretion in E. coli, it is generally assumed that it
proceeds by the same mechanism (Saier, M. H., Jr., Werner, P. K.
and Muller, M. 1989, Microbiol. Rev 53:333-366; Overhoff, B.,
Klein, M., Spies, M. and Freudl, R., 1991, Mol. Gen. Genet.
228:417-423). One difference between the two sets of secreted
proteins is the length of their signal peptides which tend to be up
to 20 amino acids longer in gram-positive than their corresponding
gram-negative counterparts. Thus, the general strategy for the
expression of heterologous proteins in gram-positive organisms such
as Bacillus subtilis involves mating the target protein to the
secretory apparatus of the host (Mountain, A., 1989, Bacillus, C.
Harwood, ed., Plenum Press, New York, 73-114). Standard protocols
using the above techniques are known in the art and were used for
the over-expression of recombinant asparaginase by Bacillus
subtilis.
Example 1
Cultivation of Flammulina velutipes
[0119] All media and equipment were autoclaved prior to use and
standard sterile techniques were applied throughout the procedure.
Flammulina velutipes was maintained on standard agar plates (30.0 g
L-1 glucose-monohydrate; 4.5 g L-1 asparagine-monohydrate; 1.5 g
L.sup.-1 KH.sub.2PO.sub.4; 0.5 g L.sup.-1 MgSO.sub.4; 3.0 g
L.sup.-1 yeast extract; 15.0 g L.sup.-1 agar agar; 1.0 mL L.sup.-1
trace metal solution containing 0.005 g L.sup.1
CuSO.sub.4.5H.sub.2O, 0.08 g L.sup.-1 FeCl.sub.3.6H.sub.2O, 0.09 g
L.sup.-1 ZnSO.sub.4.7H.sub.2O, 0.03 g L.sup.-1 MnSO.sub.4H.sub.2O
and 0.4 g L.sup.-1 EDTA. The pH of the medium was adjusted to pH 6
with 1 M NaOH prior to sterilisation. Precultures were prepared by
homogenisation of a 10.times.10 mm agar plug with mycelium of
Flammulina velutipes in 100 mL of sterile standard nutrition
solution using an Ultra Turrax (Miccra D-9, Art, Mullheim,
Germany). Submerged cultures were maintained at 24.degree. C. and
150 rpm. After cultivation for 5 days, 50 ml preculture were
transferred into 250 ml main culture medium consisting of minimal
medium (1.5 g L.sup.-1 KH.sub.2PO.sub.4; 0.5 g L.sup.-1 MgSO.sub.4;
1.0 ml L.sup.-1 trace metal solution) and 40 g L.sup.-1 gluten or
10 mM glutamine, respectively.
Example 2
Enzyme Preparation from Flammulina velutipes
[0120] After 18 days of cultivation, the culture was filtrated and
the extracellular enzyme-containing supernatant (200 mL) was
reverse-foamed, the asparaginase and another protein being the only
proteins left in the supernatant. The remaining liquid was
concentrated using ultra-filtration (MWCO 10,000), and both
proteins were separated via size exclusion chromatography at a
Superose 6.
[0121] Most of the hydrolytic activity originally present was
recovered indicating that this protocol yielded a useful enzyme
concentrate through two steps only.
Example 3
Hydrolysis of L-Asparagine Using Native Enzyme
[0122] 100 .mu.L of 10 mM asparagine in 0.1 M
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer (pH 7.0) were preheated at
37.degree. C. for 5 min. The reaction was started with the addition
of 50 .mu.L enzyme solution. After an incubation time of 20 min at
37.degree. C. and 400 rpm in a thermoshaker, the assay was stopped
by the addition of 20 .mu.L TCA. A control experiment was carried
out without substrate. The contents of aspartic acid were
quantitatively measured with the HPLC after OPA-derivatisation, and
the difference between sample and control was used to calculate
then enzyme's activity.
[0123] The analytical evidence indicates a fast enzymatic
hydrolysis of the substrate L-asparagine.
Example 4
Hydrolysis of L-Glutamine Using Native Enzyme
[0124] 100 .mu.L of 10 mM glutamine in 0.1 M
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer (pH 7.0) were preheated at
37.degree. C. for 5 min. The reaction was started with the addition
of 50 .mu.l enzyme solution. After an incubation time of 20 min at
37.degree. C. and 400 rpm in a thermoshaker, the assay was stopped
by the addition of 20 .mu.l TCA (Trichloroacetic acid). A control
experiment was carried out without substrate. The contents of
glutamic acid were quantitatively measured with the HPLC after
OPA-derivatisation, and the difference between sample and control
was used to calculate the enzyme's activity.
[0125] This analytical evidence indicated a useful side activity of
the asparaginase towards the substrate L-glutamine.
Example 5
Hydrolysis of L-Asparagine Using Recombinant Enzyme
[0126] 140 .mu.l of 10 mM asparagine in 0.1 M
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer (pH 7.0) were preheated at
37.degree. C. for 5 min. The reaction was started with the addition
of 10 .mu.l recombinant enzyme solution 200 times diluted with
water. After an incubation time of 10 min at 37.degree. C. and 400
rpm in a thermoshaker, the assay was stopped by heating at
95.degree. C. for 10 min. A control experiment was carried out
without substrate. The contents of aspartic acid were
quantitatively measured with the HPLC after OPA-derivatisation, and
the difference between sample and control was used to calculate the
enzyme's activity. The activity of the recombinant asparaginase
enzyme towards asparagine was calculated to be 43.3 kU
L.sup.-1.
Example 6
Hydrolysis of L-Glutamine Using Recombinant Enzyme
[0127] 140 .mu.L of 10 mM glutamine in 0.1 M
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer (pH 7.0) were preheated at
37.degree. C. for 5 min. The reaction was started with the addition
of 10 .mu.l recombinant enzyme solution 200 times diluted with
water. After an incubation time of 10 min at 37.degree. C. and 400
rpm in a thermoshaker, the assay was stopped by heating at
95.degree. C. for 10 min. Blanks were prepared without the
substrate. The contents of glutamic acid were quantitatively
measured with the HPLC after OPA-derivatisation, and the difference
between sample and blank was used to calculate enzyme's activity.
The activity of the recombinant asparaginase towards glutamine was
calculated to be 4.3 kU L.sup.-1.
[0128] Having thus described the present invention in detail, it is
to be understood that the detailed description is not intended to
limit the scope of the invention thereof.
Sequence CWU 1
1
121123PRTFlammulina velutipes 1Met Lys Ser Phe Ala Leu Phe Val Pro
Leu Ile Val Ala Ala Val Val 1 5 10 15 Asn Ser Ala Val Val Thr Phe
Ser Thr Gly Leu Gly Cys Asn Ser Val 20 25 30 Ser Gln Thr Tyr Arg
Gly Asn Gly Asn Phe Cys Ala Asp Pro Pro Gly 35 40 45 Asp Trp Ser
Ser Val Gly Phe Ser Glu Ile Gly Gly Asp Asn Arg Val 50 55 60 Thr
Val His Asn Gln Asn Ser Cys Thr Pro Ala Ser Gln Val Gly Gln 65 70
75 80 Gly Phe Gly Pro Ala Cys Trp Asn Gln Gly Ala Thr Lys Leu Arg
Ser 85 90 95 Ala Trp Val Ala Cys Pro Gly Gln Arg Leu Ala Glu Asn
Gly Thr Ile 100 105 110 Val Asp Asp Asp Gly Ala Phe Ile Asp Phe Ala
115 120 227DNAArtificialPrimer sequence 2attctagagg ccgaggcggc
cgacatg 27330DNAArtificialPrimer 3aagcagtggt atcaacgcag agtacgcggg
30423DNAArtificialPrimer 4aagcagtggt atcaacgcag agt
23518DNAArtificialPrimer 5tgtaaaacga cggccagt
18618DNAArtificialPrimer 6caggaaacag ctatgacc
18722DNAArtificialPrimer 7atgaaatctt ttgccctctt cg
22819DNAArtificialPrimer 8tcaagcaaag tgatgaagg
19930DNAArtificialPrimer 9atagaattca tgaaatcttt tgccctcttc
301027DNAArtificialPrimer 10ataggatcct caagcaaagt cgatgaa
2711477DNAFlammulina velutipes 11atgaaatctt ttgccctctt cgtccctctc
atcgttgctg ctgtcgtcaa cagcgccgtg 60gtcacctttt ccaccggcct tggctgcaac
tctgtctcgc agacctaccg tggcaactgc 120aacttctgcg ctgacccacc
cggcggtagg tctacttcac attgatccct atagctcgat 180gctcatctca
tctactagac tggagctcag tcggcttttc tgagatcgga ggcgacaacc
240gcgtcaccgt tcataaccag aacagctgca cccccgcttc gcaggtcggc
caaggctttg 300gaccggcctg ctggaaccaa ggcgctacca agcttcgttc
tgcttgggtt gcgtgccctg 360gacagaggtg agtcggttct ctgcagcttc
ttcttttggt ttactaacga tgccactaga 420ctcgctgaga acggtaccat
cgtcgacgac gacggcgcct tcatcgactt tgcttga 47712372DNAFlammulina
velutipes 12atgaaatctt ttgccctctt cgtccctctc atcgttgctg ctgtcgtcaa
cagcgccgtg 60gtcacctttt ccaccggcct tggctgcaac tctgtctcgc agacctaccg
tggcaactgc 120aacttctgcg ctgacccacc cggcgactgg agctcagtcg
gcttttctga gatcggaggc 180gacaaccgcg tcaccgttca taaccagaac
agctgcaccc ccgcttcgca ggtcggccaa 240ggctttggac cggcctgctg
gaaccaaggc gctaccaagc ttcgttctgc ttgggttgcg 300tgccctggac
agagactcgc tgagaacggt accatcgtcg acgacgacgg cgccttcatc
360gactttgctt ga 372
* * * * *
References